Gary J. Gorbsky - US grants

DESCRIPTION (adapted from applicant's abstract): The kinetochore mediates the movement of chromosomes along the microtubules of the mitotic spindle. The Cyert antibody, which is directed to an incompletely characterized, phosphorylated epitope, binds to the kinetochores of chromosomes in prometaphase in a unprecedented and intriguing manner. Chromosomes moving toward the metaphase plate express the phospho-epitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This differential expression of the epitope may reflect a biochemical pathway regulating chromosome movement and the progression of mitosis. Two competing hypotheses for the molecular nature of the epitope recognized by the Cyert antibody will be tested. First, the antibody recognizes a regulatory phosphorylation on a protein that powers or guides chromosome movement. Second, the epitope recognizes an enzymatic intermediate of a phosphatase that is differentially regulated at kinetochores. The aims of this application are: (1) to examine how expression of the phospho-epitope changes in relation to chromosome movement and mitotic progression; (2) to determine how kinases and phosphatases regulate that expression; (3) to identify the proteins that contain the epitope; and (4) to understand how positional and mechanical signals in the mitotic spindle are converted to biochemical information that regulates chromosome movement and progression through mitosis. These aims will be achieved by immunochemical isolation, fractionation, and molecular characterization of kinetochore proteins. These efforts will be complemented by the analysis of chromosome behavior in live cells, immunolabeling studies, micromanipulation of chromosomes, and the microinjection of antibodies and inhibitors.

The objective of this application is to establish a state-of-the-art facility for the execution of cellular and subcellular "knockouts" of protein function through the technique of Chromophore-Assisted Laser Inactivation (CALI). In this technique the chromophore Malachite green is covalently coupled to an antibody. The Malachite green-antibody conjugate is microinjected into living cells. Controlled laser pulses from a nitrogen pumped dye laser are applied. At the wavelengths used, only the chromophore absorbs the laser energy. Proteins bound by the Malachite green-labeled antibody are inactivated by free radical damage. Because antibodies are used as targeting probes, the CALI technique provides a high degree of molecular specificity. Because inactivation occurs only upon laser irradiation, the technique provides excellent temporal control and the ability to image cells at high resolution before and after inactivation. Finally, because the laser can be focused on the entire cell or onto a small subregion, the echnique provides excellent spatial resolution in disrupting cellular pathways. The members of the major user group intend to apply this approach in four areas of mammalian cell biology: 1) signaling components of the M phase checkpoint (Gorbsky), 2) the role of SCAMPS and other vesicle trafficking proteins (Castle), 3) signaling and assembly of focal adhesions, 4) activation of the Map kinase pathway in G1 and mitosis (Weber). Currentiy each laboratory has been microinjecting function-blocking antibodies into living cells as a means of studying cellular signaling and physiology. However, most antibodies are neutral; i.e they have no inherent function-blocking ability. The CALI technique uses these neutral antibodies as knockout reagents to examine the role of target proteins in vivo. The protein ablation microscope workstation is intended to complement other knockout strategies (anti- sense, mouse gene knockouts) and become a pivotal resource for the community of researchers seeking a simple, inexpensive, and effective way to study the effects of inactivating specific proteins in living mammalian cells.

DESCRIPTION: (provided by applicant) The spindle checkpoint is a signaling pathway that normally promotes the proper segregation of chromosomes in M phase. If in mitosis the chromosomes are not properly aligned on the mitotic spindle, the spindle checkpoint halts cell cycle progression prior to the metaphase-to-anaphase transition. This delay allows more time for chromosomes to attach the mtitotic spindle microtubules and move to their proper positions at the midplane of the cell. Individual cells can be artificially forced to override the spindle checkpoint by the injection of antibodies to proteins of the spindle checkpoint or by transfection of cDNA's encoding mutant, dominant-negative spindle checkpoint proteins. These interventions induce massive imbalances in chromosome segregation that can be directly lethal or cause apoptosis. Tumor cells often exhibit defective checkpoint responses compared to normal somatic cells. The relative weakness of tumor cells to induce cell cycle checkpoints in response to damage may be one mechanism by which anti-cancer therapies show some selectivity for tumors. Drugs that specifically target cell cycle checkpoints may be useful in further sensitizing tumor cell population to therapy. There are currently no cell permeable drugs that can inactivate the spindle checkpoint. The project proposed here is to develop and institute a high throughput screen for drugs that can penetrate cell membranes and inactivate the spindle checkpoint. Several of the protein components of the spindle checkpoint pathway have no other functions. Thus it is reasonable that specific small molecule inhibitors of this pathway can be found. Once effective drugs have been identified, the specific proteins of the spindle checkpoint pathway that are targeted will be determined. It is anticipated that these drugs will be clinically useful for anti-cancer therapy, particularly when used in combination with chemotherapeutic anti-microtubule drugs such as the vinca alkaloids and the taxanes which are themselves potent inducers of the spindle checkpoint.

[unreadable] DESCRIPTION (provided by applicant): The objective of this application is to establish a novel state-of-the-art facility termed the Cell Dynamics Microscope for the observation and manipulation of molecules within living cells using fluorescence microscopy. The envisioned workstation is centered on a Zeiss Axiovert 200M inverted microscope with two major accessories. The first is a Perkin Elmer Ultraview Live Cell Imager for imaging fluorescence in living cells with minimal photobleaching and phototoxicity. The second accessory is a Digital Diaphragm laser illumination system from Photonic Instruments. This new instrument allows intense laser illumination of any subregion, set of subregions or pattern within the field of view. Setting of subregions or patterns is controlled by drawing or importing patterns on a computer monitor image of the target. This ability permits unprecedented control of illumination for photochemical techniques including fluorescence recovery after photobleaching (FRAP), Fluorescence loss in photobleaching (FLIP), fluorescence resonance energy transfer (FRET), photoactivation of caged compounds, direct photoablation, dyesensitized photoablation (sometimes called chromophore-assisted laser inactivation or CALl), and ion imaging. The members of the major user group will apply these techniques in several areas: Microtubule and Kinetochore Dynamics in Mitosis (Gorbsky), Protein Translocation in Photoreceptor Cells (McGinnis), Microtubules in Insulin-mediated Glut4 Translocation (Olson), NF-KB Dynamics in Bladder Epithelium (Saban), and Polycystin-2 in Primary Cilia and Mitotic Spindles (Tsiokas). These projects will be supplemented by nine projects from a group of additional users. The analyses performed with this new instrument will complement the molecular studies ongoing in each laboratory. The Cell Dynamics Microscope will become a pivotal resource for the community of researchers seeking elegant and userfriendly methods to observe and manipulate target molecules within living cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chromosome instability (CIN) is an important component in several human health problems including cancer, birth defects, and infertility. The Gorbsky lab discovered a new source of CIN that was termed cohesion fatigue. Cohesion fatigue is the progressive, asynchronous separation of sister chromatids in cells delayed at metaphase. The overall goals of this project are to map the downstream consequences of cohesion fatigue, the mechanisms by which chromatids surrender cohesion, and the upstream pathways that modulate the cell sensitivity to cohesion fatigue. In Aim 1, advanced microscopy at both the single cell level and population level will be used to track the chromosome abnormalities that arise from cohesion fatigue. Cohesion fatigue has the potential to simultaneously generate the two types of gross chromosome aberrations that often arise during oncogenesis, changes in whole chromosome number (aneuploidy) and large segmental chromosome duplications, deletions, and translocations. In addition, cohesion fatigue is highly likely to lead to the formation of micronuclei, which have been implicated as sites of massive DNA damage. Aim 2 will determine the mechanisms of cohesin release during cohesion fatigue through experiments that lock individual joints of the cohesin protein complex. In addition, quantitative mass spectrometry will be used to analyze cohesin components that are removed or altered in their post-translational modifications during cohesion fatigue. Aim 3 will map the upstream pathways that regulate sensitivity to cohesion fatigue, concentrating on defects in transformed cells that may exacerbate CIN. Transformed cells often exhibit defects in cell cycle regulators, and cohesion genes are among the most often mutated in human tumors. Thus, transformed cells may be highly susceptible to cohesion fatigue. This aim will test how alterations of cell cycle regulators induce metaphase delays and how these delays synergize with transformation-associated defects in spindle microtubule dynamics and chromosome cohesion to promote cohesion fatigue. Aim 4 extends the analysis of cohesion fatigue to budding yeast to examine the conservation of cohesion fatigue regulators in mitosis and to test specific hypotheses about how cohesion fatigue contributes to premature loss of cohesion between homologous chromosomes during meiosis. Recent evidence implicates decay in chromosome cohesion as a contributor to the maternal age effect, whereby the oocytes of older women show a greatly increased incidence of aneuploidy. In mammalian gametes, cohesion fatigue may be an important causative factor in meiotic aneuploidy, contributing to birth defects and infertility.

Abstract The Aim of the Imaging Core is to provide enabling technology in histology and microscopy for all five Junior Investigators and Pilot Project Investigators in this Developmental Biology COBRE.

Abstract Cell division requires a complex network of dozens of pathways whose interactions drive key transitions. One of the most critical is the transition from metaphase to anaphase and mitotic exit. The Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase, comprises the central target node of this critical decision point. APC/C activity is sensitive to chromosome congression to the metaphase plate. At metaphase, the APC/C ubiquitylates target mitotic regulators for destruction in the proteasome and induction of chromatid separation and mitotic exit. While regulatory pathways for the APC/C have been identified, complete understanding of how it is tuned to program the metaphase-anaphase transition after chromosome alignment remains unclear. Recent work has implicated the Spindle and Kinetochore Associated (Ska) protein complex as a key element. The studies proposed will clarify the molecular mechanisms by which Ska collaborates with and controls other mitotic regulators, particularly protein phosphatases, governing the temporal and spatial activation of the APC/C to drive the metaphase-anaphase transition. Metaphase, itself, is generally brief, but the Gorbsky laboratory discovered that delays, even short ones, can cause partial or complete chromatid separation, a phenomenon termed ?cohesion fatigue.? Cohesion fatigue may be remarkably common as a source of both numerical aneuploidy and large chromosome deletions, duplications and translocations, particularly in cells transformed by activated oncogenes and the loss of tumor suppressors. The Gorbsky laboratory is taking a broad approach to study all potential inputs that contribute to cohesion fatigue. The laboratory is also mapping the short term and long term consequences of cohesion fatigue to determine how it promotes chromosome missegregation and damage. Finally, complete understanding of cell division and its transitions can only occur if all components and regulators of the process are identified. While the mitotic parts list is already large, continued reports of new components indicate that it is not yet complete in vertebrates. Using bioinformatic guidance that has proven highly effective, the Gorbsky lab is testing candidate mitotic regulators and characterizing their functions in cell division in normal and transformed cells.