Fred Chang - US grants

DESCRIPTION: This investigator has identified a novel gene, cdc12, in S. pombe that appears to play an important role in the assembly and placement of the contractile ring. He hypothesizes that during interphase, cdc12 exists in a large multiprotein complex that travels along microtubules to the middle of the cell to mark the future site of the cleavage furrow. During mitosis, the cdc12 protein is hypothesized to participate in actin ring assembly through an interaction with the actin-binding protein profilin. Dr. Chang's immediate goal is to test these hypotheses. Three specific aims are proposed: (1) to determine the function of the interaction between cdc12 and cdc3 (profilin); (2) to identify and characterize additional proteins that interact with cdc12p; and (3) to investigate the spatial aspects of ring formation by examining ring formation in living cells.

phosphoproteins; phosphorylation; molecular site; biomedical resource;

DESCRIPTION (provided by applicant): Regulation of microtubule organizing centers (MTOCs) contributes to the reorganization of the microtubule cytoskeleton during the cell cycle and in development. Fission yeast Schizosaccharomyces pombe cells use three types of MTOCs for building different microtubule structures through the cell cycle. During interphase, microtubule bundles are organized by multiple interphase microtubule organizing centers (iMTOCs) on the nuclear envelope. iMTOCs are required for nuclear positioning and may contain proteins involved in microtubule nucleation, bundling and attachment to the nuclear envelope. Establishment of iMTOCs occurs during cytokinesis, concurrent with the disassembly of the equatorial MTOC (eMTOC) located at the cell division site. Here, we will determine mechanisms that orchestrate the breakdown of the eMTOC and the formation of iMTOCs. Our specific aims are: 1) to determine the roles of a J-domain protein rsp1p and an MTOC protein coi1p in regulation of Emtoc disassembly. 2) To determine the function of motile particles (satellites) containing MTOC components. We will test a model that satellites transport MTOC components from the disassemblying eMTOC to the nuclear envelope, where they contribute to iMTOC formation. 3) To identify nuclear envelope proteins that attaches iMTOCs and interphase MT bundles to the outer nuclear envelope for nuclear positioning. This work will provide insights into the regulation of microtubules of many cell types such as epithelial and muscle cells. As centrosomal defects are prevalent in cancer, MTOC regulation is now an important area in cancer research.

DESCRIPTION (provided by applicant): Regulation of microtubule organizing centers (MTOCs) contributes to the reorganization of the microtubule cytoskeleton during the cell cycle and in development. Fission yeast Schizosaccharomyces pombe cells use three types of MTOCs for building different microtubule structures through the cell cycle. During interphase, microtubule bundles are organized by multiple interphase microtubule organizing centers (iMTOCs) on the nuclear envelope. iMTOCs are required for nuclear positioning and may contain proteins involved in microtubule nucleation, bundling and attachment to the nuclear envelope. Establishment of iMTOCs occurs during cytokinesis, concurrent with the disassembly of the equatorial MTOC (eMTOC) located at the cell division site. Here, we will determine mechanisms that orchestrate the breakdown of the eMTOC and the formation of iMTOCs. Our specific aims are: 1) to determine the roles of a J-domain protein rsp1p and an MTOC protein coi1p in regulation of Emtoc disassembly. 2) To determine the function of motile particles (satellites) containing MTOC components. We will test a model that satellites transport MTOC components from the disassemblying eMTOC to the nuclear envelope, where they contribute to iMTOC formation. 3) To identify nuclear envelope proteins that attaches iMTOCs and interphase MT bundles to the outer nuclear envelope for nuclear positioning. This work will provide insights into the regulation of microtubules of many cell types such as epithelial and muscle cells. As centrosomal defects are prevalent in cancer, MTOC regulation is now an important area in cancer research.

DESCRIPTION (provided by applicant): Microtubules are cytoskeletal elements responsible for the spatial organization of cells. They carry out critical cellular processes such as chromosome segregation, cell polarization and cell migration. The dynamic behaviors of MT plus ends - their growth, shrinkage, targeting and attachment -- are modulated by a bevy of MT-associated factors. Here, we study the molecular mechanisms that regulate the MT cytoskeleton, using the fission yeast Schizosaccharomyces pombe as a model organism. Our studies focus on elucidating the mechanisms that regulate the transitions between MT growth and shrinkage, namely MT rescue and catastrophe. Our aims are: 1) to characterize CLASP, a candidate MT rescue factor that stabilizes MTs within the mitotic spindle and interphase MT bundles; 2) to determine why MT catastrophe events occur at specific cortical sites at cell tips; 3) to investigate possible roles of actin in the nucleus, focusing on its effect on chromosome segregation during mitosis. PUBLIC HEALTH RELEVANCE: Microtubules are dynamic filaments responsible for cell division and determination of cell shape and function. In this proposal we will determine how the growth and shrinkage of microtubules are regulated. These studies will provide greater understanding of how mitotic spindles work and will be relevant for human diseases such as cancer.

Intellectual Merit: Cells typically divide at a specific, predictable location. Positioning of the cell division plane in cytokinesis is a fundamental and universal cellular process that has broad relevance to cell morphogenesis, development, tissue architecture, and stem cells. The aims of this research are to determine the molecular mechanisms governing this process. This project utilizes the sea urchin embryo as a potent animal cell model for symmetric cell division. Cell division site determination involves multiple processes. The cell initially positions and orients the nucleus in late interphase along a reproducible axis relative to its cell shape. In this process, the nucleus must somehow sense the shape of the cell. The axis of the interphase nucleus sets the position of the spindle in mitosis, which then specifies the site of cleavage in cytokinesis. The project will focus on the mechanisms of how the nucleus is positioned, how cell shape is sensed, and how the spindle signals to the cortex to specify the division plane. The researchers have recently developed a powerful new approach to study the effect of cell geometry in a systematic manner by manipulating the shape of sea urchin cells by introducing them into micro-fabricated chambers of different shapes (such as rectangles, triangles, etc. The project will include live cell experiments focusing on the identifying the location of and specific molecular motor responsible for nuclear positioning coupled with computational modeling of how the division plane is determined in cells manipulated into different shapes.

Broader Impacts: The researchers continue longstanding efforts to increase the diversity of scientists by broadening participation of underrepresented minorities in science. Efforts will be directed both in the laboratory and in national activities. One of the PI's on this collaborative project is a cell biologist who is also a Native American (Cherokee) who has a long record of involvement in working with a diverse pool of students and in national programmatic efforts. The researchers share a laboratory at the MBL, Woods Hole for about two months each summer where they host undergraduates (mostly from underrepresented groups) recruited nationally at the Society for Advancement of Chicanos and Native Americans in Science (SACNAS) annual conference, from Boston College, and from the NSF funded REU summer program at the MBL. These students are mentored to participate in a number of activities whose aim is to better equip them, should they choose to pursue a scientific career. At the national level, the PI is continuing in a leadership role in programs whose goal is to increase the diversity of scientists including leading NSF supported efforts for SACNAS, for the American Society for Cell Biology and serving in a regular capacity to effect policy change, evaluate programs, and contribute to national activities by service to the AAAS, the NSF, and to colleges, universities and scientific societies.

How cells control cell size remains a question of great interest in biology. Although individual components related to this process are known, their integration into a rational functional system has not been fully achieved. In this collaborative project, investigators from the US (Columbia University) and the UK (John Innes Institute) combine experiments and theory to address the precise control of fission yeast cell size. The preliminary theoretical model and experimentation has allowed the framing of a novel hypothesis on the role of a specific protein, termed Cdr2, in this process. Control of cell size and cell division is necessary in all living organisms, and understanding the basic dynamics of cell size control offers both new knowledge and insight into diseases where cell size control is impaired. The project provides interdisciplinary training opportunities at the interface between physics (including theory), quantitative experimental methods and cell biology.

To understand the machinery that regulates cell size and cell division requires the integration of theory and experiment, which is a goal of this project. The specific focus is on Cdr2, which is a peripheral membrane binding protein that specifically targets the node area in the middle of the cell. The population of this area "reports" the status of cell size to other proteins that prompt the cell to divide. This project will examine a novel hypothesis that predicts that when Cdr2 reaches a certain threshold concentration at the node region, unbound Cdr2 becomes available to inhibit the downstream regulatory factor, Wee1 kinase. In the absence of free Cdr2, Wee1 inhibits the cell cycle control factors Cdk1/Cyclin B to block entry into mitosis. Thus the inhibition of Wee1 by Cdr2 triggers the entry into mitosis.

This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.

? DESCRIPTION (provided by applicant): Regulation of dynamic microtubules (MTs) is critical for cellular processes such as cell division, cell migration, cell polarity, organelle movements and nuclear positioning. Understanding conserved mechanisms of MT regulation are highly relevant to human biology and diseases such as cancer, neuronal diseases and wound healing. In this grant, we aim to elucidate fundamental conserved mechanisms responsible for MT regulation by studying MT plus end tracking proteins (+TIPs). These proteins coat the MT plus end and control the assembly, stability and disassembly of these polymers. In general, how these proteins work collectively to regulate MT dynamics in vivo is a pressing question in this active field. We study MT regulation using in vivo and in vitro approaches, using the fission yeast Schizosaccharomyces pombe as a tractable, simple model system. The XMAP215/Alp14 family of +TIPs is emerging as one of the most important of the +TIPs. These tubulin-binding proteins function as MT polymerases that add tubulin onto the ends of growing MT. Our preliminary results suggest that it has additional functions in MT nucleation and disassembly. Our specific aims are: 1) To define how Alp14 works with gamma tubulin complex for MT nucleation; 2) To determine how Alp14 and the kinesin-8 Klp5/6 regulate the disassembly of MTs; 3) To elucidate the molecular mechanisms of how XMAP215/Alp14 proteins interact with tubulin to regulate MT dynamics.

Intellectual Merit: Cells typically divide at a specific, predictable location. Positioning of the cell division plane in cytokinesis is a fundamental and universal cellular process that has broad relevance to cell morphogenesis, development, tissue architecture, and stem cells. The aims of this research are to determine the molecular mechanisms governing this process. This project utilizes the sea urchin embryo as a potent animal cell model for symmetric cell division. Cell division site determination involves multiple processes. The cell initially positions and orients the nucleus in late interphase along a reproducible axis relative to its cell shape. In this process, the nucleus must somehow sense the shape of the cell. The axis of the interphase nucleus sets the position of the spindle in mitosis, which then specifies the site of cleavage in cytokinesis. The project will focus on the mechanisms of how the nucleus is positioned, how cell shape is sensed, and how the spindle signals to the cortex to specify the division plane. The researchers have recently developed a powerful new approach to study the effect of cell geometry in a systematic manner by manipulating the shape of sea urchin cells by introducing them into micro-fabricated chambers of different shapes (such as rectangles, triangles, etc. The project will include live cell experiments focusing on the identifying the location of and specific molecular motor responsible for nuclear positioning coupled with computational modeling of how the division plane is determined in cells manipulated into different shapes.

Broader Impacts: The researchers continue longstanding efforts to increase the diversity of scientists by broadening participation of underrepresented minorities in science. Efforts will be directed both in the laboratory and in national activities. One of the PI's on this collaborative project is a cell biologist who is also a Native American (Cherokee) who has a long record of involvement in working with a diverse pool of students and in national programmatic efforts. The researchers share a laboratory at the MBL, Woods Hole for about two months each summer where they host undergraduates (mostly from underrepresented groups) recruited nationally at the Society for Advancement of Chicanos and Native Americans in Science (SACNAS) annual conference, from Boston College, and from the NSF funded REU summer program at the MBL. These students are mentored to participate in a number of activities whose aim is to better equip them, should they choose to pursue a scientific career. At the national level, the PI is continuing in a leadership role in programs whose goal is to increase the diversity of scientists including leading NSF supported efforts for SACNAS, for the American Society for Cell Biology and serving in a regular capacity to effect policy change, evaluate programs, and contribute to national activities by service to the AAAS, the NSF, and to colleges, universities and scientific societies.

Project Summary/ Abstract The cytoplasm is a crowded subcellular environment that is packed with organelles, proteins, nucleic acids and other large macromolecules, as well as water and small molecules. How cell biological processes function in this milieu remains poorly understood. Macromolecules present in the cytoplasm are thought to exert physical forces that contribute to cytoplasmic organization, phase separation, and osmotic pressure. Cellular density, which is the concentration of cellular components such as proteins and nucleic acids, is a key predictor of these macromolecular crowding effects. Recent evidence from our lab and others reveals that density and macromolecular crowding effects are not constant but actually change during the cell cycle, as well in various physiological and disease states, and during development. However, little is known about how these changes impact cellular physiology and mechanics. Thus, cellular density and the effects of macromolecular crowding represent critical but understudied aspects of cellular physiology that likely impact most cellular processes. The general goals are to elucidate physical- and molecular- based mechanisms responsible for cellular processes responsible for cell growth and division: mitosis, microtubule dynamics, nuclear size control, chromosome mobility and cell wall assembly. A general thrust of the investigations is to determine how the biophysical properties of the cytoplasm and nucleoplasm impact these diverse cellular processes. In particular, our studies will address how intracellular osmotic pressures generated by macromolecules act to dampen microtubule dynamics, inflate the nucleus, modulate the mechanics of the mitotic spindle, and regulate chromosome motility for DNA repair. Approaches include innovative live cell assays for the biophysical properties of living cells (e.g. microrheology and quantitative phase imaging) and quantitative cell biology approaches in the fission yeast Schizosaccharomyces pombe. These studies will establish a foundation for the emerging field of cellular density and will contribute to our understanding of a fundamental but understudied aspect of cell biology. This work will significantly impact our understanding of mechanisms governing cell growth and division that are relevant for biomedical applications including cancer, aging and fungal pathogenesis.

This project elucidates how the size of the nucleus in the cell is determined. It has been appreciated for over a hundred years that nuclear size scales with cell size, so that larger cells have larger nuclei. As cells grow, the nucleus grows at the same rate. This size scaling relationship is one of the fundamental rules of life, but the mechanisms responsible for nuclear size regulation are still poorly understood. Deciphering such mechanisms will give insights into why it is important for cells to regulate nuclear size and will provide general principles of how size regulation of cells and organelles is accomplished. In general, this work will add to our knowledge of fundamental cellular processes that are used to build living cells. As a broader outcome, this project will introduce to the larger public the importance of physical forces and quantitative measurements in cell biology research through development of educational modules and a scientific exhibit. <br/><br/>In this project, a quantitative model for nuclear size scaling will be developed. This model proposes that nuclear size is specified by physical factors such as osmotic pressure and membrane tension. Specifically, nuclear size may be determined largely by a balance of colloid osmotic pressures within the nucleoplasm and cytoplasm, which are determined by the numbers of osmotically active macromolecules present in each compartment. The general approach is to use theoretical modeling coupled with quantitative experiments on the fission yeast Schizosaccharomyces pombe as a model organism. In S. pombe, the nucleus behaves as an “ideal osmometer” whose size corresponds quantitatively to its osmotic environment. This model will be tested in experiments in which physical parameters such as osmotic pressure are varied. The mechanism of nuclear size homeostasis in which abnormal nuclear sizes are gradually corrected will be elucidated. Mechanisms that couple nuclear volume growth and nuclear envelope surface area will be examined. This osmotic model of nuclear size promises to greatly impact the general understanding of organelle size regulation.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.