Jeffrey E. Segall - US grants

Amoeboid chemotaxis is an important phenomenon in which signal transduction and cell motility interact. It may be critical to movement of cancer cells away from the original in metastasis as well as the ability of white blood cells such as to respond rapidly to infection. Dictyostelium discoideum amoebae provide a powerful model system for analyzing amoeboid motility and chemotaxis using a genetic approach. They have a habloid genome which allbws expression of be analyzed genetically, and are easily cultivited. The ultimate goal of this study is a dissection of amoeboid motility and chemotaxis by analyzing the biochemical and behavioral properties of mutants. Mutants will be generated by chemical mutagenesis. Dictyostelium mutants will be selected using an established selection chamber and screening assay. The mutants that are selected will be analyzed in terms of their biochemistry and behavior. Chemoattractant binding, production of intracellular messengers (such as IP3, calcium, and cGMP), and actin/myosin association with the cytoskeleton will assayed. Computer-controlled image analysis will be used to determine single cell speed, turning, orientation and shape in response to both stable chemoattractant gradients and rapid changes in concentration. Genetic studies will determine complementation groups and insure that the biochemical defect is associated with the behavioral alteration. The wild-type copies of the mutant genes will be cloned by complementing selected mutants with a library of genomic DNA in a Dictyostelium transformation vector. Chemotactic transformants will be selected on the basis of morphology or using the selection chamber. Recovery of the transformation vector from chemotactic transformants will lead to the identification of wild-type sequences that can complement the mutation. Tbe long term objective is to identify the biochemical processes responsible for particular behavioral responses.

9304992 Segall A new method (termed the micropipet assay) has been developed to demonstrate that Saccharomyces cerevisiae cells direct growth of mating projections towards higher concentrations of mating pheromone. Current work studying yeast mating has focussed on components that are absolutely required for mating formation. The micropipet assay allows analysis of a new aspect of this pathway, gradient sensing, and thus provides the potential for identifying proteins specifically involved in this process. The work described here focuses on the signal transduction pathway that allows yeast cells to determine the direction of an external spatial gradient of mating factor. The first objective is to use this new method to assay mutants in genes potentially involved in gradient sensing. The second objective is to identify new mutants specifically defective in this orientation. The first objective will utilize the micropipet assay to characterize mutants in genes potentially involved in sensing gradients of mating factor. For example, mutants in the mating pathway, bud site selection, and cytoskeletal proteins could have altered abilities to sense and orient in spatial gradients. The second objective, identification of mutants defective in orientation in spatial gradients, will involve a two stage process. The first stage is a genetic screen for mutants with altered partner selection during mating. The phenotype expected for cells that no longer can determine the direction of spatial gradients of mating factor is a more random selection of mating partners. A simple, visual color assay of colonies on a plate will be used to identify mutants that are still able to mate, but whose choice of mating partner is no longer determined by the amount of mating factor produced by that partner. Strains that reproducibly show reduced discrimination in this assay will then be directly assayed using the micropipet assay for orientation. Strains with reduced orientati on in the micropipet assay will be grouped into complementation groups, with the eventual goal of cloning the genes via complementation with a genomic library. %%% This research is expected to provide insight into the sensing of spatial gradients of chemicals in a wide variety of organisms. ***

MCB9728324 Segall, Jeffrey This project is to understand the role of mitogen-activated protein kinases (MAP kinases) in chemotaxis and signaling responses of the amoeboid cells of the slime mold, Dictyostelium discoideum. This organism will be used as a biochemically and genetically accessible model system to delineate the molecular events by which MAP kinases activate adenylyl cyclase and give rise to changes in cell motility. The project will focus on the role of one kinase, designated DdERK2, which the principal investigator has already partially characterized. The project will now identify and characterize potential target proteins that DdERK2 phosphorylates. Approaches will include both an in vitro biochemical kinase assay and a yeast two-hybrid genetic assay. The functions of the target proteins identified by either approach will be tested by analysis of the phenotypes that result when the genes encoding the proteins are disrupted. Identification and detailed analysis of the proteins should provide an understanding of the sequential events in this signaling pathway, both in Dictyostelium and presumably other organisms.

DESCRIPTION: (Adapted from the investigator's abstract) The proteins ErbB1 and ErbB2 have been found to be important indicators of poor prognosis in breast cancer. However, the mechanisms by which these molecules contribute to metastasis have not been established. In particular, determination of whether there is a rate limiting step for metastasis which is determined by these molecules is unknown. Identification of the stage of metastasis that is enhanced by ErbB1 and ErbB2 together with the molecular mechanism is critical for determining approaches for treatment and prognosis of breast cancer. He has developed new methods for evaluating tumor cells in the primary tumor, blood and target organs, and will use these methods for evaluating tumor cells in the primary tumor, blood and target organs, and will use these methods to provide important information regarding how ErbB1 and ErbB2 function in metastasis. The objective of this proposal is to determine whether chemotaxis stimulated by ErbB1 and ErbB2 is rate-limiting for metastasis. The first specific aim will be to complete development of a metastasis assay that will provide information of blood burden, cell arrest in the lungs and metastasis formation in the lungs. A pair of well-characterized rat mammary adenocarcinoma cell lines using orthotopic implantation in a syngeneic host are being used to develop the assay. The assay will then be applied to human mammary adenocarcinoma cell lines implanted in nude mice. The second specific aim will be to use this assay to evaluate the contributions of ErbB1 and ErbB2 to metastasis in terms of chemotaxis and mitogenesis. The third specific aim will evaluate the roles of the rho family of small G proteins in metastasis.

[unreadable] DESCRIPTION (provided by applicant): The second Gordon Conference on Gradient Sensing and Directed Cell Migration will be held January 28 - February 2, 2007 at the Crowne Plaza, Ventura, California. This meeting will focus directly on mechanisms of eukaryotic chemotaxis. Because chemotaxis is critical for allergies and infectious disease, neurological diseases, development, immunology, wound healing, and tumor cell metastasis, the meeting is designed to bring together the leading chemotaxis researchers in these fields for cross-fertilization of concepts and approaches. This strategy is manifested by organizing each topic according to stage in the process; for example, researchers in different fields who are focused on chemotaxis signaling networks are grouped together in a single session. The session topics are chosen to focus attention on the critical steps in chemotactic signaling, providing attendees with an integrated overview of the entire chemotaxis process over the course of the entire meeting. The goal of the meeting is to advance our understanding of these mechanisms by bringing together modelers with the leading experts in the study of cell polarization in chemotaxis in systems including leukocytes, neurons, fibroblasts, tumor cells, yeast, Dictyostelium, and Drosophila. We have planned nine sessions with roughly 34 plenary talks, and each session will be chaired by an expert in the topic who will lead the discussion. Considerable time is planned to encourage extensive discussion. The program will provide opportunities for investigators at all levels to participate, including those currently funded through the NIH, including NIGMS, NINDS, NHLBI, NICHD, NCI, and NIAID. The program will also provide opportunity for 10 "short talk" presentations, which will be selected by the session chairs from submitted abstracts and posters. The final list of conferees (including session chairs, speakers and participants) will be chosen so as to represent researchers from all sectors. We will encourage the participation of junior investigators, post-doctoral trainees and new investigators to the field in all aspects of the meeting. In addition, we will foster the involvement of women, diverse groups of minorities and handicapped individuals who are interested in studying chemotaxis. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The tools and resources are now available for the development of screens for genes that enhance metastasis in vivo. Such screens will identify new targets for drug development that may complement therapies currently under development or in clinical use. In addition, the information provided by such screens will aid in reducing the number of potential targets that are identified through microarray studies by indicating which gene products are likely to be driving metastasis. We propose to combine high throughput gene expression and suppression technologies with in vivo metastasis methodology to accelerate in vivo screening by a factor of up to 50. The screen will indicate the contributions of proteins to the steps of primary tumor growth, intravasation, and lung colonization. The first aim will focus on production of pools of cells expressing or suppressing selected proteins. The second aim will evaluate detection technologies for measuring construct distributions in a pool. The third aim will determine the appropriate formation of pools to be screened. The fourth and fifth aims will perform an initial screen and validate candidates identified in the screen. This R21 application will enable the development of the screen and demonstrate feasibility; providing a paradigm for evaluating gene function for the most critical feature of tumor cell malignancy - the ability to metastasize. [unreadable] [unreadable] ASSESSMENT: [unreadable] [unreadable] [unreadable]