Bruce L. Goode - US grants

Many cellular processes require coordinate changes in the organization of actin filaments and microtubules. These include changes in cell shape, cell movement, establishment of cell polarity, spindle formation and nuclear migration. Yet the molecular mechanisms governing these changes and the biochemical nature of the actin-microtubule interrelationship are largely unknown. The specific aim of this proposal is to identify factors in yeast that associate with both the actin and microtubule networks, with the goal of understanding how actin and microtubule organization is coordinately regulated in vivo. The strategy will be to use actin and microtubule affinity chromatography to purity proteins and/or protein complexes that bind to both actin and microtubules, raise antibodies to purified factors, clone the genes encoding these factors, and study their in vivo functions by genetic approaches. Yeast is an ideal organism in which to study the actin-microtubule functional relationship because it provides the unique opportunity to integrate biochemical and genetic strategies to address protein function.

DESCRIPTION:Regulation of actin dynamics is a fundamental requirement in all eukaryotes. Endocytosis, cell motility and cell morphogenesis all rely on rapid remodeling of actin networks, which must be temporally and spatially regulated. The goal of our research is to define the molecular mechanisms governing actin assembly and organization. We are exploring this issue using the budding yeast Saccharomyces cerevisiae as a model organism to study conserved components of the cytoskeleton. Previously, we identified and characterized the yeast homologue of coronin, a ubiquitous actin associated protein with poorly defined cellular functions. We now demonstrate that coronin associates in vivo with the aboutctin- aboute1ated protein (Arp) 2/3 complex, a central nucleator of actin filament assembly and branching in eukaryotic cells. Further, purified coronin binds directly to the Arp2/3 complex and promotes actin filament debranching and turnover. This activity is consistent with our previously observed genetic interactions between coronin and cofilin and may define a conserved cellular function of coronin. The Arp2/3 complex has 7 different sub-units and is regulated by a large number of activators and other factors, including the Wiskott-Aldrich Syndrome (WAS) protein. We have identified a new activator of the Arp2/3 complex, Abplp. Our preliminary results suggest that WASp and Abplp activate Arp2/3 complex by distinct mechanisms, and we will directly test this hypothesis. Further, we have in vivo evidence that there are two distinct pathways in cells for regulating Arp2/3 complex, one that is WASp-dependent and one that is Abplp dependent. We will take a combined genetic and biochemical approach to dissect these in vivo pathways and to uncouple the activities and interactions of the Arp2/3 complex. This will allow us to determine how Arp2/3 complex activities are regulated in different cellular processes. The specific aims are: (1) Characterize the coroninArp2/3 complex activity and test its functional significance in vivo, (2) Dissect pathways for differential regulation of Arp2/3 complex functions in vivo and define their underlying biochemical basis, (3) Uncouple Arp2/3 complex activities and in vivo functions using a large collection of arc35 mutant alleles, and (4) Define the mechanisms 01 Arp2/3 complex activation by WASp and Abplp.

DESCRIPTION (provided by applicant): In all eukaryotes, actin is assembled into highly dynamic thin filaments, which are organized into networks that provide polarity and force to drive different cellular processes (e.g., cell migration, cytokinesis, muscle contraction, and endocytosis). The proper regulation of actin assembly and actin cytoskeletal function is impaired in many disease states, particularly those relevant to heart, lung, and blood research. Mutations in human WASp (Wiskott Aldrich Syndrome protein), a stimulator of actin related protein (Arp) 2/3 complex-medicated actin assembly, cause defects in neutrophil cell motility and loss of immune function. In asthma, calcium levels rise in response to histamine release and induce smooth muscle contraction in respiratory tracts of the lungs. Defects in actin itself are linked directly to dilated cardiomyopathy and heart failure, and mutations in key actin regulators (e.g., cofilin) lead to muscle denervation and dystrophy. Thus, understanding the biochemical basis of actin assembly is an important first step in defining how these pathological processes disrupt normal function in these tissues. Studies in the budding yeast Saccharomyces cerevisiae have been instrumental in dissecting the functions of key actin regulators, because combined genetic and biochemical approaches can be used. The objectives of this research career award (RCA) are to expand the specific aims of an existing R01 (Regulation of Actin Assembly in Budding Yeast) by introducing two new microscopy tools to study the structure and mechanism of action of Arp2/3 complex. Total internal reflection fluorescence (TIRF) microscopy will be used to study actin filament polymerization and branching by Arp2/3 complex and its regulation by WASp, Abp1, and coronin in real time. Electron microscopy and single particle image analysis will be used to study the structures of mutant Arp2/3 complexes with impaired activities and in vivo defects. The funding of this proposal would significantly enhance the ability of the PI to accomplish these goals by reducing his teaching and administrative duties. The interdisciplinary nature of this research program necessitates central involvement of the PI in training of students and postdocs in techniques and areas in which they are inexperienced. The PI will be directly involved in the integration of TIRF microscopy and single particle imaging into his research program.

[unreadable] DESCRIPTION (provided by applicant): Immune cells are complex and dynamic systems. A key example, T-cell activation requires the formation of an immunological synapse between the T-cell and its target. Formation and signaling across this synapse involves the spatially and temporally coordinated assembly of transmembrane receptors, lipid rafts, soluble proteins and the local reorganization of the cytoskeleton. Even subtle defects in individual molecules can disrupt this process with dire consequences for immune system function. Modern optical microscopy, in particular in its combination with genetically- encoded, fluorescence-based reporters, has been a key experimental tool to follow the molecular processes at the immunological synapse. However, until now we have been limited to observing this process. It is our goal to develop molecular tools that allow us to manipulate the immunological synapse with the same spatial and temporal resolution, with which we can now observe it. Specifically we propose to design and test genetically-encoded, light-switched versions of the Wiskott-Aldrich Syndrome Protein (WASP), a key regulator of actin skeleton reorganization at the immunological synapse. Our design is based on coupling the conformational equilibria of WASP and the small bacterial photoreceptor PYP (Photoactive Yellow Protein) so that light activation alleviates WASP's auto-inhibition and thus stimulates actin polymerization via activation of the Arp2/3 complex. The Wiskott-Alrdrich syndrome protein is a key molecular player in target recognition by immune cells, cell migration during wound healing and dendrite growth during neuronal development and remodeling. Consequently malfunction of this protein leads to a series of debilitating diseases including, but not limited to the eponymous Wiskott-Aldrich syndrome. We are proposing to develop new research tools to better understand the function of this protein and to help develop cures for the diseases caused by its malfunction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long-term goal is to understand how cell architecture and function are regulated by dynamic rearrangements of the actin cytoskeleton. Remodeling of actin networks in response to intracellular and extracellular signals is essential for fundamental biological processes, including cell division, cell migration, cell morphogenesis, intracellular transport, and endocytosis. The specific focus of our lab's research is to determine how multiple actin-associated proteins with diverse activities function in concert to elicit changes in actin dynamics and organization. We are addressing open questions about the mechanisms and functions of these proteins using budding yeast Saccharomyces cerevisiae as a model organism for our in vivo studies. We take a multi-disciplinary approach to solving these problems: biochemical, genetic, structural, and cell biological, combined with microscopic imaging of individual actin filament dynamics in real time in vitro and electron microscopy and single particle analysis to solve protein structures. In this proposal, we seek to understand how three highly conserved proteins, coronin, Arp2/3 complex, and cofilin, function together to control the formation and rapid disassembly of actin filaments. Our work during the last funding period identified two novel cellular functions for yeast coronin in regulating actin dynamics, one Arp2/3 complex-dependent and one cofilin-dependent. Our goal is to use this knowledge as a starting point to arrive at a more complete understanding of these new mechanisms and functions. The Specific Aims are: (1) How does coronin toggle between oligomerizing to bundle F-actin and associating with Arp2/3 complex to regulate actin assembly? (2) How does coronin affect Arp2/3 complex-dependent actin nucleation and branching? (3) How does coronin amplify the effects of cofilin in promoting actin filament disassembly? Defining the molecular basis of these cellular events is critical not only for understanding normal human physiology, but also for determining how defects in the specific genes that control these processes lead to disease states arising from mis-regulation of cytoskeletal architecture, including cancer, birth defects, heart disease, and neurodegenerative disorders.

DESCRIPTION (provided by applicant): Our long-term goal is to gain a highly mechanistic understanding of how the actin cytoskeleton directs cell polarity and cell morphogenesis. All living cells have internal and external structures tailored to and critical for their distinctive physiological functions. Further, cell architecture can be changed rapidly in response to various cues. The mechanisms underlying these events remain poorly understood and represent a major challenge for cell biologists to define. Recently, a conserved family of proteins called formins has emerged as crucial regulators of actin assembly and remodeling in cells, often functioning directly downstream of Rho GTPases. Formins are large multi-domain proteins that play essential roles in cell polarity, cell division, cell migration, endocytosis, and cell adhesion in a wide range of organisms. Formins directly nucleate actin assembly by a novel mechanism and remain processively attached to the growing end of the filament, protecting the end from capping proteins while guiding insertion of new actin subunits. While the last five years have seen rapid progress in elucidating formin protein structure, mechanism and function, comparatively little is known about how formin activities are regulated spatially and temporally in cells. In this proposal, we will address this question using the budding yeast Saccharomyces cerevisiae as a model organism. Whereas mammals have 15 different formin genes, S. cerevisiae has only two (Bni1 and Bnr1), and hence offers a simplified model to dissect formin regulation. Yeast also allows us to take a multidisciplinary approach, combining genetics, biochemistry, and live cell imaging. Bni1 and Bnr1 have distinct localization and dynamics, and assemble two distinct sets of actin cables. These cables serve as polarized tracks required for targeted secretion and polarized cell growth. We will determine how one of these formins (Bnr1) is regulated in vivo, which will provide key mechanistic insights into the molecular basis of cell polarity and cell morphogenesis. The Specific Aims of the proposal are: (1) How is Bnr1 anchored at the bud neck, activated/released from an autoinhibited state, and then retrieved from actin filament ends for new rounds of actin assembly? (2) How are the activities and cellular functions of a novel Bnr1-regulator (Bud14) controlled by its in vivo binding partners (Kel1 and Kel2)? Defining the molecular basis of these events is critical not only for understanding normal human cell biology and physiology, but also for determining how mutations in the genes encoding morphogenetic determinants give rise to disease states including cancer, birth defects, and neurodegenerative disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our goal is to understand how formin activities are regulated in vivo spatially and temporally. Given the potent effects formins have on actin assembly, they must be tightly controlled in the environment of a living cell. Our hypothesis is that through controlled local activation and attenuation of formins, their activities are harnessed to produce actin networks with highly specialized architectures and functions. What are these regulatory mechanisms? We are approaching this question using the model organism S. cerevisiae. The two S. cerevisiae formins Bni1 and Bnr1 assemble sets of highly dynamic actin cables, which in turn direct myosin-dependent polarized cell growth and morphogenesis. To elucidate the pathways controlling Bni1 and Bnr1 activities, we are using a set of affinity tags on known cell polarity factors and formin ligands to isolate the native protein complexes they form, and tandem mass spectrometry to identify the components of these complexes. In parallel, we are performing biochemical and genetic experiments to determine precisely how these proteins affect formin activities and contribute to cell polarity.

This Major Research Instrumentation (MRI) award supports the acquisition by Brandeis University of an integrated live fluorescence imaging system that greatly increases the speed and resolution at which dynamic events can be imaged in cells and in solution. The system will be equipped with both spinning disk confocal and epifluorescence modes, and will be tailored for photoactivation microscopy, an elegant approach to label small populations of fluorescent molecules acutely in space and/or time. The instrument will use a newly developed setup in which the spinning disk confocal modality is equipped with two cameras, enabling maximum speed for simultaneous detection of multiple fluorophores. The epifluorescence modality will push the limits of fast fluorescence imaging by taking advantage of the most recent developments in LED-based illumination and rapid sCMOS camera acquisition. These new technologies will permit the study of extremely rapid biological events (with durations of a few seconds and velocities of micrometers per second) that until now have been extremely difficult to measure directly. Thus, the proposed instrument will allow a new level of spatial (by labeling a set of molecules starting at a particular location) and temporal (by labeling a sparse population of molecules at any given time) investigation into research problems ranging from cytoskeletal dynamics, membrane traffic and signal transduction to sensory processing and synapse formation.

Cellular processes essential for life are driven by complex molecular machines that move through the cell, interact with each other, and execute their functions in seconds. Recent technological breakthroughs in microscopy have greatly improved the speed and resolution at which we can directly see these dynamic molecular events. Researchers at Brandeis across the Biology, Biochemistry, and Physics departments and at the University of Massachusetts, Boston will use the new imaging system to tackle a broad range of scientific problems, ranging from how a neuron forms synapses to how simple biological molecules self-assemble into complex force-generating machines. This instrument will train undergraduates, graduate students and postdoctoral fellows, fostering interdisciplinary collaborations across science departments at Brandeis and in the Boston science community. As part of this training, we will take advantage of the instrument to establish a new project laboratory in live-cell imaging at Brandeis for our undergraduate and Masters students who may not be members of a research laboratory and therefore would not otherwise have access to an advanced instrument. This instrument will integrate education, training and research to provide new fundamental insights into the dynamics and interactions of molecules in cells and in the test tube.

DESCRIPTION (provided by applicant): The goal of this research is to determine how the assembly dynamics and architecture of the actin cytoskeleton are controlled by formins and the mechanisms regulating their activities in vivo. We are studying this question in budding yeast, where the formins Bni1 and Bnr1 assemble actin 'cables' that play an essential role in polarized cell growth. Our lab recently discovered four novel modes of formin regulation mediated by the polarity factors Bud6, Bud14, Smy1, and Hof1. Remarkably, each of these proteins binds to the formin-homology FH2 domain of Bnr1, but has distinct effects on Bnr1 activity in vitro and distinct Bnr1-dependent mutant actin cable phenotypes in vivo. Further, we have identified novel in vivo ligands of Bud6 and Bud14 that function with them in regulating Bnr1-mediated actin cable assembly. The proposed research will define the cellular functions and mechanisms of these proteins, and how their combined effects coordinate the proper assembly of actin cables with a characteristic length, architecture, and dynamics that is tailored to their function. This work will provide a deeper understanding of the molecular activities and interactions that underlie cell polarity and morphogenesis. The project uses a multi-disciplinary approach, combining genetics, live-cell imaging, biochemistry, and novel multi-wavelength single molecule TIRF in vitro microscopy. The Aims are to: (1) Elucidate the specific roles and mechanisms of Bud6 in regulating Bnr1- mediated actin cable assembly; (2) Test the hypothesis that Bud14 and Smy1 provide distinct modes of formin temporal regulation required for maintaining actin cable length, dynamics, and architecture; and (3) Determine how the functions of multiple FH2-binding regulators are coordinated in vitro and in vivo.

DESCRIPTION (provided by applicant): The goal of this research is to determine how the dynamics of the actin and microtubule cytoskeletons are coordinated by a group of three interacting mammalian proteins: APC, Dia1, and EB1. This work will define the functions and mechanisms of these proteins, and provide a deeper understanding of the activities and interactions that underlie such processes as cell migration and morphogenesis. This project uses bulk biochemical experiments combined with a novel multi-wavelength single molecule biophysics method tailored to elucidate the mechanisms of complex, multi-component regulatory systems in vitro. In addition, the mechanisms deduced from the experiments in vitro will be tested in vivo to verify that they are important for relevant biological functions of these proteins in living cells (e.g., directed cell migration). The Specific Aims are: (1) Test two key hypotheses about the mechanism by which Dia1 and APC synergize in promoting actin assembly, involving formation of proposed physical complexes among components; (2) Test the additional hypothesis that Dia1 and APC synergize to stimulate actin assembly by a rocket launcher mechanism; and (3) Define the mechanisms by which EB1 alone and EB1 plus microtubules regulate and/or organize APC/Dia-induced actin assembly.

DESCRIPTION (provided by applicant): The goal of this proposal is to close the gap in our understanding of how actin filament network remodeling, disassembly and turnover are regulated, and thereby clarify the mechanisms driving cell motility, cell morphogenesis, endocytosis, phagocytosis, and cytokinesis. A number of actin binding proteins besides ADF/cofilin have been genetically implicated in promoting actin turnover, but their specific functional roles, interactions, and mechanisms have remained elusive. This has left the molecular basis for dynamic remodeling and depolymerization of cellular actin arrays obscure. The research proposed here will first address how densely branched actin networks assembled by Arp2/3 complex such as those found at the leading edge of motile cells, sites of endocytosis, and trailing motile vesicles, organelles and pathogens are rapidly debranched and disassembled. These experiments will focus on the function and mechanism of a novel ADF/cofilin structural homologue we recently characterized, GMF, which binds to Arp2/3 complex and stimulates debranching (Gandhi et al., 2010), and how GMF activity is enhanced in the presence of coronin. This will include examining how GMF and coronin affect the conformation of Arp2/3 complex, and testing the hypothesis that ATP hydrolysis on Arp2 and Arp3 facilitates debranching by GMF and coronin. Further, we will explore the possibility that there is an analogy between the nucleotide state of Arp2/3 regulating coronin and GMF interactions in debranching and the nucleotide state of actin regulating ADF/cofilin and coronin interactions to promote severing. Second, we will address the basic mechanisms for stimulating actin filament severing and disassembly. These experiments will focus initially on our newly proposed roles for coronin in severing ADP-actin filaments and in spatially and temporally controlling ADF/cofilin severing effects (Gandhi et al., 2009). We will also address the contributions of two other conserved actin disassembly factors (Aip1 and twinfilin) in actin disassembly. Finally, we will use in vivo studies to determine how these five conserved disassembly factors (GMF, coronin, ADF/cofilin, Aip1, and twinfilin) act in concert to promote actin cytoskeleton remodeling and turnover in living cells. To achieve these goals, we will combine genetics, reconstituted in vitro motility assays, and novel multi- wavelength single molecule TIRF microscopy tailored to elucidate the mechanisms of multi-component regulatory systems. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo in motile cells to verify their biological importance and to assess their contributions to cell motility, in vivo actin turnover dynamics, and actin network ultrastructure (including branching) determined by cryo-electron tomography and correlative light and electron microscopy (CLEM). The Aims of the proposal are to: 1) Determine how the branched actin filament networks assembled by Arp2/3 complex are rapidly debranched by GMF and coronin. 2) Define the roles of coronin, Aip1 and twinfilin in actin filament disassembly. 3) Test the importance of debranching and turnover mechanisms for cell motility and in vivo actin network organization and dynamics.

The goal of this research is to determine how the disassembly, turnover, and resculpting of cellular actin filament arrays is regulated by the concerted actions of GMF, Coronin, Srv2/CAP, Twinfilin, Abp1, Cofilin, and AIP1. Each of these core components of the eukaryotic actin machinery is conserved from yeast to humans and has been genetically implicated in promoting actin turnover and remodeling. However, the specific roles and mechanisms of each protein and how they function in groups are only beginning to be understood. The proposed research will elucidate how these factors interact and work together in novel multi-component mechanisms to control actin filament debranching, coalescence, severing, capping, and depolymerization. This will define the molecular basis of diverse biological processes that rely on characteristic actin network geometries and turnover rates, including cell motility, endocytosis, and intracellular transport of vesicles, organelles, and pathogens. To achieve these goals, we will combine genetics, biochemical assays, in vitro multi-wavelength single molecule TIRF microscopy, and live cell imaging approaches. Mechanisms elucidated at the single molecule level in vitro will be tested in vivo using separation-of-function alleles for their importance in diverse biological processes. These include: yeast endocytosis and endosomal fusion with vacuoles, yeast actin cable turnover mediating intracellular transport, actin-based motility of Listeria in infected mammalian cells, and turnover of filopodia, stress fibers, and leading edge networks in mammalian cells. The Aims are: (1) Test the hypothesis that actin filament networks are collaboratively debranched by mechanisms involving GMF, Coronin, Cofilin, and Srv2/CAP. (2) Test the hypothesis that Srv2/CAP and Abp1 interact to form a megadalton-sized complex that remodels actin networks by a novel mechanism of filament sliding and coalescence into bundles. (3) Test the hypothesis that novel mechanisms of actin filament end depolymerization mediated by Twinfilin and Srv2/CAP control the disassembly and length of cellular actin structures. (4) Test the hypothesis that differences in actin filament decoration and spatial organization govern how actin networks are remodeled and turned over.

? DESCRIPTION (provided by applicant): The goal of this research is to determine how both the dynamics and spatial organization of the actin and microtubule cytoskeletons are coordinated by a group of five interacting mammalian proteins: APC, Dia1, EB1, CLIP-170, and capping protein. This work will define the functions and mechanisms of these proteins in microtubule-actin cross-regulation, and will thereby provide a deeper understanding of the molecular activities and interactions that underlie such processes as cell migration, cell adhesion, and cell and tissue morphogenesis. This project uses bulk biochemical experiments combined with novel multi-wavelength single molecule fluorescence methods that we have tailored to directly observe the mechanisms of complex multi- component regulatory systems in vitro. Further, the mechanisms deduced from the experiments in vitro will be tested in vivo to confirm that they are important for specific cellular functions of these proteins. The Specific Aims are: (1) Test the hypothesis that the microtubule plus end-binding protein EB1 directly regulates nucleation of actin filaments by APC-Dia1; (2) Test the hypothesis that the rate and duration of actin filament elongation is controlled by dynamic binding interactions of Dia1, capping protein, microtubules, and/or EB1 at barbed ends; and (3) Define the roles of APC, Dia1, EB1, and CLIP-170 in controlling microtubule plus end dynamics and in triggering ultrafast actin polymerization from microtubule plus ends.

? DESCRIPTION (provided by applicant): The goal of this research is to determine how the assembly dynamics and architecture of the actin cytoskeleton are controlled by formins and the cellular mechanisms regulating their activities. We are studying this question in budding yeast, where formins assemble actin cables of a characteristic length, architecture, and dynamics required for polarized cell growth. Further, we extend this work to mammalian formin regulation. The proposal focuses on several new multi-component mechanisms discovered during the previous funding cycle, which control formin-mediated actin nucleation, or the duration and speed of formin-mediated actin filament elongation events. The project combines genetics, live- cell imaging, biochemistry, and novel multi-wavelength single molecule TIRF microscopy. The Aims are: (1) Test the hypothesis that formin-mediated actin cable nucleation is spatially and temporally controlled by the combinatorial effects of Bud6, profilin, Tpm1, and Tpm2; (2) Test the hypothesis that actin cable length, velocity, and architecture are controlled by dynamic interplay at filament barbed ends involving formins, Bud14-Kel1-Kel2 complex, capping protein, and Smy1; and (3) Test the hypothesis that human CLIP-170 interacts with mDia1 to form a novel barbed end- tracking complex that supports ultrafast actin filament elongation in vitro and in vivo.

Project Summary The overall goal of the NIGMS-funded research in my lab is to define the molecular and cellular mechanisms underlying dynamic rearrangements of the actin cytoskeleton, and to explore how these mechanisms are harnessed in vivo (in yeast and animal cells) to control diverse actin-based processes such as cell motility, endocytosis, intracellular transport, and cell morphogenesis. Genetic and biochemical research has been rapidly producing a ?molecular parts list? for the actin cytoskeleton, and many of the components have been characterized individually for their biochemical effects on actin filaments and their genetic effects on cellular actin organization and function. However, it is becoming clear that most of these proteins do not function alone, but rather in groups to perform their biological roles, and thus, new approaches are needed to define how they work in concert to perform their cellular functions. Our lab is tackling this problem using advanced single molecule TIRF microscopy to directly observe multi-component actin regulatory mechanisms in real time, and testing these mechanisms using genetic, cell biological, biochemical, and structural approaches. Through this approach, we have made fundamental new insights into actin regulation. For instance, we defined the first collaborative actin nucleation mechanisms of formins (with Bud6 & APC). We discovered that formins and Capping Protein can bind simultaneously at filament ends to accelerate each other?s dissociation. We showed that Cofilin, AIP1, and Coronin work together via an ordered mechanism to sever and disassemble F-actin. We discovered that Srv2/CAP works in conjunction with Cofilin and Twinfilin to depolymerize filament ends. In parallel, we have combined genetics, cellular imaging, and separation-of-function mutants to dissect the contributions of these mechanisms to actin-based processes in yeast and mammalian cells. Moving forward, we will ask the following questions: what are the complete regulatory cycles of the two yeast formins (Bni1 and Bnr1)? How is Arp2/3 complex-mediated actin nucleation balanced by its inhibitors (Coronin and GMF) and activators (Las17/WASP and Abp1)? How is actin nucleation at the leading edge of motile cells controlled by interactions among IQGAP1, APC and formins? How do interactions at filament ends between Capping Protein and formins (and their in vivo binding partners) control actin network growth? How do the filament severing and depolymerization mechanisms (Cofilin, AIP1, Coronin, Twinfilin, and Srv2/CAP) drive net disassembly of actin under the assembly-promoting conditions of the cytosol? Are there actin-associated proteins that accelerate the nucleotide state transition on F-actin to promote disassembly? In addition, we will introduce new technologies and directions to our research, including in vitro reconstitution of cellular actin structures, cryo-EM to study protein structure, cell-free extracts to genetically-biochemically dissect actin mechanisms, and a systems-level approach to determine how genetic disruptions in individual actin regulators affect the cellular levels, localization, and functions of the remaining actin-associated proteins.

Project Summary/Abstract The goals of this training program entitled ?Predoctoral Training in Cross-disciplinary Molecular and Cellular Biology? (CMCB) at Brandeis University are to produce rigorous, quantitative scientists with expertise in multiple disciplines, to provide trainees with the skills needed to succeed in diverse science-related careers, and to help trainees explore and pursue their career interests in an informed manner. This new program combines the complementary strengths of two prior Brandeis T32 programs: ?Genetic and Biochemical Mechanisms of Regulation, T32GM007122?, expiring after >40 years of NIGMS support, and ?Quantitative Biology, T32EB009419?, expiring after 10 years of NIBIB support. While both training programs had strong track records of student research productivity and career outcomes, we significantly rethought and revised core elements of training (based on student and faculty feedback) to better prepare students for a future in which interdisciplinary research is increasingly crucial. Innovations include: (1) increased quantitative training, through courses and an annual workshop, (2) revising the timing and goal of the qualifying exam, both to improve the training value and to better serve students from diverse scientific and personal backgrounds, (3) earlier implementation of Individual Development Plans and Thesis Committee Meetings to accelerate trainee career development and research progress, (4) introducing a secondary research advisor in a complementary discipline to facilitate interdisciplinary training, (5) a two week professional Externship in Year 4+, (6) new program self-assessment mechanisms, including semi-annual trainee feedback and the creation of an External Advisory Committee, and (7) formal training in and oversight of mentoring for all training faculty. Trainee appointments will be made at the end of Year 1, after students have completed one year of coursework, four 9- week laboratory rotations, and chosen a lab. In Year 2, trainees serve as teaching assistants for one course per semester, take a Proseminar course to help them craft their thesis research plan and defend it at their qualifying exam (end of Year 2), and to develop a career Individual Development Plan (IDP). In Year 3+, trainees take a final elective and focus on their research. They present their work at annual Departmental talks, have annual Thesis Committee Meetings focused on career planning and research progress. In Year 4+, they engage in a two-week career Externship and serve as mentors at the annual Quantitative Analysis workshop. Program outcomes and success will be measured by: (1) sustained research impact (reflected in trainee publications), (2) development of independent scientific thinking and communication (assessed through thesis proposals, annual Departmental talks and Thesis Committee Meetings), (3) active trainee engagement in their own career development (reflected in annual IDPs, discussions at Thesis Committee Meetings, and the new Externship program), and (4) trainee placement and long-term success in science-related careers. There are 29 CMCB training faculty, and 12 slots are requested (~6 trainees/year, with trainees supported in Years 2 and 3).