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WO2001011341A2 - Systeme criblage de cellules - Google Patents

Systeme criblage de cellules Download PDF

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Publication number
WO2001011341A2
WO2001011341A2 PCT/US2000/021426 US0021426W WO0111341A2 WO 2001011341 A2 WO2001011341 A2 WO 2001011341A2 US 0021426 W US0021426 W US 0021426W WO 0111341 A2 WO0111341 A2 WO 0111341A2
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Prior art keywords
cells
cell
mitotic
protein
image
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PCT/US2000/021426
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English (en)
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WO2001011341A3 (fr
Inventor
Keith Olson
Oleg Lapets
Gary Bright
Randall O. Shopoff
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Cellomics, Inc.
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Application filed by Cellomics, Inc. filed Critical Cellomics, Inc.
Priority to AU66228/00A priority Critical patent/AU6622800A/en
Publication of WO2001011341A2 publication Critical patent/WO2001011341A2/fr
Publication of WO2001011341A3 publication Critical patent/WO2001011341A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition

Definitions

  • This invention is in the field of fluorescence-based cell and molecular biochemical assays for drug discovery.
  • Drug discovery is a long, multiple step process involving identification of specific disease targets, development of an assay based on a specific target, validation of the assay, optimization and automation of the assay to produce a screen, high throughput screening of compound libraries using the assay to identify "hits", hit validation and hit compound optimization.
  • the output of this process is a lead compound that goes into pre-clinical and, if validated, eventually into clinical trials.
  • the screening phase is distinct from the assay development phases, and involves testing compound efficacy in living biological systems.
  • drug discovery is a slow and costly process, spanning numerous years and consuming hundreds of millions of dollars per drug created.
  • Figure 12 is a flow chart defining the processing steps in the High Throughput mode of the System for Cell Based Screening.
  • Biosensors Macromolecules consisting of a biological functional domain and a luminescent probe or probes that report the environmental changes that occur either internally or on their surface.
  • a class of luminescently labeled macromolecules designed to sense and report these changes have been termed "fluorescent-protein biosensors".
  • the protein component of the biosensor provides a highly evolved molecular recognition moiety.
  • a fluorescent molecule attached to the protein component in the proximity of an active site transduces environmental changes into fluorescence signals that are detected using a system with an appropriate temporal and spatial resolution such as the cell scanning system of the present invention. Because the modulation of native protein activity within the living cell is reversible, and because fluorescent-protein biosensors can be designed to sense reversible changes in protein activity, these biosensors are essentially reusable.
  • Figure 3 shows a schematic drawing of a preferred camera assembly.
  • a digital cable 30 transports digital signals to the computer.
  • the standard optical configurations described above use microscope optics to directly produce an enlarged image of the specimen on the camera sensor in order to capture a high resolution image of the specimen. This optical system is commonly referred to as 'wide field' microscopy. Those skilled in the art of microscopy will recognize that a high resolution image of the specimen can be created by a variety of other optical systems, including, but not limited to, standard scanning confocal detection of a focused point or line of illumination scanned over the specimen (Go et al.
  • a high throughput system is directly coupled with the HCS either on the same platform or on two separate platforms connected electronically (e.g. via a local area network).
  • This embodiment of the invention referred to as a dual mode optical system, has the advantage of increasing the throughput of a HCS by coupling it with a HTS and thereby requiring slower high resolution data acquisition and analysis only on the small subset of wells that show a response in the coupled HTS.
  • an exemplified two platform dual mode optical system consists of two light optical instruments, a high throughput platform 60 and a high content platform 65 ⁇ which read fluorescent signals emitted from cells cultured in microtiter plates or microwell arrays on a microplate, and communicate with each other via an electronic connection 64.
  • the high throughput platform 60 analyzes all the wells in the whole plate either in parallel or rapid serial fashion.
  • the HTS software residing on the system's computer 62, controls the high throughput instrument, and results are displayed on the monitor 61.
  • the HCS software residing on it's computer system 67, controls the high content instrument hardware 65, optional devices (e.g. plate loader, environmental chamber, fluid dispenser), analyzes digital image data from the plate, displays results on the monitor 66 and manages data measured in an integrated database.
  • the two systems can also share a single computer, in which case all data would be collected, processed and displayed on that computer, without the need for a local area network to transfer the data.
  • Microtiter plates are transferred from the high throughput system to the high content system 63 either manually or by a robotic plate transfer device, as is well known in the art (Beggs (1997), supra; Mcaffrey (1996), supra).
  • the dual mode optical system utilizes a single platform system ( Figure 7). It consists of two separate optical modules, an HCS module 203 and an HTS module 209 that can be independently or collectively moved so that only one at a time is used to collect data from the microtiter plate 201.
  • the microtiter plate 201 is mounted in a motorized X,Y stage so it can be positioned for imaging in either HTS or HCS mode. After collecting and analyzing the HTS image data as described below, the HTS optical module 209 is moved out of the optical path and the HCS optical module 203 is moved into place.
  • the cell screening system further comprises a fluid delivery device for use with the live cell embodiment of the method of cell screening (see below).
  • Figure 8 exemplifies a fluid delivery device for use with the system of the invention. It consists of a bank of 12 syringe pumps 701 driven by a single motor drive. Each syringe 702 is sized according to the volume to be delivered to each well, typically between 1 and 100 ⁇ L. Each syringe is attached via flexible tubing 703 to a similar bank of connectors which accept standard pipette tips 705. The bank of pipette tips are attached to a drive system so they can be lowered and raised relative to the microtiter plate 706 to deliver fluid to each well.
  • the present invention provides a method for analyzing cells comprising providing an anay of locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecule in the cells; converting the fluorescent signals into digital data; and utilizing the digital data to determine the distribution, environment or activity of the fluorescent reporter molecule within the cells.
  • the method of the present invention is based on the high affinity of fluorescent or luminescent molecules for specific cellular components.
  • the affinity for specific components is governed by physical forces such as ionic interactions, covalent bonding (which includes chimeric fusion with protein-based chromophores, fluorophores, and lumiphores), as well as hydrophobic interactions, electrical potential, and, in some cases, simple entrapment within a cellular component.
  • the luminescent probes can be small molecules, labeled macromolecules, or genetically engineered proteins, including, but not limited to green fluorescent protein chimeras.
  • the luminescent probes can be synthesized within the living cell or can be transported into the cell via several non-mechanical modes including diffusion, facilitated or active transport, signal-sequence-mediated transport, and endocytotic or pinocytotic uptake.
  • Mechanical bulk loading methods which are well known in the art, can also be used to load luminescent probes into living cells (Barber et al. (1996), Neuroscience Letters 207:17-20; Bright et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), pp. 153-173).
  • cells can be genetically engineered to express reporter molecules, such as GFP, coupled to a protein of interest as previously described (Chalfie and Prasher U.S. Patent No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science 20:448-455).
  • the luminescent probes accumulate at their target domain as a result of specific and high affinity interactions with the target domain or other modes of molecular targeting such as signal-sequence-mediated transport.
  • Fluorescently labeled reporter molecules are useful for determining the location, amount and chemical environment of the reporter. For example, whether the reporter is in a lipophilic membrane environment or in a more aqueous environment can be determined (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomolecular Structure 24:405-434; Giuliano and Taylor (1995), Methods in Neuroscience 27:1-16). The pH environment of the reporter can be determined (Bright et al. (1989), J.
  • fluorescent reporter molecules exhibit a change in excitation or emission spectra, some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a loss (quenching) or appearance of fluorescence, while some report rotational movements (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol. Structure 24:405-434; Giuliano et al. (1995), Methods in Neuroscience 27: 1-16).
  • a preferred embodiment is provided to analyze cells that comprises operator-directed parameters being selected based on the assay being conducted, data acquisition by the cell screening system on the distribution of fluorescent signals within a sample, and interactive data review and analysis.
  • the operator enters information 100 that describes the sample, specifies the filter settings and fluorescent channels to match the biological labels being used and the information sought, and then adjusts the camera settings to match the sample brightness.
  • the software allows selection of various parameter settings used to identify nuclei and cytoplasm, and selection of different fluorescent reagents, identification of cells of interest based on mo ⁇ hology or brightness, and cell numbers to be analyzed per well. These parameters are stored in the system's for easy retrieval for each automated run.
  • the software dynamically displays the scan status, including the number of cells analyzed, the current well being analyzed, images of each independent wavelength as they are acquired, and the result of the screen for each well as it is determined.
  • the plate 4 ( Figure 1) is scanned in a se ⁇ entine style as the software automatically moves the motorized microscope XY stage 3 from well to well and field to field within each well of a 96-well plate.
  • Those skilled in the programming art will recognize how to adapt software for scanning of other microplate formats such as 24, 48, and 384 well plates.
  • the scan pattern of the entire plate as well as the scan pattern of fields within each well are programmed.
  • the system adjusts sample focus with an autofocus procedure 104 ( Figure 9) through the Z axis focus drive 5, controls filter selection via a motorized filter wheel 19, and acquires and analyzes images of up to four different colors ("channels" or "wavelengths").
  • the autofocus procedure is called at a user selected frequency, typically for the first field in each well and then once every 4 to 5 fields within each well.
  • the autofocus procedure calculates the starting Z-axis point by inte ⁇ olating from the pre-calculated plane focal model. Starting a programmable distance above or below this set point, the procedure moves the mechanical Z-axis through a number of different positions, acquires an image at each position, and finds the maximum of a calculated focus score that estimates the contrast of each image. The Z position of the image with the maximum focus score determines the best focus for a particular field.
  • Those skilled in the art will recognize this as a variant of automatic focusing methods as described in Harms et al. in Cytometry 5 (1984), 236-243, Groen et al.
  • images are acquired of a primary marker 105 ( Figure 9) (typically cell nuclei counterstained with DAPI or PI fluorescent dyes) which are segmented ("identified") using an adaptive thresholding procedure.
  • the adaptive thresholding procedure 106 is used to dynamically select the threshold of an image for separating cells from the background.
  • the staining of cells with fluorescent dyes can vary to an unknown degree across cells in a microtiter plate sample as well as within images of a field of cells within each well of a microtiter plate. This variation can occur as a result of sample preparation and/or the dynamic nature of cells.
  • a global threshold is calculated for the complete image to separate the cells from background and account for field to field variation.
  • the software determines if the object meets the criteria for a valid cell nucleus 111 by measuring its mo ⁇ hological features (size and shape). For each valid cell, the XYZ stage location is recorded, a small image of the cell is stored, and features are measured 112.
  • the cell scanning method of the present invention can be used to perform many different assays on cellular samples by applying a number of analytical methods simultaneously to measure features at multiple wavelengths.
  • An example of one such assay provides for the following measurements:
  • the shape of the cell nucleus for color 1 is described by three shape features: a) perimeter squared area b) box area ratio c) height width ratio
  • the area of the cytoplasmic mask 7.
  • Feature 10 describes a screen used for counting of DNA or RNA probes within the nuclear region in colors 2-4.
  • probes are commercially available for identifying chromosome-specific DNA sequences (Life Technologies, Gaifhersburg, MD; Genosys, Woodlands, TX; Biotechnologies, Inc., Richmond, CA; Bio 101, Inc., Vista, CA) Cells are three-dimensional in nature and when examined at a high magnification under a microscope one probe may be in-focus while another may be completely out-of-focus.
  • the cell screening method of the present invention provides for detecting three-dimensional probes in nuclei by acquiring images from multiple focal planes.
  • the software moves the Z-axis motor drive 5 ( Figure 1) in small steps where the step distance is user selected to account for a wide range of different nuclear diameters.
  • an image is acquired.
  • the maximum gray-level intensity from each pixel in each image is found and stored in a resulting maximum projection image.
  • the maximum projection image is then used to count the probes.
  • the above method works well in counting probes that are not stacked directly above or below another one.
  • users can select an option to analyze probes in each of the focal planes acquired. In this mode, the scanning system performs the maximum plane projection method as discussed above, detects probe regions of interest in this image, then further analyzes these regions in all the focal plane images.
  • the system After measuring cell features 112 ( Figure 9), the system checks if there are any unprocessed objects in the current field 113. If there are any unprocessed objects, it locates the next object 110 and determines whether it meets the criteria for a valid cell nucleus 111, and measures its features. Once all the objects in the current field are processed, the system determines whether analysis of the cureent plate is complete 114; if not, it determines the need to find more cells in the curcent well 115. If the need exists, the system advances the XYZ stage to the next field within the cunent well 109 or advances the stage to the next well 116 of the plate.
  • reports can be generated on one or more statistics of the measured features.
  • Users can generate a graphical report of data summarized on a well-by-well basis for the scanned region of the plate using an interactive report generation procedure 120.
  • This report includes a summary of the statistics by well in tabular and graphical format and identification information on the sample.
  • the report window allows the operator to enter comments about the scan for later retrieval.
  • Multiple reports can be generated on many statistics and be printed with the touch of one button. Reports can be previewed for placement and data before being printed.
  • the above-recited embodiment of the method operates in a single high resolution mode refened to as the high content screening (HCS) mode.
  • HCS high content screening
  • the HCS mode provides sufficient spatial resolution within a well (on the order of 1 ⁇ m) to define the distribution of material within the well, as well as within individual cells in the well.
  • the high degree of information content accessible in that mode comes at the expense of speed and complexity of the required signal processing.
  • a high throughput system is directly coupled with the HCS either on the same platform or on two separate platforms connected electronically (e.g. via a local area network).
  • This embodiment of the invention referred to as a dual mode optical system, has the advantage of increasing the throughput of an HCS by coupling it with an HTS and thereby requiring slower high resolution data acquisition and analysis only on the small subset of wells that show a response in the coupled HTS.
  • High throughput 'whole plate' reader systems are well known in the art and are commonly used as a component of an HTS system used to screen large numbers of compounds (Beggs et al. (1997), supra; McCaffrey et al. (1996), supra ).
  • the HTS of the present invention is earned out on the microtiter plate or microwell anay by reading many or all wells in the plate simultaneously with sufficient resolution to make determinations on a well-by-well basis. That is, calculations are made by averaging the total signal output of many or all the cells or the bulk of the material in each well. Wells that exhibit some defined response in the HTS (the 'hits') are flagged by the system. Then on the same microtiter plate or microwell anay, each well identified as a hit is measured via HCS as described above.
  • the dual mode process involves:
  • High throughput processing 306 is first performed on the microtiter plate or microwell array by acquiring and analyzing the signal from each of the wells in the plate.
  • the processing performed in high throughput mode 307 is illustrated in Figure 12 and described below.
  • Wells that exhibit some selected intensity response in this high throughput mode (“hits") are identified by the system.
  • the system performs a conditional operation 308 that tests for hits. If hits are found, those specific hit wells are further analyzed in high content (micro level) mode 309.
  • the processing performed in high content mode 312 is illustrated in Figure 13.
  • the system updates 310 the informatics database 311 with results of the measurements on the plate. If there are more plates to be analyzed 313 the system loads the next plate 303; otherwise the analysis of the plates terminates 314.
  • the system begins the HTS acquisition and analysis 401.
  • the HTS optical module is selected by controlling a motorized optical positioning device 402 on the dual mode system.
  • data from a primary marker on the plate is acquired 403 and wells are isolated from the plate background using a masking procedure 404. Images are also acquired in other fluorescence channels being used 405. The region in each image conesponding to each well 406 is measured 407.
  • a feature calculated from the measurements for a particular well is compared with a predefined threshold or intensity response 408. and based on the result the well is either flagged as a "hit" 409 or not. The locations of the wells flagged as hits are recorded for subsequent high content mode processing. If there are wells remaining to be processed 410 the program loops back 406 until all the wells have been processed 411 and the system exits high throughput mode. Following HTS analysis, the system starts the high content mode processing
  • This binary image also called a mask in the art, is used to determine if the field contains objects 507.
  • the mask is labeled with a blob labeling method whereby each object (or blob) has a unique number assigned to it. If objects are found in the field, images are acquired for all other active channels 508. otherwise the stage is advanced to the next field 514 in the current well. Each object is located in the image for further analysis 509. Mo ⁇ hological features, such as area and shape of the objects, are used to select objects likely to be cell nuclei 510. and discard (do no further processing on) those that are considered artifacts. For each valid cell nucleus, the XYZ stage location is recorded, a small image of the cell is stored, and assay specific features are measured 511.
  • the system then performs multiple tests on the cells by applying several analytical methods to measure features at each of several wavelengths. After measuring the cell features, the systems checks if there are any unprocessed objects in the current field 512. If there are any unprocessed objects, it locates the next object 509 and determines whether it meets the criteria for a valid cell nucleus 510, and measures its features. After processing all the objects in the cunent field, the system deteremines whether it needs to find more cells or fields in the current well 513. If it needs to find more cells or fields in the current well it advances the XYZ stage to the next field within the current well 515. Otherwise, the system checks whether it has any remaining hit wells to measure 515. If so, it advances to the next hit well 503 and proceeds through another cycle of acquisition and analysis, otherwise the HCS mode is finished 516.
  • a method of kinetic live cell screening is provided.
  • the previously described embodiments of the invention are used to characterize the spatial distribution of cellular components at a specific point in time, the time of chemical fixation.
  • these embodiments have limited utility for implementing kinetic based screens, due to the sequential nature of the image acquisition, and the amount of time required to read all the wells on a plate. For example, since a plate can require 30 - 60 minutes to read through all the wells, only very slow kinetic processes can be measured by simply preparing a plate of live cells and then reading through all the wells more than once. Faster kinetic processes can be measured by taking multiple readings of each well before proceeding to the next well, but the elapsed time between the first and last well would be too long, and fast kinetic processes would likely be complete before reaching the last well.
  • the kinetic live cell extension of the invention enables the design and use of screens in which a biological process is characterized by its kinetics instead of, or in addition to, its spatial characteristics.
  • a response in live cells can be measured by adding a reagent to a specific well and making multiple measurements on that well with the appropriate timing.
  • This dynamic live cell embodiment of the invention therefore includes apparatus for fluid delivery to individual wells of the system in order to deliver reagents to each well at a specific time in advance of reading the well. This embodiment thereby allows kinetic measurements to be made with temporal resolution of seconds to minutes on each well of the plate.
  • the acquisition control program is modified to allow repetitive data collection from sub-regions of the plate, allowing the system to read other wells between the time points required for an individual well.
  • Figure 8 describes an example of a fluid delivery device for use with the live cell embodiment of the invention and is described above.
  • This set-up allows one set of pipette tips 705, or even a single pipette tip, to deliver reagent to all the wells on the plate.
  • the bank of syringe pumps 701 can be used to deliver fluid to 12 wells simultaneously, or to fewer wells by removing some of the tips 705.
  • the temporal resolution of the system can therefore be adjusted, without sacrificing data collection efficiency, by changing the number of tips and the scan pattern as follows.
  • the data collection and analysis from a single well takes about 5 seconds. Moving from well to well and focusing in a well requires about 5 seconds, so the overall cycle time for a well is about 10 seconds.
  • the overall time to collect a kinetic data set from the plate is then simply the time to perform a single scan of the plate, multiplied by the number of time points required. Typically, 1 time point before addition of compounds and 2 or 3 time points following addition should be sufficient for screening pu ⁇ oses.
  • Figure 14 shows the acquisition sequence used for kinetic analysis.
  • the start of processing 801 is configuration of the system, much of which is identical to the standard HCS configuration.
  • the operator must enter information specific to the kinetic analysis being performed 802, such as the sub-region size, the number of time points required, and the required time increment.
  • a sub-region is a group of wells that will be scanned repetitively in order to accumulate kinetic data.
  • the size of the sub-region is adjusted so that the system can scan a whole sub-region once during a single time increment, thus minimizing wait times.
  • the optimum sub-region size is calculated from the setup parameters, and adjusted if necessary by the operator.
  • the system then moves the plate to the first sub-region 803.
  • the system After processing each well in a sub-region, the system checks to see if all the wells in the sub-region have been processed 806 ( Figure 14), and cycles through all the wells until the whole region has been processed. The system then moves the plate into position for fluid addition, and controls fluidic system delivery of fluids to the entire sub-region 807. This may require multiple additions for sub-regions which span several rows on the plate, with the system moving the plate on the X,Y stage between additions. Once the fluids have been added, the system moves to the first well in the sub-region 808 to begin acquisition of time points. The data is acquired from each well 809 and as before the system cycles through all the wells in the sub-region 810.
  • cell screening methods and machine readable storage medium comprising a program containing a set of instructions for causing a cell screening system to execute procedures for defining the distribution and activity of specific cellular constituents and processes.
  • the cell screening system comprises a high magnification fluorescence optical system with a stage adapted for holding cells and a means for moving the stage, a digital camera, a light source for receiving and processing the digital data from the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • This "aspect of the invention comprises programs that instruct the cell screening system to define the distribution and activity of specific cellular constituents and processes, using the luminescent probes, the optical imaging system, and the pattern recognition software of the invention.
  • Prefened embodiments of the machine readable storage medium comprise programs consisting of a set of instructions for causing a cell screening system to execute the procedures set forth in Figures 9, 11, 12, 13, 14 or 15.
  • Another preferred embodiment comprises a program consisting of a set of instructions for causing a cell screening system to execute procedures for detecting the distribution and activity of specific cellular constituents and processes.
  • the cellular processes include, but are not limited to, nuclear translocation of a protein, cellular mo ⁇ hology, apoptosis, receptor intemalization, and protease-induced translocation of a protein.
  • the cell screening methods are used to identify compounds that modify the various cellular processes.
  • the cells can be contacted with a test compound, and the effect of the test compound on a particular cellular process can be analyzed.
  • the cells can be contacted with a test compound and a known agent that modifies the particular cellular process, to determine whether the test compound can inhibit or enhance the effect of the known agent.
  • the methods can be used to identify test compounds that increase or decrease a particular cellular response, as well as to identify test compounds that affects the ability of other agents to increase or decrease a particular cellular response.
  • the locations containing cells are analyzed using the above methods at low resolution in a high throughput mode, and only a subset of the locations containing cells are analyzed in a high content mode to obtain luminescent signals from the luminescently labeled reporter molecules in subcellular compartments of the cells being analyzed.
  • Regulation of transcription of some genes involves activation of a transcription factor in the cytoplasm, resulting in that factor being transported into the nucleus where it can initiate transcription of a particular gene or genes.
  • This change in transcription factor distribution is the basis of a screen for the cell-based screening system to detect compounds that inhibit or induce transcription of a particular gene or group of genes.
  • a general description of the screen is given followed by a specific example.
  • the distribution of the transcription factor is determined by labeling the nuclei with a DNA specific fluorophore like Hoechst 33423 and the transcription factor with a specific fluorescent antibody.
  • an image of the nuclei is acquired in the cell-based screening system and used to create a mask by one of several optional thresholding methods, as described supra.
  • the mo ⁇ hological descriptors of the regions defined by the mask are compared with the user defined parameters and valid nuclear masks are identified and used with the following method to extract transcription factor distributions.
  • Each valid nuclear mask is eroded to define a slightly smaller nuclear region.
  • the original nuclear mask is then dilated in two steps to define a ring shaped region around the nucleus, which represents a cytoplasmic region.
  • the average antibody fluorescence in each of these two regions is determined, and the difference between these averages is defined as the NucCyt Difference.
  • Figure 10A illustrates an unstimulated cell with its nucleus 200 labeled with a blue fluorophore and a transcription factor in the cytoplasm 201 labeled with a green fluorophore.
  • Figure 10B illustrates the nuclear mask 202 derived by the cell-based screening system.
  • Figure 10C illustrates the cytoplasm 203 of the unstimulated cell imaged at a green wavelength.
  • Figure 10D illustrates the nuclear mask 202 is eroded (reduced) once to define a nuclear sampling region 204 with minimal cytoplasmic distribution.
  • the nucleus boundary 202 is dilated (expanded) several times to form a ring that is 2-3 pixels wide that is used to define the cytoplasmic sampling region 205 for the same cell.
  • Figure 10E further illustrates a side view which shows the nuclear sampling region 204 and the cytoplasmic sampling region 205. Using these two sampling regions, data on nuclear translocation can be automatically analyzed by the cell-based screening system on a cell by cell basis.
  • Figure 10F-J illustrates the strategy for determining nuclear translocation in a stimulated cell.
  • Figure 10F illustrates a stimulated cell with its nucleus 206 labeled with a blue fluorophore and a transcription factor in the cytoplasm 207 labeled with a green fluorophore.
  • the nuclear mask 208 in Figure 10G is derived by the cell based screening system.
  • Figure 10H illustrates the cytoplasm 209 of a stimulated cell imaged at a green wavelength.
  • Figure 101 illustrates the nuclear sampling region 211 and cytoplasmic sampling region 212 of the stimulated cell.
  • Figure 10J further illustrates a side view which shows the nuclear sampling region 211 and the cytoplasmic sampling region 212.
  • a specific application of this method has been used to validate this method as a screen.
  • a human cell line was plated in 96 well microtiter plates. Some rows of wells were titrated with IL-1, a known inducer of the NF-KB transcription factor. The cells were then fixed and stained by standard methods with a fluorescein labeled antibody to the transcription factor, and Hoechst 33423. The cell-based screening system was used to acquire and analyze images from this plate and the NucCyt Difference was found to be strongly conelated with the amount of agonist added to the wells as illustrated in Figure 16.
  • NucCyt Difference gives consistent results over a wide range of cell densities and reagent concentrations, and can therefore be routinely used to screen compound libraries for specific nuclear translocation activity.
  • the same method can be used with antibodies to other transcription factors, or GFP-transcription factor chimeras, or fluorescently labeled transcription factors introduced into living or fixed cells, to screen for effects on the regulation of transcription factor activity.
  • Figure 18 is a representative display on a PC screen of data which was obtained in accordance with Example 1.
  • Graph 1 180 plots the difference between the average antibody fluorescence in the nuclear sampling region and cytoplasmic sampling region, NucCyt Difference verses Well #.
  • Graph 2 181 plots the average fluorescence of the antibody in the nuclear sampling region, NP1 average, versus the Well #.
  • Graph 3 182 plots the average antibody fluorescence in the cytoplasmic sampling region, LIP1 average, versus Well #.
  • the software permits displaying data from each cell.
  • Figure 18 shows a screen display 183. the nuclear image 184. and the fluorescent antibody image 185 for cell #26.
  • NucCyt Difference refened to in graph 1 180 of Figure 18 is the difference between the average cytoplasmic probe (fluorescent reporter molecule) intensity and the average nuclear probe (fluorescent reporter molecule) intensity.
  • NP1 average refened to in graph 2 181 of Figure 18 is the average of cytoplasmic probe (fluorescent reporter molecule) intensity within the nuclear sampling region.
  • L1P1 average referred to in graph 3 182 of Figure 18 is the average probe (fluorescent reporter molecule) intensity within the cytoplasmic sampling region.
  • this aspect of the invention can be performed using other transcription factors that translocate from the cytoplasm to the nucleus upon activation.
  • activation of the c-fos transcription factor was assessed by defining its spatial position within cells. Activated c-fos is found only within the nucleus, while inactivated c-fos resides within the cytoplasm.
  • 3T3 cells were plated at 5000-10000 cells per well in a Polyfiltronics 96- well plate. The cells were allowed to attach and grow overnight.
  • the cells were rinsed twice with 100 ⁇ l serum-free medium, incubated for 24-30 hours in serum-free MEM culture medium, and then stimulated with platelet derived growth factor (PDGF-BB) (Sigma Chemical Co., St. Louis, MO) diluted directly into serum free medium at concentrations ranging from 1-50 ng/ml for an average time of 20 minutes.
  • PDGF-BB platelet derived growth factor
  • HBSS IX Hanks buffered saline solution
  • HBSS HBSS
  • 50 ⁇ l of the dilution was applied to each well.
  • Cells were incubated in the presence of primary antibody for one hour at room temperature, and then incubated for one hour at room temperature in a light tight container with goat anti-rabbit secondary antibody conjugated to ALEXATM 488 (Molecular Probes), diluted 1:500 from a 100 ⁇ g/ml stock in HBSS.
  • Hoechst DNA dye (Molecular Probes) was then added at a 1 :1000 dilution of the manufacturer's stock solution (10 mg/ml).
  • the cells were then washed with HBSS, and the plate was sealed prior to analysis with the cell screening system of the invention.
  • the data from these experiments demonstrated that the methods of the invention could be used to measure transcriptional activation of c-fos by defining its spatial position within cells.
  • transcription factors include, but are not limited to fos and jun homologs, NF-KB (nuclear factor kappa from B cells), NFAT (nuclear factor of activated T-lymphocytes), and STATs (signal transducer and activator of transcription) factors (For example, see Strehlow, I., and Schindler, C. 1998. J. Biol. Chem. 273:28049-28056; Chow, et al. 1997 Science.
  • indicator cells are treated with test compounds and the distribution of luminescently labeled transcription factor is measured in space and time using a cell screening system, such as the one disclosed above.
  • the luminescently labeled transcription factor may be expressed by or added to the cells either before, together with, or after contacting the cells with a test compound.
  • the transcription factor may be expressed as a luminescently labeled protein chimera by transfected indicator cells.
  • the luminescently labeled transcription factor may be expressed, isolated, and bulk-loaded into the indicator cells as described above, or the transcription factor may be luminescently labeled after isolation.
  • the transcription factor is expressed by the indicator cell, which is subsequently contacted with a luminescent label, such as an antibody, that detects the transcription factor.
  • kits for analyzing transcription factor activation, comprising an antibody that specifically recognizes a transcription factor of interest, and instructions for using the antibody for carrying out the methods described above.
  • the transcription factor-specific antibody, or a secondary antibody that detects the transcription factor antibody is luminescently labeled.
  • the kit contains cells that express the transcription factor of interest, and or the kit contains a compound that is known to modify activation of the transcription factor of interest, including but not limited to platelet derived growth factor (PDGF) and serum, which both modify fos activation; and interleukin l(IL-l) and tumor necrosis factor (TNF), which both modify NF-KB activation.
  • PDGF platelet derived growth factor
  • TNF tumor necrosis factor
  • the kit comprises a recombinant expression vector comprising a nucleic acid encoding a transcription factor of interest that translocates from the cytoplasm to the nucleus upon activation, and instructions for using the expression vector to identify compounds that modify transcription factor activation in a cell of interest.
  • the kits contain a purified, luminescently labeled transcription factor.
  • the transcription factor is expressed as a fusion protein with a luminescent protein, including but not limited to green fluorescent protein, luceriferase, or mutants or fragments thereof.
  • the kit further contains cells that are transfected with the expression vector, an antibody or fragment that specifically bind to the transcription factor of interest, and/or a compound that is known to modify activation of the transcription factor of interest (as above).
  • the cytoplasm to nucleus screening methods can also be used to analyze the activation of any protein kinase that is present in an inactive state in the cytoplasm and is transported to the nucleus upon activation, or that phosphorylates a substrate that translocates from the cytoplasm to the nucleus upon phosphorylation.
  • protein kinases examples include, but are not limited to extracellular signal-regulated protein kinases (ERKs), c-Jun amino-terminal kinases (JNKs), Fos regulating protein kinases (FRKs), p38 mitogen activated protein kinase (p38MAPK), protein kinase A (PKA), and mitogen activated protein kinase kinases (MAPKKs).
  • ERKs extracellular signal-regulated protein kinases
  • JNKs c-Jun amino-terminal kinases
  • FRKs Fos regulating protein kinases
  • p38MAPK p38 mitogen activated protein kinase
  • PKA protein kinase A
  • MAPKKs mitogen activated protein kinase kinases
  • protein kinase activity is assayed by monitoring translocation of a luminescently labeled protein kinase substrate from the cytoplasm to the nucleus after being phosphorylated by the protein kinase of interest.
  • the substrate is non-phosphorylated and cytoplasmic prior to phosphorylation, and is translocated to the nucleus upon phosphorylation by the protein kinase.
  • the protein kinase itself translocates from the cytoplasm to the nucleus in this embodiment.
  • indicator cells are treated with test compounds and the distribution of luminescently labeled protein kinase or protein kinase substrate is measured in space and time using a cell screening system, such as the one disclosed above.
  • the luminescently labeled protein kinase or protein kinase substrate may be expressed by or added to the cells either before, together with, or after contacting the cells with a test compound.
  • the protein kinase or protein kinase substrate may be expressed as a luminescently labeled protein chimera by transfected indicator cells.
  • the luminescently labeled protein kinase or protein kinase substrate may be expressed, isolated, and bulk-loaded into the indicator cells as described above, or the protein kinase or protein kinase substrate may be luminescently labeled after isolation.
  • the protein kinase or protein kinase substrate is expressed by the indicator cell, which is subsequently contacted with a luminescent label, such as a labeled antibody, that detects the protein kinase or protein kinase substrate.
  • protein kinase activity is assayed by monitoring the phosphorylation state (ie: phosphorylated or not phosphorylated) of a protein kinase substrate.
  • phosphorylation state ie: phosphorylated or not phosphorylated
  • phosphorylation state is monitored by contacting the cells with an antibody that binds only to the phosphorylated form of the protein kinase substrate of interest (For example, as disclosed in U.S. Patent No. 5,599,681).
  • a biosensor of phosphorylation is used.
  • a luminescently labeled protein or fragment thereof can be fused to a protein that has been engineered to contain (a) a phosphorylation site that is recognized by a protein kinase of interest; and (b) a nuclear localization signal that is unmasked by the phosphorylation.
  • a biosensor will thus be translocated to the nucleus upon phosphorylation, and its translocation can be used as a measure of protein kinase activation.
  • kits for analyzing protein kinase activation, comprising a primary antibody that specifically binds to a protein kinase, a protein kinase substrate, or a phosphorylated form of the protein kinase substrate of interest and instructions for using the primary antibody to identify compounds that modify protein kinase activation in a cell of interest.
  • the primary antibody, or a secondary antibody that detects the primary antibody is luminescently labeled.
  • the kit further comprises cells that express the protein kinase of interest, and/or a compound that is known to modify activation of the protein kinase of interest, including but not limited to dibutyryl cAMP (modifies PKA), forskolin (PKA), and anisomycin (p38MAPK).
  • a compound that is known to modify activation of the protein kinase of interest including but not limited to dibutyryl cAMP (modifies PKA), forskolin (PKA), and anisomycin (p38MAPK).
  • kits comprise an expression vector encoding a protein kinase or a protein kinase substrate of interest that translocates from the cytoplasm to the nucleus upon activation and instructions for using the expression vector to identify compounds that modify protein kinase activation in a cell of interest.
  • the kits contain a purified, luminescently labeled protein kinase or protein kinase substrate.
  • the protein kinase or protein kinase substrate of interest is expressed as a fusion protein with a luminescent protein.
  • the present invention comprises a machine readable storage medium comprising a program containing a set of instructions for causing a cell screening system to execute the methods disclosed for analyzing transcription factor or protein kinase activation, wherein the cell screening system comprises an optical system with a stage adapted for holding a plate containing cells, a digital camera, a means for directing fluorescence or luminescence emitted from the cells to the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • Example 2 Automated Screen for Compounds that Modify Cellular Morphology Changes in cell size are associated with a number of cellular conditions, such as hypertrophy, cell attachment and spreading, differentiation, growth and division, necrotic and programmed cell death, cell motility, mo ⁇ hogenesis, tube formation, and colony formation.
  • cellular hypertrophy has been associated with a cascade of alterations in gene expression and can be characterized in cell culture by an alteration in cell size, that is clearly visible in adherent cells growing on a coverslip.
  • Cell size can also be measured to determine the attachment and spreading of adherent cells.
  • Cell spreading is the result of selective binding of cell surface receptors to substrate ligands and subsequent activation of signaling pathways to the cytoskeleton.
  • Cell attachment and spreading to substrate molecules is an important step for the metastasis of cancer cells, leukocyte activation during the inflammatory response, keratinocyte movement during wound healing, and endothehal cell movement during angiogenesis.
  • Compounds that affect these surface receptors, signaling pathways, or the cytoskeleton will affect cell spreading and can be screened by measuring cell size.
  • Total cellular area can be monitored by labeling the entire cell body or the cell cytoplasm using cytoskeletal markers, cytosolic volume markers, or cell surface markers, in conjunction with a DNA label.
  • cytoskeletal markers cytosolic volume markers, or cell surface markers
  • cell surface markers examples of such labels (many available from Molecular Probes (Eugene, Oregon) and Sigma Chemical Co. (St. Louis,
  • D1ICI6 Dihexadecyl tetramethylindocarbocyanine perchlorate
  • Biotin labeling of surface proteins followed by fluorescent strepavidin labeleing Protocols for cell staining with these various agents are well known to those skilled in the art.
  • Cells are stained live or after fixation and the cell area can be measured.
  • live cells stained with D1ICI6 have homogeneously labeled plasma membranes, and the projected cross-sectional area of the cell is uniformly discriminated from background by fluorescence intensity of the dye.
  • Live cells stained with cytosolic stains such as CMFDA produce a fluorescence intensity that is proportional to cell thickness.
  • cell labeling is dimmer in thin regions of the cell, total cell area can be discriminated from background.
  • Fixed cells can be stained with cytoskeletal markers such as ALEXATM 488 phalloidin that label polymerized actin. Phalloidin does not homogeneously stain the cytoplasm, but still permits discrimination of the total cell area from background.
  • a screen to analyze cellular hypertrophy is implemented using the following strategy.
  • Primary rat myocytes can be cultured in 96 well plates, treated with various compounds and then fixed and labeled with a fluorescent marker for the cell membrane or cytoplasm, or cytoskeleton, such as an antibody to a cell surface marker or a fluorescent marker for the cytoskeleton like rhodamine-phalloidin, in combination with a DNA label like Hoechst. After focusing on the Hoechst labeled nuclei, two images are acquired, one of the Hoechst labeled nuclei and one of the fluorescent cytoplasm image.
  • the nuclei are identified by thresholding to create a mask and then comparing the mo ⁇ hological descriptors of the mask with a set of user defined descriptor values.
  • Each non-nucleus image (or "cytoplasmic image") is then processed separately.
  • the original cytoplasm image can be thresholded, creating a cytoplasmic mask image.
  • Local regions containing cells are defined around the nuclei. The limits of the cells in those regions are then defined by a local dynamic threshold operation on the same region in the fluorescent antibody image.
  • a sequence of erosions and dilations is used to separate slightly touching cells and a second set of mo ⁇ hological descriptors is used to identify single cells.
  • the area of the individual cells is tabulated in order to define the distribution of cell sizes for comparison with size data from normal and hypertrophic cells.
  • Responses from entire 96-well plates (measured as average cytoplasmic area/cell) were analyzed by the above methods, and the results demonstrated that the assay will perform the same on a well-to-well, plate-to-plate, and day-to-day basis
  • the aggregate whole nucleus area is the number of nonzero pixels in the nuclear mask.
  • the average whole nucleus area is the aggregate whole nucleus area divided by the total number of nuclei.
  • the aggregate cytoplasm intensity is the sum of the intensities of all pixels in the cytoplasmic mask.
  • the cytoplasmic area per nucleus is the total cytoplasmic area divided by the total nucleus count.
  • the cytoplasmic intensity per nucleus is the aggregate cytoplasm intensity divided by the total nucleus count.
  • the average cytoplasm intensity is the aggregate cytoplasm intensity divided by the cytoplasm area.
  • the cytoplasm nucleus ratio is the total cytoplasm area divided by the total nucleus area.
  • one or more fluorescent antibodies to other cellular proteins can be included. Images of these additional labeled proteins can be acquired and stored with the above images, for later review, to identify anomalies in the distribution and mo ⁇ hology of these proteins in hypertrophic cells. This example of a multi -parametric screen allows for simultaneous analysis of cellular hypertrophy and changes in actin or myosin distribution.
  • Cell spreading is a measure of the response of cell surface receptors to substrate attachment ligands. Spreading is proportional to the ligand concentration or to the concentration of compounds that reduce receptor-ligand function.
  • One example of selective cell-substrate attachment is prostate carcinoma cell adhesion to the extracellular matrix protein collagen. Prostate carcinoma cells metastasize to bone via selective adhesion to collagen.
  • PC3 human prostate carcinoma cells were cultured in media with appropriate stimulants and are passaged to collagen coated 96 well plates.
  • Ligand concentration can be varied or inhibitors of cell spreading can be added to the wells.
  • Examples of compounds that can affect spreading are receptor antagonists such as integrin- or proteoglycan-blocking antibodies, signaling inhibitors including phosphatidyl inositol-3 kinase inhibitors, and cytoskeletal inhibitors such as cytochalasin D.
  • receptor antagonists such as integrin- or proteoglycan-blocking antibodies
  • signaling inhibitors including phosphatidyl inositol-3 kinase inhibitors
  • cytoskeletal inhibitors such as cytochalasin D.
  • the size of cells under these various conditions can be distinguished above background levels.
  • the number of cells per field is determined by measuring the number of nuclei stained with the Hoechst DNA dye.
  • the area per cell is found by dividing the cytoplasmic area (phalloidin image) by the cell number (Hoechst image).
  • the size of cells is proportional to the ligand-receptor function. Since the area is determined by ligand concentration and by the resultant function of the cell, drug efficacy, as well as drug potency, can be determined by this cell-based assay. Other measurements can be made as discussed above for cellular hypertrophy.
  • the methods for analyzing cellular mo ⁇ hology can be used in a combined high throughput-high content screen.
  • the high throughput mode scans the whole well for an increase in fluorescent phalloidin intensity.
  • a threshold is set above which both nuclei (Hoechst) and cells (phalloidin) are measured in a high content mode.
  • an environmental biosensor (examples include, but are not limited to, those biosensors that are sensitive to calcium and pH changes) is added to the cells, and the cells are contacted with a compound. The cells are scanned in a high throughput mode, and those wells that exceed a pre-determined threshold for luminescence of the biosensor are scanned in a high content mode.
  • kits for analyzing cellular mo ⁇ hology, comprising a luminescent compound that can be used to specifically label the cell cytoplasm, membrane, or cytoskeleton (such as those described above), and instructions for using the luminescent compound to identify test stimuli that induce or inhibit changes in cellular mo ⁇ hology according to the above methods.
  • the kit further comprises a luminescent marker for cell nuclei.
  • the kit comprises at least one compound that is known to modify cellular mo ⁇ hology, including, but not limited to integrin- or proteoglycan- blocking antibodies, signaling inhibitors including phosphatidyl inositol-3 kinase inhibitors, and cytoskeletal inhibitors such as cytochalasin D.
  • the present invention comprises a machine readable storage medium comprising a program containing a set of instructions for causing a cell screening system to execute the disclosed methods for analyzing cellular mo ⁇ hology
  • the cell screening system comprises an optical system with a stage adapted for holding a plate containing cells, a digital camera, a means for directing fluorescence or luminescence emitted from the cells to the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • the following example is a screen for activation of a G-protein coupled receptor
  • GPCR as detected by the translocation of the GPCR from the plasma membrane to a proximal nuclear location.
  • This example illustrates how a high throughput screen can be coupled with a high-content screen in the dual mode System for Cell Based
  • G-protein coupled receptors are a large class of 7 trans-membrane domain cell surface receptors. Ligands for these receptors stimulate a cascade of secondary signals in the cell, which may include, but are not limited to, Ca ++ transients, cyclic AMP production, inositol triphosphate (IP ) production and phosphorylation. Each of these signals are rapid, occuring in a matter of seconds to minutes, but are also generic. For example, many different GPCRs produce a secondary Ca "1"1" signal when activated. Stimulation of a GPCR also results in the transport of that GPCR from the cell surface membrane to an internal, proximal nuclear compartment.
  • FIG. 19 illustrates a dual mode screen for activation of a GPCR.
  • Cells carrying a stable chimera of the GPCR with a blue fluorescent protein (BFP) would be loaded with the acetoxymethylester form of Fluo-3, a cell permeable calcium indicator (green fluorescence) that is trapped in living cells by the hydrolysis of the esters. They would then be deposited into the wells of a microtiter plate 601.
  • BFP blue fluorescent protein
  • the wells would then be treated with an anay of test compounds using a fluid delivery system, and a short sequence of Fluo-3 images of the whole microtiter plate would be acquired and analyzed for wells exhibiting a calcium response (i.e., high throughput mode).
  • the images would appear like the illustration of the microtiter plate 601 in Figure 19.
  • a small number of wells, such as wells C4 and E9 in the illustration, would fluoresce more brightly due to the Ca ++ released upon stimulation of the receptors.
  • the locations of wells containing compounds that induced a response 602. would then be transfened to the HCS program and the optics switched for detailed cell by cell analysis of the blue fluorescence for evidence of GPCR translocation to the perinuclear region.
  • the bottom of Figure 19 illustrates the two possible outcomes of the analysis of the high resolution cell data.
  • the camera images a sub-region 604 of the well area 603. producing images of the fluorescent cells 605.
  • well C4 the uniform distribution of the fluorescence in the cells indicates that the receptor has not internalized, implying that the Ca "1"1" response seen was the result of the stimulation of some other signalling system in the cell.
  • the cells in well E9 606 on the other hand, clearly indicate a concentration of the receptor in the perinuclear region clearly indicating the full activation of the receptor. Because only a few hit wells have to be analyzed with high resolution, the overall throughput of the dual mode system can be quite high, comparable to the high throughput system alone.
  • the following is an example of a screen to measure the kinetics of intemalization of a receptor.
  • the stimulation of a GPCR results in the intemalization of the receptor, with a time course of about 15 min.
  • Simply detecting the endpoint as internalized or not, may not be sufficient for defining the potency of a compound as a GPCR agonist or antagonist.
  • 3 time points at 5 min intervals would provide information not only about potency during the time course of measurement, but would also allow extrapolation of the data to much longer time periods.
  • the sub-region would be defined as two rows, the sampling interval as 5 minutes and the total number of time points 3.
  • HCS human glucocorticoid receptor
  • hGR human glucocorticoid receptor
  • Plasmid construct A eukaryotic expression plasmid containing a coding sequence for a green fluorescent protein - human glucocorticoid receptor (GFP -hGR) chimera was prepared using GFP mutants (Palm et al., Nat. Struct. Biol 4:361 (1997). The construct was used to transfect a human cervical carcinoma cell line (HeLa). Cell preparation and transfection.
  • GFP -hGR green fluorescent protein - human glucocorticoid receptor
  • HeLa cells (ATCC CCL-2) were trypsinized and plated using DMEM containing 5% charcoal/dextran-treated fetal bovine serum (FBS) (HyClone) and 1% penicillin-streptomycin (C-DMEM) 12-24 hours prior to transfection and incubated at 37°C and 5% CO 2 . Transfections were performed by calcium phosphate co-precipitation (Graham and Van der Eb, Virology 52:456, 1973; Sambrook et al., (1989). Molecular Cloning: A Laboratory Manual, Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) or with Lipofectamine (Life Technologies, Gaithersburg, MD).
  • FBS fetal bovine serum
  • C-DMEM penicillin-streptomycin
  • the medium was replaced, prior to transfection, with DMEM containing 5% charcoal/dextran-treated FBS.
  • Cells were incubated with the calcium phosphate-DNA precipitate for 4-5 hours at 37°C and 5% CO 2 , washed 3-4 times with DMEM to remove the precipitate, followed by the addition of C-DMEM.
  • Lipofectamine transfections were performed in serum-free DMEM without antibiotics according to the manufacturer's instructions (Life Technologies, Gaithersburg, MD). Following a 2-3 hour incubation with the DNA-liposome complexes, the medium was removed and replaced with C-DMEM. All transfected cells in 96-well microtiter plates were incubated at 33°C and 5% CO 2 for 24-48 hours prior to drug treatment. Experiments were performed with the receptor expressed transiently in HeLa cells.
  • Dexamethasone induction of GFP-hGR translocation To obtain receptor- ligand translocation kinetic data, nuclei of transfected cells were first labeled with 5 ⁇ g/ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20 minutes at 33°C and 5% CO 2 . Cells were washed once in Hank's Balanced Salt Solution (HBSS) followed by the addition of 100 nM dexamethasone in HBSS with 1% charcoal/dextran-treated FBS.
  • HBSS Hank's Balanced Salt Solution
  • transfected HeLa cells were first washed with DMEM and then incubated at 33°C and 5% CO for 1 h in the presence of 0 - 1000 nM dexamethasone in DMEM containing 1% charcoal/dextran- treated FBS.
  • Cells were analyzed live or they were rinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS, stained with Hoechst 33342, and washed before analysis. The intracellular GFP-hGR fluorescence signal was not diminished by this fixation procedure.
  • this translocation ratio was calculated from data obtained from at least 200 cells at each concentration of dexamethasone tested. Drug-induced translocation of GFP-hGR from the cytoplasm to the nucleus was therefore correlated with an increase in the translocation ratio.
  • Figure 20 schematically displays the drug-induced cytoplasm 253 to nucleus 252 translocation of the human glucocorticoid receptor.
  • the upper pair of schematic diagrams depicts the localization of GFP-hGR within the cell before 250 (A) and after 251 (B) stimulation with dexamethasone.
  • the drug induces a large portion of the cytoplasmic GFP-hGR to translocate into the nucleus. This redistribution is quantified by determining the integrated intensities ratio of the cytoplasmic and nuclear fluorescence in treated 255 and untreated 254 cells.
  • the lower pair of fluorescence micrographs show the dynamic redistribution of GFP-hGR in a single cell, before 254 and after 255 treatment.
  • the HCS is performed on wells containing hundreds to thousands of transfected cells and the translocation is quantified for each cell in the field exhibiting GFP fluorescence. Although the use of a stably transfected cell line would yield the most consistently labeled cells, the heterogeneous levels of GFP-hGR expression induced by transient transfection did not interfere with analysis by the cell screening system of the present invention.
  • the cell screening system scans each well of the plate, images a population of cells in each, and analyzes cells individually.
  • two channels of fluorescence are used to define the cytoplasmic and nuclear distribution of the GFP-hGR within each cell. Depicted in Figure 21 is the graphical user interface of the cell screening system near the end of a GFP-hGR screen.
  • the user interface depicts the parallel data collection and analysis capability of the system.
  • the windows labeled “Nucleus” 261 and “GFP-hGR” 262 show the pair of fluorescence images being obtained and analyzed in a single field.
  • the window labeled “Color Overlay” 260 is formed by pseudocoloring the above images and merging them so the user can immediately identify cellular changes.
  • the 96 well plate depicted in the lower window of the screen 267 shows which wells have met a set of user-defined screening criteria. For example, a white-colored well 269 indicates that the drug-induced translocation has exceeded a predetermined threshold value of 50%. On the other hand, a black-colored well 270 indicates that the dmg being tested induced less than 10% translocation. Gray-colored wells 268 indicate "hits" where the translocation value fell between 10% and 50%. Row “E” on the 96 well plate being analyzed 266 shows a titration with a dmg known to activate GFP-hGR translocation, dexamethasone. This example screen used only two fluorescence channels.
  • Two additional channels (Channels 3 263 and 4 264) are available for parallel analysis of other specific targets, cell processes, or cytotoxicity to create multiple parameter screens.
  • the user has total access to image and calculated data ( Figure 22).
  • the comprehensive data analysis package of the cell screening system allows the user to examine HCS data at multiple levels. Images 276 and detailed data in a spread sheet 279 for individual cells can be viewed separately, or summary data can be plotted. For example, the calculated results of a single parameter for each cell in a 96 well plate are shown in the panel labeled Graph 1 275.
  • the user can display the entire data set for a particular cell that is recalled from an existing database. Shown here are the image pair 276 and detailed fluorescence and mo ⁇ hometric data from a single cell (Cell #118, gray line 277).
  • the large graphical insert 278 shows the results of dexamethasone concentration on the translocation of GFP-hGR. Each point is the average of data from at least 200 cells.
  • the calculated EC 50 for dexamethasone in this assay is 2 nM.
  • FIG. 23 shows kinetic data for the dexamethasone- induced translocation of GFP-hGR in several cells within a single field.
  • Human HeLa cells transfected with GFP-hGR were treated with 100 nM dexamethasone and the translocation of GFP-hGR was measured over time in a population of single cells.
  • the graph shows the response of transfected cells 285. 286, 287, and 288 and non- transfected cells 289. These data also illustrate the ability to analyze cells with different expression levels.
  • Apoptosis is a complex cellular program that involves myriad molecular events and pathways. To understand the mechanisms of drag action on this process, it is essential to measure as many of these events within cells as possible with temporal and spatial resolution. Therefore, an apoptosis screen that requires little cell sample preparation yet provides an automated readout of several apoptosis-related parameters would be ideal.
  • a cell-based assay designed for the cell screening system has been used to simultaneously quantify several of the mo ⁇ hological, organellar, and macromolecular hallmarks of paclitaxel-induced apoptosis.
  • the cells chosen for this study were mouse connective tissue fibroblasts (L-929; ATCC CCL-1) and a highly invasive glioblastoma cell line (SNB- 19; ATCC CRL-2219) (Welch et al., In Vitro Cell. Dev. Biol. 31:610, 1995).
  • the day before treatment with an apoptosis inducing drug 3500 cells were placed into each well of a 96-well plate and incubated overnight at 37°C in a humidified 5% CO 2 atmosphere.
  • the culture medium was removed from each well and replaced with fresh medium containing various concentrations of paclitaxel (0 - 50 ⁇ M) from a 20 mM stock made in DMSO.
  • DMSO DMSO
  • the maximal concentration of DMSO used in these experiments was 0.25%.
  • the cells were then incubated for 26 h as above.
  • each well received fresh medium containing 750 nM MitoTracker Red (Molecular Probes; Eugene, OR) and 3 ⁇ g/ml Hoechst 33342 DNA-binding dye (Molecular Probes) and was incubated as above for 20 min.
  • Each well on the plate was then washed with HBSS and fixed with 3.7% formaldehyde in HBSS for 15 min at room temperature.
  • the average nuclear area ( ⁇ m ) was calculated by dividing the total nuclear area in a field by the number of nuclei detected.
  • the average nuclear perimeter ( ⁇ m) was calculated by dividing the sum of the perimeters of all nuclei in a field by the number of nuclei detected in that field. Highly convoluted apoptotic nuclei had the largest nuclear perimeter values.
  • the average nuclear brightness was calculated by dividing the integrated intensity of the entire field of nuclei by the number of nuclei in that field. An increase in nuclear brightness was conelated with increased DNA content.
  • the average cellular brightness was calculated by dividing the integrated intensity of an entire field of cells stained with MitoTracker dye by the number of nuclei in that field. Because the amount of MitoTracker dye that accumulates within the mitochondria is proportional to the mitochondrial potential, an increase in the average cell brightness is consistent with an increase in mitochondrial potential.
  • the average cellular brightness was also calculated by dividing the integrated intensity of an entire field of cells stained with Bodipy FL phallacidin dye by the number of nuclei in that field. Because the phallotoxins bind with high affinity to the polymerized form of actin, the amount of Bodipy FL phallacidin dye that accumulates within the cell is proportional to actin polymerization state.
  • Figure 24 shows the changes paclitaxel induced in the nuclear mo ⁇ hology of L-929 cells. Increasing amounts of paclitaxel caused nuclei to enlarge and fragment 293, a hallmark of apoptosis. Quantitative analysis of these and other images obtained by the cell screening system is presented in the same figure. Each parameter measured showed that the L-929 cells 296 were less sensitive to low concentrations of paclitaxel than were SNB-19 cells 297. At higher concentrations though, the L-929 cells showed a response for each parameter measured. The multiparameter approach of this assay is useful in dissecting the mechanisms of drug action.
  • the area, brightness, and fragmentation of the nucleus 298 and actin polymerization values 294 reached a maximum value when SNB-19 cells were treated with 10 nM paclitaxel (Figure 24; top and bottom graphs).
  • mitochondrial potential 295 was minimal at the same concentration of paclitaxel ( Figure 24; middle graph).
  • the fact that all the parameters measured approached control levels at increasing paclitaxel concentrations (>10 nM) suggests that SNB-19 cells have low affinity drag metabolic or clearance pathways that are compensatory at sufficiently high levels of the drag. Contrasting the drag sensitivity of SNB-19 cells 297, L-929 showed a different response to paclitaxel 296.
  • fibroblastic cells showed a maximal response in many parameters at 5 ⁇ M paclitaxel, a 500-fold higher dose than SNB-19 cells. Furthermore, the L-929 cells did not show a sha ⁇ decrease in mitochondrial potential 295 at any of the paclitaxel concentrations tested. This result is consistent with the presence of unique apoptosis pathways between a normal and cancer cell line. Therefore, these results indicate that a relatively simple fluorescence labeling protocol can be coupled with the cell screening system of the present invention to produce a high-content screen of key events involved in programmed cell death.
  • Example 7 Protease induced translocation of a signaling enzyme containing a disease-associated sequence from cytoplasm to nucleus.
  • Plasmid construct A eukaryotic expression plasmid containing a coding sequence for a green fluorescent protein - caspase (Cohen (1997), Biochemical J.
  • GFP mutants The construct is used to transfect eukaryotic cells.
  • Cell preparation and transfection Cells are trypsinized and plated 24 h prior to transfection and incubated at 37°C and 5% CO 2 .
  • Transfections are performed by methods including, but not limited to calcium phosphate coprecipitation or lipofection. Cells are incubated with the calcium phosphate-DNA precipitate for 4-5 hours at 37°C and 5% CO 2 , washed 3-4 times with DMEM to remove the precipitate, followed by the addition of C-DMEM.
  • Lipofectamine transfections are performed in serum- free DMEM without antibiotics according to the manufacturer's instmctions. Following a 2-3 hour incubation with the DNA-liposome complexes, the medium is removed and replaced with C-DMEM
  • DMEM fetal calf serum
  • Kinetic data are collected by acquiring fluorescence image pairs (Caspase-GFP and Hoechst 33342-labeled nuclei) from fields of living cells at 1 min intervals for 30 min after the addition of compound. Likewise, image pairs are obtained from each well of the fixed time point screening plates 1 h after the addition of compound. In both cases, the image pairs obtained at each time point are used to define nuclear and cytoplasmic regions in each cell. Translocation of Caspase-GFP is calculated by dividing the integrated fluorescence intensity of Caspase- GFP in the nucleus by the integrated fluorescence intensity of the chimera in the cytoplasm or as a nuclear-cytoplasmic difference of GFP fluorescence.
  • this translocation ratio is calculated from data obtained from at least 200 cells at each concentration of compound tested. Drag-induced translocation of Caspase-GFP from the cytoplasm to the nucleus is therefore conelated with an increase in the translocation ratio.
  • Molecular interaction libraries including, but not limited to those comprising putative activators or inhibitors of apoptosis-activated enzymes are use to screen the indicator cell lines and identify a specific ligand for the DAS, and a pathway activated by compound activity.
  • Example 8 Identification of novel steroid receptors from DAS Two sources of material and/or information are required to make use of this embodiment, which allows assessment of the function of an uncharacterized gene.
  • disease associated sequence bank(s) containing cDNA sequences suitable for transfection into mammalian cells can be used. Because every RADE or differential expression experiment generates up to several hundred sequences, it is possible to generate an ample supply of DAS.
  • information from primary sequence database searches can be used to place DAS into broad categories, including, but not limited to, those that contain signal sequences, seven trans-membrane motifs, conserved protease active site domains, or other identifiable motifs. Based on the information acquired from these sources, method types and indicator cell lines to be transfected are selected. A large number of motifs are already well characterized and encoded in the linear sequences contained within the large number genes in existing genomic databases.
  • Information from the DAS identification experiment is used as the basis for selecting the relevant biological processes, (for example, look at the DAS from a tumor line for cell cycle modulation, apoptosis, metastatic proteases, etc.) 2) Sorting of DNA sequences or DAS by identifiable motifs (ie. signal sequences, 7- transmembrane domains, conserved protease active site domains, etc.) This initial grouping will determine fluorescent tagging strategies, host cell lines, indicator cell lines, and banks of bioactive molecules to be screened, as described supra.
  • ligate DAS into an expression vector designed for this pu ⁇ ose ligate DAS into an expression vector designed for this pu ⁇ ose.
  • Generalized expression vectors contain promoters, enhancers, and terminators for which to deliver target sequences to the cell for transient expression. Such vectors may also contain antibody tagging sequences, direct association sequences, chromophore fusion sequences like GFP, etc. to facilitate detection when expressed by the host.
  • Transiently transfect cells with DAS containing vectors using standard transfection protocols including: calcium phosphate co-precipitation, liposome mediated, DEAE dextran mediated, polycationic mediated, viral mediated, or electroporation, and plate into microtiter plates or microwell anays. Alternatively, transfection can be done directly in the microtiter plate itself.
  • DAS shown to possess a motif(s) suggestive of transcriptional activation potential are utilized to identify novel steroid receptors.
  • Defining the fluorescent tags for this experiment involves identification of the nucleus through staining, and tagging the DAS by creating a GFP chimera via insertion of DAS into an expression vector, proximally fused to the gene encoding GFP.
  • a single chain antibody fragment with high affinity to some portion of the expressed DAS could be constructed using technology available in the art (Cambridge Antibody Technologies) and linked to a fluorophore (FITC) to tag the putative transcriptional activator/receptor in the cells. This alternative would provide an external tag requiring no DNA transfection and therefore would be useful if distribution data were to be gathered from the original primary cultures used to generate the DAS.
  • Plasmid construct A eukaryotic expression plasmid containing a coding sequence for a green fluorescent protein - DAS chimera is prepared using GFP mutants. The constmct is used to transfect HeLa cells. The plasmid, when transfected into the host cell, produces a GFP fused to the DAS protein product, designated GFP- DASpp. Cell preparation and transfection. HeLa cells are trypsinized and plated using DMEM containing 5% charcoal dextran-treated fetal bovine serum (FBS) (Hyclone) and 1% penicillin-streptomycin (C-DMEM) 12-24 hours prior to transfection and incubated at 37°C and 5% CO 2 .
  • FBS fetal bovine serum
  • C-DMEM penicillin-streptomycin
  • Transfections are performed by calcium phosphate coprecipitation or with Lipofectamine (Life Technologies).
  • the medium is replaced, prior to transfection, with DMEM containing 5% charcoal/dextran-treated FBS.
  • Cells are incubated with the calcium phosphate-DNA precipitate for 4-5 hours at 37°C and 5% CO 2 , and washed 3-4 times with DMEM to remove the precipitate, followed by the addition of C-DMEM.
  • Lipofectamine transfections are performed in semm-free DMEM without antibiotics according to the manufacturer's instructions. Following a 2-3 hour incubation with the DNA-liposome complexes, the medium is removed and replaced with C-DMEM. All transfected cells in 96-well microtiter plates are incubated at 33°C and 5% CO 2 for 24-48 hours prior to drag treatment. Experiments are performed with the receptor expressed transiently in HeLa cells.
  • nuclei of transfected cells are first labeled with 5 ⁇ g/ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20 minutes at 33°C and 5% CO 2 .
  • Cells are washed once in Hank's Balanced Salt Solution (HBSS). The cells are analyzed live or they are rinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS, stained with Hoechst 33342, and washed before analysis.
  • Hoechst 33342 Molecular Probes
  • image acquisition and analysis are performed using the cell screening system of the present invention.
  • the intracellular GFP-DASpp fluorescence signal is collected by acquiring fluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nuclei) from field cells.
  • the image pairs obtained at each time point are used to define nuclear and cytoplasmic regions in each cell. Data demonstrating dispersed signal in the cytoplasm would be consistent with known steroid receptors that are DNA transcriptional activators.
  • GFP-DASpp Screening for induction of GFP-DASpp translocation.
  • a screen of various ligands is performed using a series of steroid type ligands including, but not limited to: estrogen, progesterone, retinoids, growth factors, androgens, and many other steroid and steroid based molecules.
  • Image acquisition and analysis are performed using the cell screening system of the invention.
  • the intracellular GFP-DASpp fluorescence signal is collected by acquiring fluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nuclei) from fields cells.
  • the image pairs obtained at each time point are used to define nuclear and cytoplasmic regions in each cell.
  • Translocation of GFP-DASpp is calculated by dividing the integrated fluorescence intensity of GFP-DASpp in the nucleus by the integrated fluorescence intensity of the chimera in the cytoplasm or as a nuclear-cytoplasmic difference of GFP fluorescence.
  • a translocation from the cytoplasm into the nucleus indicates a ligand binding activation of the DASpp thus identifying the potential receptor class and action. Combining this data with other data obtained in a similar fashion using known inhibitors and modifiers of steroid receptors, would either validate the DASpp as a target, or more data would be generated from various sources.
  • an automated method for identifying compounds that modify microtubule stmcture is provided.
  • indicator cells are treated with test compounds and the distribution of luminescent microtubule-labeling molecules is measured in space and time using a cell screening system, such as the one disclosed above.
  • the luminescent microtubule-labeling molecules may be expressed by or added to the cells either before, together with, or after contacting the cells with a test compound.
  • living cells express a luminescently labeled protein biosensor of microtubule dynamics, comprising a protein that labels microtubules fused to a luminescent protein.
  • Appropriate microtubule- labeling proteins for this aspect of the invention include, but are not limited to and ⁇ tubulin isoforms, and MAP4.
  • Prefened embodiments of the luminescent protein include, but are not limited to green fluorescent protein (GFP) and GFP mutants.
  • the method involves transfecting cells with a microtubule labeling luminescent protein, wherein the microtubule labeling protein can be, but is not limited to, ⁇ -tubulin, ⁇ -tubulin, or micro tubule-associated protein 4 (MAP4).
  • a microtubule labeling luminescent protein can be, but is not limited to, ⁇ -tubulin, ⁇ -tubulin, or micro tubule-associated protein 4 (MAP4).
  • MAP4 micro tubule-associated protein 4
  • MAP4 is fused to a modified version of the Aequorea victoria green fluorescent protein (GFP).
  • GFP Aequorea victoria green fluorescent protein
  • a DNA constmct has been made which consists of a fusion between the EGFP coding sequence (available from Clontech) and the coding sequence for mouse MAP4.
  • MAP4 is a ubiquitous microtubule-associated protein that is known to interact with microtubules in inte ⁇ hase as well as mitotic cells (Olmsted and Murofushi, (1993), MAP4. In "Guidebook to the Cytoskeleton and Motor Proteins.” Oxford University Press. T. Kreis and R.
  • GFP-MAP4 does not dismpt microtubule function or integrity (Olson et al., 1995). Similar constmcts can be made using ⁇ -tubulin or ⁇ -tubulin via standard techniques in the art. These chimeras will provide a means to observe and analyze microtubule activity in living cells during all stages of the cell cycle.
  • the luminescently labeled protein biosensor of microtubule dynamics is expressed, isolated, and added to the cells to be analyzed via bulk loading techniques, such as microinjection, scrape loading, and impact-mediated loading.
  • bulk loading techniques such as microinjection, scrape loading, and impact-mediated loading.
  • ⁇ and ⁇ tubulin isoforms, MAP4, MAP2 and/or tau can all be used.
  • the protein biosensor is expressed by the cell, and the cell is subsequently contacted with a luminescent label, such as a labeled antibody, that detects the protein biosensor, endogenous levels of a protein antigen, or both.
  • a luminescent label that detects ⁇ and ⁇ tubulin isoforms, MAP4, MAP2 and/or tau, can be used.
  • GFP mutants are available, all of which would be effective in this invention, including, but not limited to, GFP mutants which are commercially available (Clontech, California).
  • the MAP4 constmct has been introduced into several mammalian cell lines (BHK-21, Swiss 3T3, HeLa, HEK 293, LLCPK) and the organization and localization of tubulin has been visualized in live cells by virtue of the GFP fluorescence as an indicator of MAP4 localization.
  • the constmct can be expressed transiently or stable cell lines can be prepared by standard methods. Stable HeLa cell lines expressing the EGFP-MAP4 chimera have been obtained, indicating that expression of the chimera is not toxic and does not interfere with mitosis.
  • Possible selectable markers for establishment and maintenance of stable cell lines include, but are not limited to the neomycin resistance gene, hygromycin resistance gene, zeocin resistance gene, puromycin resistance gene, bleomycin resistance gene, and blastacidin resistance gene.
  • the utility of this method for the monitoring of microtubule assembly, disassembly, and reanangement has been demonstrated by treatment of transiently and stably transfected cells with microtubule drags such as paclitaxel, nocodazole, vincristine, or vinblastine.
  • the present method provides high-content and combined high throughput-high content cell-based screens for anti-microtubule drags, particularly as one parameter in a multi-parametric cancer target screen.
  • the EGFP-MAP4 construct used herein can also be used as one of the components of a high-content screen that measures multiple signaling pathways or physiological events.
  • a combined high throughput and high content screen is employed, wherein multiple cells in each of the locations containing cells are analyzed in a high throughput mode, and only a subset of the locations containing cells are analyzed in a high content mode.
  • the high throughput screen can be any screen that would be useful to identify those locations containing cells that should be further analyzed, including, but not limited to, identifying locations with increased luminescence intensity, those exhibiting expression of a reporter gene, those undergoing calcium changes, and those undergoing pH changes.
  • the present invention may be applied to clinical diagnostics, the detection of chemical and biological warfare weapons, and the basic research market since fundamental cell processes, such as cell division and motility, are highly dependent upon microtubule dynamics.
  • Image data can be obtained from either fixed or living indicator cells.
  • To extract mo ⁇ hometric data from each of the images obtained the following method of analysis is used: 1. Threshold each nucleus and cytoplasmic image to produce a mask that has value 0 for each pixel outside a nucleus or cell boundary.
  • Microtubule mo ⁇ hology is defined using a set of classifiers to quantify aspects of microtubule shape, size, aggregation state, and polymerization state.
  • These classifiers can be based on approaches that include co-occunence matrices, texture measurements, spectral methods, stmctural methods, wavelet transforms, statistical methods, or combinations thereof. Examples of such classifiers are as follows:
  • a classifier to quantify microtubule length and width using edge detection methods such as that discussed in Kolega et al. ((1993). Biolmaging 1:136- 150), which discloses a non-automated method to determine edge strength in individual cells), to calculate the total edge strength within each cell.
  • the total edge strength can be divided by the cell area to give a "microtubule mo ⁇ hology" value. Large microtubule mo ⁇ hology values are associated with strong edge strength values and are therefore maximal in cells containing distinct microtubule stractures. Likewise, small microtubule mo ⁇ hology values are associated with weak edge strength and are minimal in cells with depolymerized microtubules.
  • the physiological range of microtubule mo ⁇ hology values is set by treating cells with either the microtubule stabilizing drag paclitaxel (10 ⁇ M) or the microtubule depolymerizing drag nocodazole (10 ⁇ g/ml).
  • a classifier to quantify apparent interconnectivity, or branching (or both), of the microtubules.
  • kits for analyzing microtubule stability, comprising an expression vector comprising a nucleic acid that encodes a microtubule labeling protein and instmctions for using the expression vector for carrying out the methods described above.
  • the expression vector further comprises a nucleic acid that encodes a luminescent protein, wherein the microtubule binding protein and the luminescent protein thereof are expressed as a fusion protein.
  • the kit may contain an antibody that specifically binds to the microtubule-labeling protein.
  • the kit includes cells that express the microtubule labeling protein.
  • the cells are transfected with the expression vector.
  • kits further contain a compound that is known to dismpt microtubule stmcture, including but not limited to curacin, nocodazole, vincristine, or vinblastine.
  • the kits further comprise a compound that is known to stabilize microtubule stmcture, including but not limited to taxol (paclitaxel), and discodermolide.
  • the present invention comprises a machine readable storage medium comprising a program containing a set of instmctions for causing a cell screening system to execute the disclosed methods for analyzing microtubule stability
  • the cell screening system comprises an optical system with a stage adapted for holding a plate containing cells, a digital camera, a means for directing fluorescence or luminescence emitted from the cells to the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • the cell screening system comprises an optical system with a stage adapted for holding a plate containing cells, a digital camera, a means for directing fluorescence or luminescence emitted from the cells to the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • the cell cycle of proliferating cells is characterized by four distinct phases: mitosis and the Gl, S and G2 components of inte ⁇ hase (Mercer, J Cell Biochem Suppl, 1998. 30-31: p. 50-4; Neufeld and Edgar, Cun Opin Cell Biol, 1998. 10(6): p. 784-90).
  • Mitosis is the process by which cells divide, and is separated into prophase, metaphase, anaphase and telophase. Each phase is precisely orchestrated such that the products of each cell division are two sister cells with intact genomes and a full complement of cytoplasmic organelles.
  • Cell division is a tightly regulated process involving a variety of proteins from converging intracellular signaling cascades (Kohn, Mol Biol Cell, 1999.
  • Chromatin stmcture is also remodeled during this time, triggered by post- translational modification of the core histone proteins.
  • One such modification is the phosphorylation of histone H3 (Goto,et al., J Biol Chem, 1999. 274(36): p. 25543- 25549; Van Hooser et al., J Cell Sci., 1998. l l l(Pt 23): p. 3497-506; Wei et al., Proc Natl Acad Sci U S A., 1998. 95(13): p. 7480-4; Wei et al., Cell., 1999. 97(1): p. 99- 109).
  • an automated method for measuring mitotic index comprising providing an anay of locations comprising multiple cells, wherein the cells possess at least a first luminescently labeled reporter molecule that reports on the mitotic index of the cells, and a second luminescently labeled reporter molecule that locates cells; -imaging or scanning multiple cells in each of the locations containing multiple cells to obtain luminescent signals from the first and second luminescently-labeled reporter molecules;
  • the luminescent signals into digital data; and -utilizing the digital data to automatically make measurements, wherein the measurements are used to automatically calculate changes in the distribution, environment or activity of the first and second luminescent reporter molecules on or within the cells, wherein the calculated changes indicate the mitotic index of the cells.
  • the phrase "the cells possess one or more luminescent reporter molecules” means that the luminescent reporter molecule may be expressed as a luminescent reporter molecule by the cells, added to the cells as a luminescent reporter molecule, or luminescently labeled by contacting the cell with a luminescently labeled molecule that binds to the reporter molecule, such as a dye or antibody, that binds to the reporter molecule.
  • the first luminescently labeled reporter molecule reports on histone H3 phosphorylation.
  • the methods of the present invention can also be used for various diagnostic pu ⁇ oses.
  • mitotic index itself is also used as a means for characterizing and diagnosing cancerous cells in the clinic.
  • the cells to be analyzed are derived from a patient, and the calculated changes in the distribution, environment or activity of the first and second luminescent reporter molecules are used to identify the presence of cancerous cells in the patient, by looking for cells with increased mitotic index relative to control cells.
  • the cells on the anay of locations include skin fibroblasts derived from a patient to be tested for Alzheimer's disease, and the calculated changes in the distribution, environment or activity of the first and second luminescent reporter molecules are used to diagnose Alzheimer's disease in the patient, by looking for cells with increased mitotic index relative to control cells.
  • the method further comprises contacting the cells with a test stimulus, wherein the calculated changes indicate the effect of the test stimulus on the mitotic index of the cells.
  • the contacting of the cells with the test stimulus can occur before, after, or simultaneously with the possession of the luminescent reporter molecules by the cells.
  • the cells are contacted with a control compound known to modify the mitotic index of the cells, and utilizing the calculated changes to determine whether the test stimulus inhibited the control compound from modifying the mitotic index of the cells.
  • the contacting of the cells with the control compound can occur before, after, or simultaneously with the contacting of the cells with the test stimulus.
  • the methods of the present invention are useful both for identifying compounds that modify the cellular mitotic index, and for various cell-based tests that utilize mitotic index as a diagnostic tool.
  • the methods can also be used for more detailed cell cycle staging, in combination with luminescent reporter molecules comprising other cell cycle markers, including but not limited to a generic marker for cells in Gl -phase, such as cyclin D, a generic S-phase marker such as bromodeoxyuridine (BrDU) inco ⁇ oration, or proliferating cell nuclear antigen (PCNA).
  • a generic marker for cells in Gl -phase such as cyclin D
  • a generic S-phase marker such as bromodeoxyuridine (BrDU) inco ⁇ oration
  • PCNA proliferating cell nuclear antigen
  • the present invention also provides computer readable storage media comprising a program containing a set of instmctions for causing a cell screening system to execute the methods of this aspect of the invention, wherein the cell screening system comprises an optical system with a stage adapted for holding a plate containing cells, a means for moving the stage or the optical system, a digital camera, a means for directing light emitted from the cells to the digital camera, and a computer means for receiving and processing the digital data from the digital camera.
  • the cell screening system is that disclosed in the present application.
  • kits for measuring mitotic index comprising at least a first luminescently labeled reporter molecule that reports on the mitotic index of the cells and instmctions for using the luminescent reporter molecule to carry out the methods of this aspect of the invention.
  • the kit further comprises at least a second luminescent reporter molecule that identifies cells, preferably by reporting on cell DNA content; most preferably a nucleic acid dye.
  • the first luminescently labeled reporter molecule reports on histone H3 phosphorylation.
  • the first luminescently labeled reporter molecule comprises a phospho-histone H3 antibody.
  • the kits may further contain appropriate fluorescently labeled secondary antibodies, and/or other reagents for use in carrying out the methods of the invention.
  • the method measures the total number of cells and the total number of mitotic figures within each well, and reports the total number of nuclei in the well and the mitotic index for the well.
  • mitotic figures refers to any cells in a mitotic state.
  • Such mitotic figures can be detected using any reporter molecule(s) that detect mitotic cells, including but not limited to reporters for chromatin condensation and microtubule spindle formation. In a prefened embodiment, the reporter reports on histone H3 phosphorylation.
  • the method is detailed in the flow charts of Figures 25 and 26, and first involves counting the number of cells within a field using an image of fluorescently labeled nuclei. ( Figure 25) An optional background compensation step to account for shadings in images can be carried out.
  • This image is processed, where such processing can include averaging of the nuclear image multiple times to smooth out nuclear holes (i.e.: “smoothing"), and a fixed threshold is applied to create a binary image (i.e.: “nuclear mask”). Nuclei are then counted through identification of local maxima.
  • identification of the local maxima comprises removal of border objects, filling of holes, erosion to unlink slightly touching objects, and application of a filter capable of determining local maxima within the image. The number of local maxima found is the number of nuclei in the image.
  • the mitotic figures are identified in a second fluorescence channel based on immunolabeling with a phospho-histone H3 antibody.
  • Figure 26 the image is processed, preferably by applying a background compensation filter, and a threshold is applied to generate a binary mask.
  • border objects are removed, holes are filled in the mask, and the mask is eroded to remove fluorescent bodies not associated with mitosis. Mitotic objects are then counted within the mask. After counting, the method reports the ratio of mitotic figures to total cell number. This value is refened to as the mitotic index.
  • selected wells are assessed for the optimal exposure time, which is fixed for scanning the entire plate. Fields away from the center region of the microplate can be chosen to avoid photobleaching fields scanned during formal data acquisition. These parameters are stored with the protocol, and thus no specific adjustments are needed after setup. Autoexposure in the mitotic channel requires that the field contain a representative number of labeled mitotic cells to insure optimal image acquisition. However, auto-exposure of the nuclear channel in fields with large numbers of mitotic cells will result in under-exposure of untreated wells. Therefore, auto-expose settings should be determined in a control well treated for 10-12 hours with l the EC 50 dosage for a given control compound (ie. 20nM vinblastine). After scanning the plate, the results can be visualized with a data viewer, including but not limited to that disclosed in U.S. Patent Application Serial No. 09/437,976 filed November 10, 1999, or exported to an external program, for visualization.
  • the present invention has been used to determine dose response for a variety of known anti-microtubule compounds that are known inhibitors of mitotic progression (vinblastine, paclitaxel, and nocodzaole) in four separate cell types (MDCK, LLCPK, PtK, and HeLa cells), using a protocol that is described below.
  • the data collected demonstrate the efficacy of the present method for a variety of cell type and compound combinations. For example, such tests using vinblastine on MDCK cells have demonstrated that the drag treatment enriched mitotic cells approximately 16-fold relative to untreated controls. Mitotic enrichment induced with anti-mitotic compounds is not only cell-type dependent, but also shows time and dose dependency.
  • MDCK and HeLa cells were treated with vinblastine for 10 hours at varying concentrations and the method was used to analyze the dose response. While both cells were able to respond to vinblastine treatment with increased mitotic index, the optimal concentration for maximum mitotic enrichment was cell type dependent, as was the absolute increase in mitotic index.
  • the main sources of error in utilizing the present method are the presence of multiple local maxima located within a single cell, and counting artifacts.
  • Enors in determining intracellular local maxima are usually due to dark subcellular components; i.e., cell nucleoli, and inegularly shaped cells.
  • the region sunounding the dark intracellular body can be inte ⁇ reted as a local maximum. If the dark body is located near the center of the cell, then two local maxima are detected.
  • This problem can be solved by a combination of smoothing the image more and adjusting the size of the local maxima kernel.
  • Single cells having an elliptical shape tend to be counted as multiple cells. This problem may be solved by requiring the distance between local maxima points to be greater than the average cell radius in length.
  • MDCK cells were plated at approximately 2500 cells per well in a 96-well microplate, allowed to attach, and grown overnight. Longer incubations that lead to confluency negatively impact on mitotic index.
  • mitotics were enriched using anti-microtubule drags such as paclitaxel (500 nM), colchicine (50 nM), vinblastine (500 nM), nocodazole (750 nM), and curacin A (200 nM).
  • anti-microtubule drags such as paclitaxel (500 nM), colchicine (50 nM), vinblastine (500 nM), nocodazole (750 nM), and curacin A (200 nM).
  • the cells were incubated with test compounds for 12-18 hours, or until mitotic figures were visible as rounded cells. 4. Following drag treatment, cells were fixed for 20 minutes in 4% EM-grade formaldehyde solution in IX Hanks buffered salt solution (HBSS) pH 7.20. After fixation the cells were washed once with HBSS to remove residual fixative. For 96- well microplates, the washing volume was 100-150 ⁇ l.
  • HBSS IX Hanks buffered salt solution
  • the optimal seeding density was calculated as approximately 5000 cells per well using the growth times and dmg treatment times indicated in the assay protocol. Cell densities higher than 5000 cells per well, or incubation times that lead to confluency prior to drag treatment decrease cell proliferation, resulting in lower peak mitotic index values for treated cells.
  • the method requires that the mitotic cells remain attached to the substratum during drag treatment and plate processing. An average mitotic index lower than 1% in untreated cells is an indication that cells are not retained during processing.
  • the present invention can also be used in the diagnosis of the presence of cancerous cells, and as an early diagnostic parameter for Alzheimer's disease. Such observations demonstrate the potential value of analyzing mitotic index when assessing various disease states and broaden the general applicability of the present method.
  • a fluorescent protein biosensor of profilin membrane binding is prepared by labeling purified profilin (Federov et al.(1994), J. Molec. Biol. 241 :480-482; Lanbrechts et al. (1995), Eur. J. Biochem. 230:281-286) with a probe possessing a fluorescence lifetime in the range of 2-300 ns.
  • the labeled profilin is introduced into living indicator cells using bulk loading methodology and the indicator cells are treated with test compounds. Fluorescence anisotropy imaging microscopy (Gough and Taylor (1993), J. Cell Biol.
  • Rho-RhoGDI complex translocation to the membrane is used to measure test- compound dependent movement of the fluorescent derivative of profilin between the cytoplasm and membrane for a period of time after treatment ranging from 0.1 s to 10 h.
  • Rho-RhoGDI complex translocation to the membrane is used in another embodiment.
  • indicator cells are treated with test compounds and then fixed, washed, and permeabihzed.
  • the indicator cell plasma membrane, cytoplasm, and nucleus are all labeled with distinctly colored markers followed by immunolocalization of Rho protein (Self et al. (1995), Methods in Enzymology 256:3-10; Tanaka et al. (1995), Methods in Enzymology 256:41-49) with antibodies labeled with a fourth color.
  • Each of the four labels is imaged separately using the cell screening system, and the images used to calculate the amount of inhibition or activation of translocation effected by the test compound.
  • the images of the probes used to mark the plasma membrane and cytoplasm are used to mask the image of the immunological probe marking the location of intracellular Rho protein.
  • the integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the plasma membrane integrated brightness/area by the cytoplasmic integrated brightness/area. By comparing the translocation quotient values from control and experimental wells, the percent translocation is calculated for each potential lead compound. -Arrestin translocation to the plasma membrane upon G-protein receptor activation.
  • the translocation of ⁇ -anestin protein from the cytoplasm to the plasma membrane is measured in response to cell treatment.
  • living indicator cells containing luminescent domain markers are treated with test compounds and the movement of the ⁇ -anestin marker is measured in time and space using the cell screening system of the present invention.
  • the indicator cells contain luminescent markers consisting of a green fluorescent protein ⁇ -anestin (GFP- ⁇ -axrestin) protein chimera (Barak et al. (1997), J. Biol. Chem. 272:27497-27500; Daaka et al. (1998), J. Biol Chem.
  • the indicator cells that is expressed by the indicator cells through the use of transient or stable cell transfection and other reporters used to mark cytoplasmic and membrane domains.
  • the domain marker molecules partition predominately in the plasma membrane or in the cytoplasm. In the high-content screen, these markers are used to delineate the cell cytoplasm and plasma membrane in distinct channels of fluorescence.
  • the indicator cells are treated with a test compound, the dynamic redistribution of the GFP- ⁇ -anestin is recorded as a series of images over a time scale ranging from 0.1 s to 10 h. In a prefened embodiment, the time scale is 1 h.
  • Each image is analyzed by a method that quantifies the movement of the GFP- ⁇ -anestin protein chimera between the plasma membrane and the cytoplasm.
  • the images of the probes used to mark the plasma membrane and cytoplasm are used to mask the image of the GFP- ⁇ -anestin probe marking the location of intracellular GFP- ⁇ -anestin protein.
  • the integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the plasma membrane integrated brightness/area by the cytoplasmic integrated brightness/area. By comparing the translocation quotient values from control and experimental wells, the percent translocation is calculated for each potential lead compound.
  • the output of the high- content screen relates quantitative data describing the magnitude of the translocation within a large number of individual cells that have been treated with test compounds of interest.
  • an endoplasmic reticulum to Golgi translocation high- content screen the translocation of a VSVG protein from the ts045 mutant strain of vesicular stomatitis vims (Ellenberg et al. (1997), J. Cell Biol. 138:1193-1206; Presley et al. (1997) Nature 389:81-85) from the endoplasmic reticulum to the Golgi domain is measured in response to cell treatment.
  • indicator cells containing luminescent reporters are treated with test compounds and the movement of the reporters is measured in space and time using the cell screening system of the present invention.
  • the indicator cells contain luminescent reporters consisting of a GFP-VSVG protein chimera that is expressed by the indicator cell through the use of transient or stable cell transfection and other domain markers used to measure the localization of the endoplasmic reticulum and Golgi domains.
  • the GFP-VSVG protein chimera molecules are partitioned predominately in the endoplasmic reticulum.
  • domain markers of distinct colors used to delineate the endoplasmic reticulum and the Golgi domains in distinct channels of fluorescence.
  • the dynamic redistribution of the GFP-VSVG protein chimera is recorded as a series of images over a time scale ranging from 0.1 s to 10 h. Each image is analyzed by a method that quantifies the movement of the GFP-VSVG protein chimera between the endoplasmic reticulum and the Golgi domains. To do this calculation, the images of the probes used to mark the endoplasmic reticulum and the Golgi domains are used to mask the image of the GFP-VSVG probe marking the location of intracellular GFP- VSVG protein.
  • the integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the endoplasmic reticulum integrated brightness/area by the Golgi integrated brightness/area. By comparing the translocation quotient values from control and experimental wells, the percent translocation is calculated for each potential lead compound.
  • the output of the high-content screen relates quantitative data describing the magnitude of the translocation within a large number of individual cells that have been treated with test compounds of interest at final concentrations ranging from 10 "12 M to 10 "3 M for a period ranging from 1 min to 10 h.
  • the functional localization of macromolecules in response to external stimuli is measured within living cells.
  • Glycolytic enzyme activity regulation In a prefened embodiment of a cellular enzyme activity high-content screen, the activity of key glycolytic regulatory enzymes are measured in treated cells.
  • indicator cells containing luminescent labeling reagents are treated with test compounds and the activity of the reporters is measured in space and time using cell screening system of the present invention.
  • the reporter of intracellular enzyme activity is fructose-6- phosphate, 2-kinase/fructose-2,6-bisphosphatase (PFK-2), a regulatory enzyme whose phosphorylation state indicates intracellular carbohydrate anabolism or catabolism (Deprez et al. (1997) J. Biol. Chem. 272:17269-17275; Kealer et al. (1996) EEES letters 395:225-227; Lee et al. (1996), Biochemistry 35:6010-6019).
  • the indicator cells contain luminescent reporters consisting of a fluorescent protein biosensor of PFK-2 phosphorylation.
  • the fluorescent protein biosensor is constmcted by introducing an environmentally sensitive fluorescent dye near to the known phosphorylation site of the enzyme (Deprez et al. (1997), supra; Giuliano et al. (1995), supra).
  • the dye can be of the ketocyanine class (Kessler and Wolfbeis (1991), Spectrochimica Ada 47A:187-192 ) or any class that contains a protein reactive moiety and a fluorochrome whose excitation or emission spectmm is sensitive to solution polarity.
  • the fluorescent protein biosensor is introduced into the indicator cells using bulk loading methodology.
  • ratio image data are obtained from living treated indicator cells by collecting a spectral pair of fluorescence images at each time point. To extract mo ⁇ hometric data from each time point, a ratio is made between each pair of images by numerically dividing the two spectral images at each time point, pixel by pixel. Each pixel value is then used to calculate the fractional phosphorylation of PFK-2. At small fractional values of phosphorylation, PFK-2 stimulates carbohydrate catabolism. At high fractional values of phosphorylation, PFK-2 stimulates carbohydrate anabolism.
  • Protein kinase A activity and localization of subunits are measured in response to treatment with test compounds.
  • PKA protein kinase A
  • the indicator cells contain luminescent reporters including a fluorescent protein biosensor of PKA activation.
  • the fluorescent protein biosensor is constmcted by introducing an environmentally sensitive fluorescent dye into the catalytic subunit of
  • the dye can be of the ketocyanine class (Kessler, and Wolfbeis (1991), Spectrochimica Ada 47A:187-192) or any class that contains a protein reactive moiety and a fluorochrome whose excitation or emission spectrum is sensitive to solution polarity.
  • the fluorescent protein biosensor of PKA activation is introduced into the indicator cells using bulk loading methodology.
  • living indicator cells are treated with test compounds, at final concentrations ranging from 10 "12 M to 10 "3 M for times ranging from 0.1 s to 10 h.
  • ratio image data are obtained from living treated indicator cells. To extract biosensor data from each time point, a ratio is made between each pair of images, and each pixel value is then used to calculate the fractional activation of PKA (e.g., separation of the catalytic and regulatory subunits after cAMP binding). At high fractional values of activity, PFK-2 stimulates biochemical cascades within the living cell.
  • indicator cells containing luminescent reporters are treated with test compounds and the movement of the reporters is measured in space and time using the cell screening system.
  • the indicator cells contain luminescent reporters consisting of domain markers used to measure the localization of the cytoplasmic and nuclear domains.
  • the dynamic redistribution of a PKA fluorescent protein biosensor is recorded intracellularly as a series of images over a time scale ranging from 0.1 s to 10 h. Each image is analyzed by a method that quantifies the movement of the PKA between the cytoplasmic and nuclear domains.
  • the images of the probes used to mark the cytoplasmic and nuclear domains are used to mask the image of the PKA fluorescent protein biosensor.
  • the integrated brightness per unit area under each mask is used to form a translocation quotient by dividing the cytoplasmic integrated brightness/area by the nuclear integrated brightness/area.
  • the percent translocation is calculated for each potential lead compound.
  • the output of the high-content screen relates quantitative data describing the magnitude of the translocation within a large number of individual cells that have been treated with test compound in the concentration range of 10 " M to 10 " M. High-content screens involving the induction or inhibition of gene expression RNA-based fluorescent biosensors
  • the reporter of intracellular gene expression is an oligonucleotide that can hybridize with the target mRNA and alter its fluorescence signal.
  • the oligonucleotide is a molecular beacon (Tyagi and Kramer (1996) Nat.
  • Biotechnol 14:303-308) a luminescence-based reagent whose fluorescence signal is dependent on intermolecular and intramolecular interactions.
  • the fluorescent biosensor is constmcted by introducing a fluorescence energy transfer pair of fluorescent dyes such that there is one at each end (5' and 3') of the reagent.
  • the dyes can be of any class that contains a protein reactive moiety and fluorochromes whose excitation and emission spectra overlap sufficiently to provide fluorescence energy transfer between the dyes in the resting state, including, but not limited to, fluorescein and rhodamine (Molecular Probes, Inc.).
  • a portion of the message coding for ⁇ -actin (Kislauskis et al. (1994), J. Cell Biol. 127:441-451; McCann et al. (1997), Proc. Natl. Acad. Sci. 94:5679-5684; Sutoh (1982), Biochemistry 21:3654-3661) is inserted into the loop region of a hai ⁇ in-shaped oligonucleotide with the ends tethered together due to intramolecular hybridization.
  • a fluorescence donor fluorescein
  • rhodamine fluorescence acceptor
  • the fluorescence energy transfer is maximal and therefore indicative of an unhybridized molecule.
  • the tether is broken and energy transfer is lost.
  • the complete fluorescent biosensor is introduced into the indicator cells using bulk loading methodology.
  • living indicator cells are treated with test compounds, at final concentrations ranging from 10 " M to 10 " M for times ranging from 0.1 s to 10 h.
  • ratio image data are obtained from living treated indicator cells. To extract mo ⁇ hometric data from each time point, a ratio is made between each pair of images, and each pixel value is then used to calculate the fractional hybridization of the labeled nucleotide. At small fractional values of hybridization little expression of ⁇ -actin is indicated. At high fractional values of hybridization, maximal expression of ⁇ -actin is indicated. Furthermore, the distribution of hybridized molecules within the cytoplasm of the indicator cells is also a measure of the physiological response of the indicator cells.
  • the integrated intensity from the masked insulin image is compared to a set of images containing known amounts of labeled insulin.
  • the amount of insulin bound to the cell is determined from the standards and used in conjunction with the total concentration of insulin incubated with the cell to calculate a dissociation constant or insulin to its cell surface receptor.
  • Whole cell labeling is accomplished by labeling cellular components such that dynamics of cell shape and motility of the cell can be measured over time by analyzing fluorescence images of cells.
  • small reactive fluorescent molecules are introduced into living cells. These membrane-permeant molecules both diffuse through and react with protein components in the plasma membrane. Dye molecules react with intracellular molecules to both increase the fluorescence signal emitted from each molecule and to entrap the fluorescent dye within living cells. These molecules include reactive chloromethyl derivatives of aminocoumarins, hydroxycoumarins, eosin diacetate, fluorescein diacetate, some Bodipy dye "derivatives, and tetramethylrhodamine. The reactivity of these dyes toward macromolecules includes free primary amino groups and free sulfhydryl groups.
  • the cell surface is labeled by allowing the cell to interact with fluorescently labeled antibodies or lectins (Sigma Chemical Company, St. Louis, MO) that react specifically with molecules on the cell surface.
  • fluorescently labeled antibodies or lectins Sigma Chemical Company, St. Louis, MO
  • Cell surface protein chimeras expressed by the cell of interest that contain a green fluorescent protein, or mutant thereof, component can also be used to fluorescently label the entire cell surface.
  • labeling the whole plasma membrane employs some of the same methodology described above for labeling the entire cells.
  • Luminescent molecules that label the entire cell surface act to delineate the plasma membrane.
  • subdomains of the plasma membrane, the extracellular surface, the lipid bilayer, and the intracellular surface can be labeled separately and used as components of high content screens.
  • the extracellular surface is labeled using a brief treatment with a reactive fluorescent molecule such as the succinimidyl ester or iodoacetamde derivatives of fluorescent dyes such as the fluoresceins, rhodamines, cyanines, and Bodipys.
  • the extracellular surface is labeled using fluorescently labeled macromolecules with a high affinity for cell surface molecules.
  • fluorescently labeled macromolecules with a high affinity for cell surface molecules include fluorescently labeled lectins such as the fluorescein, rhodamine, and cyanine derivatives of lectins derived from jack bean (Con A), red kidney bean (erythroagglutinin PHA-E), or wheat germ.
  • fluorescently labeled antibodies with a high affinity for cell surface components are used to label the extracellular region of the plasma membrane. Extracellular regions of cell surface receptors and ion channels are examples of proteins that can be labeled with antibodies.
  • the lipid bilayer of the plasma membrane is labeled with fluorescent molecules.
  • These molecules include fluorescent dyes attached to long chain hydrophobic molecules that interact strongly with the hydrophobic region in the center of the plasma membrane lipid bilayer.
  • fluorescent dyes include the PKH series of dyes (U.S. 4,783,401, 4,762701, and 4,859,584; available commercially from Sigma Chemical Company, St.
  • fluorescent phospholipids such as nitrobenzoxadiazole glycerophosphoethanolamine and fluorescein-derivatized dihexadecanoylglycerophosphoetha-nolamine, fluorescent fatty acids such as 5-butyl- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid and 1 -pyrenedecanoic acid (Molecular Probes, Inc.), fluorescent sterols including cholesteryl 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate and cholesteryl 1- pyrenehexanoate, and fluorescently labeled proteins that interact specifically with lipid bilayer components such as the fluorescein derivative of annexin V (Caltag Antibody Co, Burlingame, CA).
  • fluorescent phospholipids such as nitrobenzoxadiazole
  • the intracellular component of the plasma membrane is labeled with fluorescent molecules.
  • these molecules are the intracellular components of the trimeric G-protein receptor, adenylyl cyclase, and ionic transport proteins. These molecules can be labeled as a result of tight binding to a fluorescently labeled specific antibody or by the inco ⁇ oration of a fluorescent protein chimera that is comprised of a membrane-associated protein and the green fluorescent protein, and mutants thereof.
  • ligands that are transported into cells by receptor-mediated endocytosis are used to trace the dynamics of endosomal organelles.
  • labeled ligands include Bodipy FL-labeled low density lipoprotein complexes, tetramethylrhodamine transferrin analogs, and fluorescently labeled epidermal growth factor (Molecular Probes, Inc.)
  • fluorescently labeled primary or secondary antibodies Sigma Chemical Co. St. Louis, MO; Molecular Probes, Inc. Eugene, OR; Caltag Antibody Co.
  • endosomes are fluorescently labeled in cells expressing protein chimeras formed by fusing a green fluorescent protein, or mutants thereof, with a receptor whose intemalization labels endosomes.
  • Chimeras of the EGF, transferrin, and low density lipoprotein receptors are examples of these molecules.
  • antibodies against lysosomal antigens are used to label lysosomal components that are localized in specific lysosomal domains.
  • lysosomal components are the degradative enzymes involved in cholesterol ester hydrolysis, membrane protein proteases, and nucleases as well as the ATP-driven lysosomal proton pump.
  • protein chimeras consisting of a lysosomal protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the lysosomal domain.
  • these components are the degradative enzymes involved in cholesterol ester hydrolysis, membrane protein proteases, and nucleases as well as the ATP-driven lysosomal proton pump.
  • cell permeant fluorescent dyes (Molecular Probes, Inc.) with a reactive group are reacted with living cells.
  • Reactive dyes including monobromobimane, 5-chloromethylfluorescein diacetate, carboxy fluorescein diacetate succinimidyl ester, and chloromethyl tetramethylrhodamine are examples of cell permeant fluorescent dyes that are used for long term labeling of the cytoplasm of cells.
  • polar tracer molecules such as Lucifer yellow and cascade blue-based fluorescent dyes (Molecular Probes, Inc.) are introduced into cells using bulk loading methods and are also used for cytoplasmic labeling.
  • cytoplasmic antigens are many of the enzymes involved in intermediary metabolism. Enolase, phosphofructokinase, and acetyl-CoA dehydrogenase are examples of uniformly distributed cytoplasmic antigens.
  • protein chimeras consisting of a cytoplasmic protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the cytoplasm.
  • fluorescent chimeras of uniformly distributed proteins are used to label the entire cytoplasmic domain. Examples of these proteins are many of the proteins involved in intermediary metabolism and include enolase, lactate dehydrogenase, and hexokinase.
  • antibodies against cytoplasmic antigens are used to label cytoplasmic components that are localized in specific cytoplasmic sub-domains.
  • these components are the cytoskeletal proteins actin, tubulin, and cytokeratin.
  • a population of these proteins within cells is assembled into discrete stractures, which in this case, are fibrous. Fluorescence labeling of these proteins with antibody-based reagents therefore labels a specific sub-domain of the cytoplasm.
  • non-antibody-based fluorescently labeled molecules that interact strongly with cytoplasmic proteins are used to label specific cytoplasmic components.
  • fluorescent analogs of the enzyme DNAse I Fluorescent analogs of this enzyme bind tightly and specifically to cytoplasmic actin, thus labeling a sub-domain of the cytoplasm.
  • fluorescent analogs of the mushroom toxin phalloidin or the drag paclitaxel Molecular Probes, Inc.
  • Protein chimeras consisting of a cytoplasmic protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label specific domains of the cytoplasm.
  • Fluorescent chimeras of highly localized proteins are used to label cytoplasmic sub- domains. Examples of these proteins are many of the proteins involved in regulating the cytoskeleton. They include the structural proteins actin, tubulin, and cytokeratin as well as the regulatory proteins microtubule associated protein 4 and ⁇ -actinin.
  • membrane permeant nucleic-acid-specific luminescent reagents are used to label the nucleus of living and fixed cells.
  • These reagents include cyanine-based dyes (e.g., TOTO ® , YOYO ® , and BOBOTM), phenanthidines and acridines (e.g., ethidium bromide, propidium iodide, and acridine orange), indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, and 4', 6- diamidino-2-phenylindole), and other similar reagents (e.g., 7-aminoactinomycin D, hydroxystilbamidine, and the psoralens).
  • cyanine-based dyes e.g., TOTO ® , YOYO ® , and BOBOTM
  • phenanthidines and acridines e.
  • antibodies against nuclear antigens are used to label nuclear components that are localized in specific nuclear domains.
  • nuclear antigens are the macromolecules involved in maintaining DNA structure and function.
  • DNA, RNA, histones, DNA polymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear antigens.
  • protein chimeras consisting of a nuclear protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the nuclear domain.
  • these proteins are many of the proteins involved in maintaining DNA stmcture and function. Histones, DNA polymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmic proteins such as actin are examples of nuclear proteins.
  • membrane permeant mitochondrial-specific luminescent reagents are used to label the mitochondria of living and fixed cells. These reagents include rhodamine 123, tetramethyl rosamine, JC-1, and the MitoTracker reactive dyes.
  • antibodies against mitochondrial antigens are used to label mitochondrial components that are localized in specific mitochondrial domains.
  • these components are the macromolecules involved in maintaining mitochondrial DNA stmcture and function.
  • DNA, RNA, histones, DNA polymerase, RNA polymerase, and mitochondrial variants of cytoplasmic macromolecules such as mitochondrial tRNA and rRNA are examples mitochondrial antigens.
  • Other examples of mitochondrial antigens are the components of the oxidative phosphorylation system found in the mitochondria (e.g., cytochrome c, cytochrome c oxidase, and succinate dehydrogenase).
  • protein chimeras consisting of a mitochondrial protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the mitochondrial domain.
  • these components are the macromolecules involved in maintaining mitochondrial DNA stmcture and function. Examples include histones, DNA polymerase, RNA polymerase, and the components of the oxidative phosphorylation system found in the mitochondria (e.g., cytochrome c, cytochrome c oxidase, and succinate dehydrogenase).
  • membrane permeant endoplasmic reticulum-specific luminescent reagents are used to label the endoplasmic reticulum of living and fixed cells.
  • These reagents include short chain carbocyanine dyes (e.g., DiOC 6 and DiOC 3 ), long chain carbocyanine dyes (e.g., DiIC ⁇ 6 and DiIC ⁇ 8 ), and luminescently labeled lectins such as concanavalin A.
  • antibodies against endoplasmic reticulum antigens are provided.
  • endoplasmic reticulum components that are localized in specific endoplasmic reticulum domains.
  • these components are the macromolecules involved in the fatty acid elongation systems, glucose-6-phosphatase, and HMG CoA-reductase.
  • protein chimeras consisting of a endoplasmic reticulum protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the endoplasmic reticulum domain.
  • these components are the macromolecules involved in the fatty acid elongation systems, glucose-6-phosphatase, and HMG CoA-reductase.
  • membrane permeant Golgi-specific luminescent reagents are used to label the Golgi of living and fixed cells.
  • These reagents include luminescently labeled macromolecules such as wheat germ agglutinin and Brefeldin A as well as luminescently labeled ceramide.
  • Golgi components that are localized in specific Golgi domains. Examples of these components are N- acetylglucosamine phosphotransferase, Golgi-specific phosphodiesterase, and mannose-6-phosphate receptor protein.
  • protein chimeras consisting of a Golgi protein genetically fused to an intrinsically luminescent protein such as the green fluorescent protein, or mutants thereof, are used to label the Golgi domain.
  • these components are N-acetylglucosamine phosphotransferase, Golgi-specific phosphodiesterase, and mannose-6-phosphate receptor protein.
  • the signaling pathway from the cell surface to target sites within the cell involves the translocation of plasma membrane-associated proteins to the cytoplasm.
  • plasma membrane-associated proteins For example, it is known that one of the src family of protein tyrosine kinases, pp60c-src (Walker et al (1993), J. Biol. Chem. 268:19552-19558) translocates from the plasma membrane to the cytoplasm upon stimulation of fibroblasts with platelet-derived growth factor (PDGF).
  • PDGF platelet-derived growth factor
  • the targets for screening can themselves be converted into fluorescence- based reagents that report molecular changes including ligand-binding and post- translocational modifications.

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Abstract

L'invention concerne des systèmes, des techniques, des cribles, des réactifs, et des kits destinés à l'analyse de cellules d'un système optique, afin de déterminer rapidement la répartition, l'environnement, ou l'activité de molécules reporteurs marquées par fluorescence dans des cellules, et de cribler un grand nombre de composés qui affectent de manière spécifique l'indice mitotique desdites cellules.
PCT/US2000/021426 1999-08-05 2000-08-04 Systeme criblage de cellules WO2001011341A2 (fr)

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