John Pringle - US grants

Studies of cellular reproduction and differentiation are central to basic biology and should also improve understanding of important diseases such as cancer. Successful analyses of these complex processes will be most likely if they are approached in relatively simple systems, such as the unicellular eukaryote yeast, in which the full power of both classical and molecular genetic methods can be brought to bear. The proposed research will use yeast to address three distinct but interconnected problesm. First, it will attempt to resolve the "gene-number paradox" (i.e., the general discrepancy between classical genetic and molecular estimates of numbers of genes) and thus enhance the utility of genetic method for the study of cellular and developmental processes. The principal approach to this problem will be the molecular analysis of a chromosome on which few genes have been identified by classical mutational analyses. Additional genes will be identified as transcribed regions on cloned segmants of the chromosome, and the reason for their "invisibility" to classical analyses will be explored using Southern-blotting and DNA-mediated gene disruption. Second, the genetic control of the cell cycle will be studied; the results should also help elucidate the reasons for the gene-number paradox. New cell-cycle genes will be sought by analysis of cold-sensitive-lethal mutants that arrest at specific cell-cycle stages. Analysis of extragenic suppressors of such mutants and of similar high-temperature-sensitive mutants should reveal additional genes as well as evidence about interactions among cell-cycle genes and their products. The mutant phenotypes will be characterized to derive clues to the molecular functions of the gene products and to the functional organization of cell-cycle events. New genes and evidence about intergenic interactions will also be sought by screening recombinant-DNA libraries for genes whose overexpression can suppress mutations in other genes of interest. Third, the molecular basis of cellular morphogenesis will be explored. The genetic studies just described will focus on genes that control the morphogenetic processes of the cell cycle. In addition, such genes will be cloned. Clues to the molecular functions of the gene products will then be sought by sequencing the genes and by raising antibodies that can be used to localize the gene products in the cells. The results should illuminate the roles of cytoskeletal elements and of other machanisms both in cellular reproduction and in cellular morphogenesis.

The septins are a novel family of proteins recently discovered in budding and fission yeast, insects (Drosophila), and mammals (both mouse and human). The available immunolocalization and genetic data make clear that septins are involved in cytokinesis both in the yeasts and in Drosophila (despite the apparent differences in modes of cytokinesis) and suggest strongly that they also have other roles in the organization of the cell surface in nondividing cells. The long-term goal of this project is to exploit the experimental advantages of Drosophila (particularly its susceptibility to genetic analysis) to elucidate the roles of these proteins in an animal system. Such studies should provide information about the mechanisms of cytokinesis, the interactions of cytoskeletal elements with each other and with the plasma membrane, and other aspects of the organization of the cell cortex. Specific Aims for the initial project period include; (1) Clarification of the role of the septins in ordinary cytokinesis by more detailed cytological analysis of two known septins (Sep1 and Pnut) and more detailed analysis of the cytokinesis defect in mutants lacking Pnut; (2) investigation of the apparent role of Sep1 and Pnut in the cellularization of the embryo, using similar approaches; (3) attempts to generate hypotheses about the roles of septins in nondividing cells by more detailed examination of the localization of Sep1 and Pnut in such cells; (4) initial biochemical studies of Sep1 and Pnut; (5) attempts to generate mutants defective in Sep1 and additional mutations affecting Pnut, to explore the functional overlap and interactions of these proteins, and to identify interacting proteins genetically; and (6) initial studies of the functions of a third Drosophila septins (Sep2). In future project periods, the analysis of Sep2 function will be extended, the identification nd analysis of additional Drosophila septins will be undertaken, attempts to identify and analyze the functions of interacting proteins will be continued, the roles of the septins in nondividing cells will be explored in more detail, and detailed structure-function studies of the septins will be undertaken. Cytokinesis is an essential part of the normal cell cycle and as such is crucial for normal cell proliferation and tissue development; thus, defects in cytokinesis may be important in diseases, such as cancer, that involve abnormal cell proliferation and development. Moreover, the proper organization of the cell cortex is essential for the normal organization of the cytoplasm, cell-cell adhesion, and intercellular communication, all processes that are essential for normal cell function and defective in various disease states. Thus, studies of the novel septin protein family in an experimentally tractable model system should contribute significantly to understanding both of normal cell and developmental biology and of important diseases.

The development of cell shape and three-dimensional organization (cellular morphogenesis) is a process of fundamental importance in cellular and developmental biology. Defects in cellular morphogenesis are also implicated in disease states such as birth defects and cancer. The long-term goal of the research proposed here is to exploit the remarkable experimental advantages of the unicellular eukaryote Saccharomyces cerevisiae (yeast) to elucidate general principles and mechanisms of cellular morphogenesis. A feature of cellular morphogenesis in most cells is the development of cell polarity along an appropriate axis, and loss of normal polarity is an early event in the development of epithelial tumors. As yeast chooses nonrandom sites for budding and then polarizes its cytoskeleton and cell-surface growth along the axis defined by the bud site, it provides an attractive model system for investigation of cell polarization. The specific studies proposed here will use a combination of genetic, molecular biological, biochemical, and cell biological methods to investigate two sets of problems. (I) How do persistent spatial markers in the cell cortex and a GTPase signaling module (based on the Ras-like Rsrlp) determine the bipolar pattern of bud-site selection (as normally seen in diploid cells)? (II) How does a second GTPase signalingmodule (based on the Rho-like Cdc42p) control the establishment of cytoskeletal polarization toward the selected bud site? Cytoplasmic division (cytokinesis) is another important aspect of cellular morphogenesis as well as of the cellular reproduction cycle; defects in cytokinesis also appear to contribute to the development of various kinds of cancer. The septins are a novel family of proteins first discovered on the basis of their involvement in cytokinesis in yeast. They are now known to be involved in cytokinesis in insect and mammalian cells as well, and the available evidence suggests that they also have a variety of other roles in the organization of the cell surface. The studies proposed here will use a combination of genetic, molecular biological, biochemical, and cell biological methods to investigate a third set of problems: (III) How do the septins assemble and associate with the plasma membrane? What other proteins do they organize at the cell cortex? What are the roles of the septins both in ordinary cytokinesis and in the specialized process by which spores are formed?

DESCRIPTION (provided by applicant): This is a competitive renewal application to support the Genetics/Developmental Biology Training Program at Stanford University. The funding will support the 30"'through 35"'years of this long-standing program that trains graduate students to become independent researchers and teachers. This highly-successful program is comprised of 16 Ph.D. students at any given time, who work in the laboratories of 42 participating faculty members in the Departments of Genetics and Developmental Biology. Research opportunities abound in using genetics, genomics, molecular and cell biology tools to study a wide range of problems in modern biology, including developmental biology, genomic sciences, computational biology, including comparative sequencing and analysis, functional genomics, DNA, protein, and carbohydrate microarray technologies, algorithm development, statistical genetics, high-throughput genotyping and genetic analysis, evolutionary genomics, pharmacogenetics, human population genetics, and many other biological problems that benefit from a broad multidisciplinary perspective. Many projects involve development of new wet-lab as well as computational technologies and tools. Emphases of the G/DB Training Program will be to provide a broad interdisciplinary education to a wide range of Trainees, to serve to coordinate research and training activities throughout the entire campus, and to help disseminate modern biological science by preparing Trainees for the next steps in their careers. In addition to providing this training, the Genetics/Developmental Biology Training Program, in collaboration with another major program that involves most of the laboratories in the two departments, will have an ambitious program for the Minority Action Plan and education outreach. The MAP and outreach components, which are already very strong on the Stanford campus, will expand and ensure the success of our efforts to help increase diversity in our scientific ranks while also providing younger students and the general public with knowledge about science and scientists and how these impact their daily lives.

In the U.S. and around the world, coral reefs are of enormous ecological and economic importance for biodiversity preservation, shoreline protection, food production, drug discovery, and recreation. Unfortunately, coral reefs have shown extensive and increasing degradation in recent years, apparently due largely to the inability of the corals to survive the stresses imposed by increasing ocean temperature, acidity, and pollution. Coral death is typically preceded by "bleaching", in which the coral animals lose the symbiotic algae that normally live inside their cells and provide them with most (often >90%) of their energy through photosynthesis. There has been much recent attention to this problem from marine and conservation biologists, but their efforts have been severely handicapped by the lack of basic information about the molecular and cellular mechanisms that underlie coral growth and the interactions between the animal hosts and their algal symbionts. This gap is due largely to the fact that corals themselves present multiple significant difficulties for laboratory study. In other areas of biological and biomedical research, progress has come primarily from intensive studies of a small number of "model organisms" that were chosen not for their intrinsic interest, but instead for their susceptibility to intensive experimental investigation, including the application of powerful genetic methods for investigation of cell function. This project focuses on developing the small sea anemone Aiptasia as a model system for the study of coral molecular and cell biology. Aiptasia is closely related to corals and is symbiotic with similar or identical types of algae, but it has many great experimental advantages. The specific studies proposed here build on recent progress in the PI's laboratory (notably the induction of spawning and production of larvae in the laboratory) and focus on three different aspects of developing genetic methods for this system, namely (1) methods for following natural and induced variation in genetic crosses, (2) methods for obtaining RNA-mediated knockdown of gene expression, and (3) methods for transforming animals with DNA constructs that will allow disruption or tagging of individual genes.
Progress with these methods should have a major impact on investigations of basic coral biology and will be communicated to the research community through presentations at conferences, publications, and both laboratory-specific and community-maintained websites. In the process, both postdoctoral and predoctoral students will receive a diverse and sophisticated training to prepare them for independent careers in research and/or policy. In addition, the intrinsic fascination of the complex and beautiful coral-reef ecosystem will provide numerous opportunities for effective outreach to both younger students and the general public on both coral-specific and more general issues of basic science and its relationship to conservation.

Cytokinesis is the final stage in cell division in which the single mother cell is physically separated into two daughter cells, and is essential for the normal proliferation and differentiation of cells of every form of life. Historically, studies of cytokinesis have focused on just two groups of organisms: the animals, fungi and their relatives, whose cells divide by forming an inward-growing "cleavage furrow", and the land plants, whose cells divide by forming an outward-growing "cell-plate". Thus, there is currently an unresolved paradox at the heart of cell biology: by what mechanism did the common ancestor of modern cells divide, and how did that mechanism evolve into the seemingly very different mechanisms seen today? This project will exploit the experimental advantages and favorable evolutionary position of the green alga Chlamydomonas reinhardtii (which is closely related to land plants but divides by forming a cleavage furrow like animals) to help elucidate the common core mechanisms that underlay cytokinesis in ancestral cells and are still shared in modern organisms despite their superficial differences. Understanding these core mechanism will provide a better understanding not only of animal, fungal, and plant cells, but also of a broad range of other cell types that have been largely ignored by cell biologists but are of enormous ecological and economic importance. In addition to basic knowledge, this project will provide new experimental tools that should be valuable both in basic research and in applied studies (particularly in biofuels development), and it will provide opportunities for postdoctoral, graduate, undergraduate, and high-school students (including students from underrepresented groups) to learn genetics, molecular biology, and cell biology as a prelude to continuing in careers in science and engineering.

This project focuses on elucidation of the mechanisms by which Chlamydomonas cells form cleavage furrows for cell division without a contractile actomyosin ring (CAR), a structure formed of filamentous (F) actin, type II myosin, and other proteins, which has long been thought to be necessary for furrow formation in animal and fungal cells. However,it is now clear both that many cells that normally have a CAR can divide without it and that most eukaryotic cells in other phylogenetic groups divide by forming furrows but have no myosin-II, so there must another driver(s) of this process. To elucidate the mechanisms of furrowing in Chlamydomonas and the associated cytoskeletal functions, three interrelated aims will be pursued. (1) Improved methods for expressing tagged proteins of interest and for microscopic visualization of cytokinesis processes and cytoskeletal dynamics will be developed and used to clarify the details of these processes and the questions to be asked at the molecular level. (2) A combination of microscopy, biochemistry, genetics, and transcriptome analyses will be used to investigate the possible role of F-actin in the cleavage furrow and the mechanisms and roles of the recently discovered dramatic transcriptional regulatory response to actin depolymerization. Similar approaches will be used to investigate the function(s) of the single Chlamydomonas septin protein, which is also expected (from comparisons to other organisms) to be involved in cytokinesis. (3) Imaging-based methods and high-throughput platforms will be used to identify mutants defective in cytokinesis and/or cytoskeletal dynamics; characterization of these mutants should help to reveal the underlying mechanisms of furrow formation and cytoskeletal behavior through the cell cycle.

Coral reefs are profoundly important, diverse ecosystems that are threatened worldwide by environmental variation and stress. They have huge economic value as fisheries, storm barriers and destinations for ecotourism. Corals are made up of large numbers of interconnected animal hosts, called polyps that house microscopic algae inside their cells. This partnership, or symbiosis, is the foundation of the entire coral reef ecosystem. The polyps receive photosynthetic nutrients from the algae, and the algae receive nutrients from the polyps. While a great deal of attention has been focused on the environmental threats to corals, there remains only a partial understanding of the microscale cellular, molecular, and genetic mechanisms that underpin the coral-algal symbiosis. Insight into regulation of this symbiosis will provide a stronger foundation for studies of coral health and coral stress, such as coral bleaching in which the host polyps lose their symbiotic algae. This project will bring together a diverse team of coral biologists, cell biologists and geneticists to study a small sea anemone that serves as an excellent proxy for corals, which do not survive well in the lab, are slow growing, and difficult to collect because of their endangered status. In contrast, the fast-growing, weedy sea anemone Aiptasia will enable the researchers to make rapid progress on the study of coral symbiosis. This award is focused on technique development and swift dissemination of results through online communication platforms to both the scientific community and the public. A variety of genetic techniques will be developed, including gene editing in both partners to be able to test hypotheses about the involvement of specific genes in coral health and stress. To increase the effectiveness and efficiency of research efforts to understand the cellular, molecular and genetic foundations of this symbiosis, the researchers will develop and optimize approaches to rear the complete life cycle of Aiptasia in the laboratory. This award will contribute to the training of scientists and expose school-aged children and the general public to coral reef and symbiosis science.

The ecological, economic, and aesthetic importance of coral reefs is widely recognized, as are the threats they face from environmental stress. Nonetheless, the underlying molecular and cell biology of corals and their resident endosymbiotic dinoflagellates (genus Symbiodinium) that are critical for host nutrition remain poorly understood. A major reason for this paucity of knowledge is the lack of methods for mutant isolation and analysis, gene knockdown, gene knockout, and gene tagging. Without these, even powerful genomic and transcriptomic analysis largely yield correlations whose implications for causal mechanisms remain uncertain. This project focuses on development of approaches to test gene function in a rapidly developing model system, the sea anemone Aiptasia and the Symbiodinium minutum (Clade B1) strain SSB01, which can form a stable endosymbiosis with Aiptasia, and can grow well in culture. Genetic manipulation in SSB01 and in small polyps will include morpholino and CRISPR/Cas9 approaches to achieve gene knockdown, knockout and gene tagging. The researchers will identify and test compounds from natural substrates and biofilms to achieve larval settlement, and will identify and test anemone neuropeptides as a means to achieve metamorphosis of Aiptasia routinely in the laboratory. Finally, a set of interwoven communications platforms and activities will be developed to rapidly disseminate information gained in the project to the broader scientific community.