David G. Drubin - US grants

[unreadable] DESCRIPTION (provided by applicant): Cell motility, certain membrane trafficking events, and cell division all rely on regulated actin assembly. The proposed studies will reveal underlying principles governing cortical actin cytoskeleton assembly dynamics. A large collection of site-directed mutants in actin, Arp2, Arp3, cofilin, profilin and Cdc42, and null alleles of genes encoding nonessential actin-binding proteins such as Aip1, coronin and twinfilin, will be analyzed using two powerful in vivo fluorescence assays of actin assembly dynamics. For one assay, effects of the mutants on an actin-dependent endocytosis pathway consisting of a series of discrete steps will be determined. For the second assay, in vivo "treadmilling" rates will be measured using Fluorescence Recovery After Photobleaching (FRAP). Hypotheses generated from these studies will be tested using biochemical treadmilling and Arp2/3-mediated actin assembly assays. The Arp2/3 complex, with particular attention on the nucleotide and novel mechanisms of activation and regulation, will be studied in detail. A central unsolved question concerns roles for the ATPs associated with Arp2 and Arp3. Based on studies of actin, nucleotide states could influence such vital aspects of Arp2/3 complex function as its activation, branching and de-branching activities, and interactions with regulatory factors. Because Arp2 and Arp3 are closely related to actin, and because actin nucleotide-binding pocket mutants have provided valuable insights into regulation of cytoskeletal dynamics, a similar set of mutants was constructed in Arp2 and Arp3. Effects of these mutants will be tested in vivo and in a battery of in vitro tests for nucleation activity, interactions with different activators, and branching and de-branching reactions. In addition, the functional properties of four Arp2/3 activators, with a particular emphasis on the type I myosins, will be investigated. Numerous proteins that interact with each of these four activators have been identified, and the effects of these proteins, post-translational modifications, and lipids on their activating activities will be tested. The proteins under investigation are central to pathogen invasion and to metastasis of cancer cells. Aberrations in actin organization are hallmarks of cancer. Rational approaches to treatment and prevention of these disease states therefore depend on understanding principles of actin assembly regulation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Verbatim from the applicant's abstract): S. cerevisiae is an excellent organism for studies of cell polarity development due to its powerful genetics, its simplicity, the ability to observe polarity development synchronously in a cell population, and existence of numerous mutations and molecular markers for cell polarity. Studies will determine how Cdc42 GTPase controls actin organization, and how actin mediates polarized endocytosis. Thirty-seven site-directed cdc42 mutants will be characterized biochemically in permeabilized yeast and in actin-assembly competent yeast extracts to relate biochemical activities to in vivo functions, and used in genetic screens to identify components of downstream pathways. Roles for Cdc42 effector Pak kinases in morphogenesis will be elucidated by genetic and chemical-genetic strategies. Special cdc42 alleles defective in pseudohyphal growth will be studied to provide a deeper understanding of the molecular basis for fungal dimorphism, a characteristic of many fungal pathogens. Finally, how a complex set of interacting proteins links endocytic membranes to the Arp2/3 complex, and how these proteins are regulated by novel Ark kinases discovered in this laboratory, will be investigated.

DESCRIPTION: The future Chair of a Gordon Research Conference requests support for speakers and other attendees at a meeting on the actin and tubulin cytoskeletons in fungi, yeast, and plants. This series of meetings has been successful in the past; the next meeting will mark its tenth anniversary. Topics to be discussed include motility mechanisms, microtubules and mitotic regulation, strategies for cell division and determination of division planes, tip growth/polarity and morphogenesis, real time analysis of cytoskeletal dynamics, cell-cell interactions, and mechanisms of actin cytoskeleton regulation.

The actin cytoskelon has been implicated in endocytosis, yet the specific roles for actin are obscure. Moreover, few molecules that link these systems have been identified. The studies proposed here focus on one such linker protein, Hip1R, and on the associated proteins Abp1 and GAK. Each protein is a mouse homologue of a yeast protein discovered and studied in great detail in the Drubin laboratory. The proposed studies provide a unique opportunity to apply to mammalian cells principles learned from studies in yeast so that the specific roles for actin in endocytosis may be elucidated. Hip1R associates intimately with clathrin-coated vesicles and binds directly to clathrin, actin filaments, and PIP2. It is related to yeast Sla2p. Dominant negative approaches and antibody microinjections will be used to test using real time analysis and electron microscopy the in vivo importance of Hiup1R and its domains for endocytosis and for the ultrastructure of the endocytic pathway. A recently developed procedure will be used to reconstitute on lipid bilayers Hip1R with clathrin, actin, and adaptor proteins, testing for roles in clathrin assembly, pit formation, and vesicle fission. Yeast Abp1p functions in endocytosis and activates the Arp2/3 complex. Mouse Abp1 binds to dynamin through its SH3 domain and to actin filaments through its N-terminus, and co-localizes with the Arp2/3 complex in PDGF- treated cells. Biochemical, cell biological, and molecular-genetic approaches will be used to study the functions and interactions of mouse Abp1. Actin Regulating Kinases (ARK) are key regulators of actin organization and endocytosis in budding yeast, and they interact physically with both Sla2p and Abp1p. GAK is a mammalian ARK-like protein kinase that, like Hip1R, associates with clathrin coated vesicles. GAK function in vivo will be elucidated using dominant negative constructs. Moreover, conventional and chemical genetic approaches will identify GAK targets. Effects of GAK phosphorylation on its targets and how GAK is regulated will be determined. In sum, studies proposed here will benefit from a strong foundation of genetic research on the actin cytoskeleton and endocytosis in budding yeast, and will provide novel insights into the roles for actin in endocytosis and the underlying mechanisms and regulatory strategies in mammalian cells.

DESCRIPTION (provided by applicant): Support is requested to continue a multidisciplinary program of predoctoral training in cellular, biochemical, and molecular sciences under the auspices of the Department of Molecular and Cell Biology (MCB) at the University of California at Berkeley. MCB is a single Department comprised of over 90 active faculty in five Divisions: Biochemistry and Molecular Biology, Cell and Developmental Biology, Genetics and Development, Immunology, and Neurobiology. Based on this organization, MCB has developed a broad, integrated and interdisciplinary approach to training Ph.D. candidates. The 69 participating faculty listed in this application pursue diverse research problems at the molecular and cellular level, providing research opportunities and appropriate instructional environments for the training of graduate students in: (a) Biochemical sciences (metabolic regulation, enzyme purification, enzymatic catalysis, structure and function of biological macromolecules, especially proteins and nucleic acids); (b) Molecular biology (chromosome structure, regulation of gene expression, DNA replication, recombination, repair, transposition); (c) Cell biology (cellular organization and function, cytoskeletal architecture, cell movement, cell surface receptors, ion channels, signal transduction mechanisms, cell growth control); and (d) Molecular and cellular aspects of development (embryogenesis, pattern formation, cell-cell interactions, neuronal differentiation, apoptosis, and development of the immune system). While enrolled in formal coursework to strengthen and broaden their background, entering students conduct ten-week laboratory research projects in three different faculty laboratories. Based on these experiences, the new trainees select doctoral dissertation mentors. In the second year, students concentrate on research, continue to take lecture and/or seminar classes, acquire teaching experience by serving as Teaching Assistants, and undergo an Oral Qualifying Examination administered by a cross-divisional committee. In years three-to-five, students focus almost exclusively on their thesis research; however, enrollment in at least three advanced seminar courses is required. Weekly divisional colloquia, named lectureships and departmental symposia ensure that eminent scientists are brought to the Berkeley campus to expose trainees to the latest breakthroughs in their fields of interest. Applicants typically have outstanding undergraduate records in the biological, chemical or physical sciences. Admission is based on previous scholastic achievement, prior research performance, GRE scores, a statement of purpose, letters of evaluation, and formal personal interviews.

endocytosis; protein structure function; biomedical resource;

actins; cell cycle; biomedical resource; biological signal transduction;

phosphoproteins; protein structure function; posttranslational modifications; phosphorylation; intermolecular interaction; biomedical resource;

DESCRIPTION (provided by applicant): State-of-the-art imaging of whole cells and of isolated cytoskeletal elements and organelles is vital to achieving the Specific Aims of the NIH grants awarded to each of the six U.C. Berkeley cell biologists participating in this Shared Instrumentation Grant proposal. Acquisition and analysis of fluorescence images using the latest technologies has become essential to the continued success of each of these research programs. The overall aim of this proposal is to secure funds to acquire a state-of-the-art Olympus imaging system with capabilities that are currently unavailable to the applicants, or that are in such high demand that progress is being significantly hindered by competition for time on the one existing shared system. The capabilities to be provided by this imaging system include Total Internal Reflection (TIRF) microscopy, high-speed multi-color image acquisition for real time analysis of dynamic cellular processes, and deconvolution microscopy. The specific system for which funding is being requested, and how the capabilities of this system relate to the Aims of the NIH-funded research program of each investigator, are described in detail in the body of this proposal.

DESCRIPTION (provided by applicant): Discovery of a conserved pathway for endocytic internalization in a simple organism amenable to powerful genetic, genomic, biochemical and cell biological analysis created unprecedented opportunities to develop a comprehensive understanding of this complex process, and to reveal underlying molecular mechanisms. An integrated approach, combining image analysis, functional genomics, proteomics, biochemistry and theoretical modeling, will elucidate the molecular mechanisms that underlie the endocytic pathway. These studies will increase understanding of actin's role in endocytosis, and how the order and timing of events in the endocytic pathway are achieved. Understanding how dynamic actin assembly is coupled to plasma membrane- associated processes is critical for understanding human health threats including metastasis, loss of proliferation regulation, and pathogen entry into cells. Focused tests of specific hypotheses, and global investigations to reveal holistic operating principles for the pathway, will be balanced. The dynamics of at least 8 newly identified endocytic proteins will be mapped onto the previously described endocytic pathway, and quantitative imaging of endocytosis in mutants of these proteins will identify their biological functions. A screen for mutants with altered sensitivity to the endocytosed yeast killer toxin, K28, coupled with real-time analysis of K28 endocytosis, will identify new endocytic proteins and intracellular sorting mechanisms. Imaging-based analysis of endocytic protein dynamics in mutants of endocytic protein ubiquitination will reveal mechanisms that coordinate events in the pathway. Studies on the functions of the myosin/WASP and scission endocytic protein modules, rather than on individual proteins, will make approachable the otherwise daunting task of deciphering the functions of over 60 endocytic proteins. A combined biochemical, genetic and image-based approach will identify the mechanisms and protein-protein and protein-lipid interactions that recruit these protein modules to the endocytic sites, and that regulate their activities. The ultrastructure of the endocytic machinery at different stages of the endocytic pathway will be determined using cryo electron microscopy on wild-type and mutant cells. The membrane geometry and the ultrastructure of the protein coat and its associated actin filaments, will be determined. Immuno-EM will reveal how different proteins are organized at the endocytic site. Theoretical modeling combined with experimental measurements of protein function, dynamics and ultrastructural organization, will develop the first coherent and quantitative model of endocytic dynamics that can recapitulate all of the key events. Quantitative design principles from the resulting model will likely apply to other cytoskeletal and membrane trafficking processes. Hypotheses generated by theoretical modeling will be tested experimentally. ) PUBLIC HEALTH RELEVANCE: Understanding how dynamic actin assembly is coupled to plasma membrane-associated processes is critical for understanding human health threats including metastasis, loss of proliferation regulation, and pathogen entry into cells. Studies on toxin entry into yeast cells and toxin-mediated cell killing may suggest novel therapeutic strategies for treatment of bacterial infections.)

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. The budding yeast kinetochore connects spindle microtubules to centromeric DNA and is critical in mitotic spindle function. A number of kinetochore proteins are phosphorylated by mitotic kinases but what impact these modifications have on their regulation has yet to be understood. Here we use mass spectrometry to map sites of phosphorylation, as well as in vivo studies, in order to understand the functional consequences of mutating these sites. This will allow us to gain more insight into the impact of phosphorylation on kinetochore protein interactions and the regulation of the mitotic spindle and budding yeast kinetochore.

[unreadable] DESCRIPTION (provided by applicant): The conference on "Dynamic Interplay Between Cytoskeletal and Membrane Systems," to be held June 27-30, 2007 in Dijon, France, is a one-time joint American Society for Cell Biology-European Cytoskeleton Forum (ASCB-ECF) conference. The molecular events occurring in the special microenvironments at membrane-cytoskeleton interfaces underlie diverse biological processes; including cell morphogenesis and cell polarity development, intracellular trafficking, cell adhesion, cell signaling, cell motility, tumor cell invasion, and host-pathogen interactions. Understanding how these diverse biological processes are mediated by membrane-cytoskeleton interactions will require the combined expertise of investigators studying cellular membranes and investigators studying cytoskeletal proteins, as well as the use of a variety of experimental approaches. In the past, opportunities for intense and focused discussions of membrane-cytoskeleton interactions have been rare or entirely lacking. The goals of this conference are to promote among two distinct communities, cytoskeletal researchers and membrane researchers, the lively exchange of ideas and reporting of conceptual advances, and to identify the most pressing challenges and the most promising opportunities for future research. The latest results from cutting-edge research on membrane cytoskeleton interactions will be presented, and ample opportunity for formal and informal communication among all conference attendees will be provided. An especially important feature of this conference is that it will bring together leading researchers from the United States, Europe and Asia thus promoting international scientific discourse. This meeting will bring together scientists from two traditionally non-overlapping research areas, and employing diverse state-of-the-art approaches including real-time imaging, electron microscopy, biochemistry, biophysics and genetics. The unique focus of this conference will facilitate interactions and exchanges that would be lacking in less focused conferences, or diluted at larger conferences. [unreadable] [unreadable] [unreadable]

Accounting; Anthrax; Anthrax disease; Binding; Binding (Molecular Function); Biological Models; CRISP; Cell Communication; Cell Interaction; Cell-to-Cell Interaction; Cells; Cholera; Computer Retrieval of Information on Scientific Projects Database; Diphtheria; Disease; Disorder; Farm Animal; Funding; Grant; Health; Human; Human, General; Institution; Investigators; Livestock; Livestocks; Man (Taxonomy); Man, Modern; Maps; Model System; Models, Biologic; Molecular Interaction; NIH; National Institutes of Health; National Institutes of Health (U.S.); Pathology; Protein Interaction Map; Protein Interaction Mapping; Protein-Protein Interaction Map; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Route; S cerevisiae; Saccharomyces cerevisiae; Source; Toxin; United States National Institutes of Health; Viral; Work; Yeast, Baker's; Yeast, Brewer's; Yeasts; disease/disorder; gene product; killer factor; killer substance; killer toxin; microbial

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. As important elements of human cytokinesis are highly conserved in S. cerevisiae, I am exploiting yeast's experimental advantages to study cytokinesis. To isolate cytokinetic ring contraction from related processes of the cell and give greater control of experimental parameters, I am developing a cell free functional assay for cytokinetic ring contraction. Excitingly, I have found it possible to isolate intact cytokinetic apparatus from yeast. To move to a definitive list of machinery sufficient for cytokinetic ring contraction, I am seeking to identify novel factors that are associated with partially purified, intact cerevisiae cytokinesis apparatus.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Clathrin-mediated endocytosis is a fundamental process in living cells, allowing them to capture macromolecules efficiently from the extracellular environment and package them into vesicles in the cytosol. Numerous diseases can be traced to a defect in the succession of molecular reactions leading to endocytosis. In the budding yeast Saccharomyces cerevisiae, endocytosis is localized to cortical actin patches, where a dense network of actin filaments is polymerized around clathrin-coated pits. This network is regulated by a complex set of proteins which cooperate in order to generate membrane invagination. We have recently developed in our laboratory a system in yeast extracts for the assembly of actin based structures around polystyrene microbeads. We believe that the protein composition of these structures could be similar to the protein composition found in vivo in cortical actin patches. We isolated the beads with their associated actin structures, and we would like to determine the protein composition by using mass spectroscopy. These results should allow us to further analyze the role of the different components during endocytosis.

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. The Saccharomyces cerevisiae protein Stu1p has an essential role in mitotic spindle function. It is necessary for mitotic spindle formation and maintenance. Stu1p is part of a network of proteins localized at the mitotic spindle midzone. We are attempting to understand the protein-protein interactions within this spindle midzone network, to better understand the establishment and maintenance of the mitotic spindle. Since Stu1p is an essential member of this network, we are interested in understanding whether it interacts with other midzone proteins, and whether these interactions are necessary for Stu1p function.

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. Ypk1, is a well conserved protein kinase involved in endocytosis and cell growth. Interestingly, Ypk1 is highly phosphorylated in vivo. So far, three protein kinases have been identified as upstream regulators of Ypk1. However, the phosphorylated sites of this protein have not been examined in vivo. Furthermore, the downstream substrates of Ypk1 have not been identified. Recently I was able to purify Ypk1 using a TEV-myc-tagged system in yeast. Mass spec analysis of purified Ypk1p may identify Ypk1?s potential binding partners (substrates) and also phosphorylated sites of Ypk1.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recent live-cell imaging studies, coupled with a growing body of experimental genetic, biochemical and pharmacological data have expanded our knowledge of the molecular events that occur during endocytosis and the role played by actin (Toret and Drubin, 2006). In this collaboration we aim to develop a deeper understanding of endocytosis in yeast using correlated fluorescence and soft x-ray tomography to image the cellular ultrastructure of a number of different mutant endocytic phenotypes. The information obtained will not only highlight the effect of these mutations on the endocytic process and machinery, but establish the consequences of these changes on the organization of the entire cell. The cell cycle is the essential mechanism by which the cells of all living organisms from unicellular bacteria to the multicellular mammals duplicate. The most basic function of the cell cycle is to accurately duplicate the vast amount of DNA in the chromosomes and then segregate the copies precisely into genetically identical daughter cells. It has been shown that there is a general control that maintains a ratio of nuclear volume to cell volume. However, such correlation during the entire cell cycle has not been fully elucidated. In addition, it is very important to understand how other organelles are inherited during the cell cycle. Another goal of this collaboration is to use soft xray tomography to study the changes in organelle positioning and the volume and density of organelles during the phases of the cell cycle.

DESCRIPTION (provided by applicant): The discovery of a conserved pathway for endocytic internalization in budding yeast, a simple organism amenable to powerful genetic, genomic, biochemical and cell biological analysis, has created unprecedented opportunities to develop a comprehensive understanding of this complex process, and to reveal underlying molecular mechanisms. Endocytosis is a key process for specifying the molecular composition of the plasma membrane, and therefore how a cell will respond to its environment. Endocytosis also provides a portal for entry of both beneficial molecules and harmful agents into cells. Despite the importance of endocytosis for cell physiology and human health, many fundamental features of the process are obscure. How sites are initiated and what mechanisms trigger the subsequent events, which invaginate the membrane and pinch off a vesicle, are not known. In this proposal, an integrated approach, combining live-cell image analysis, genetics, mathematical modeling and biochemistry is outlined. These studies promise to elucidate molecular mechanisms that underlie the endocytic process. Two aims will be investigated. (1) How endocytic sites are initiated, matured and post-translationally regulated will be determined. The yeast Eps15-like protein, Ede1, which is among the earliest to arrive at endocytic sites, and which is among the most important proteins for endocytic site initiation and progression, will be a focal point. How Ede1 is recruited to nascent endocytic sites, and how it in turn recruits other proteins to these sites, will be investigated by a combined molecular-genetic, imaging and biochemistry approach. Roles for Ede1 phosphorylation by the novel endocytic kinase Hrr25 and for Ede1 ubiquitination, will be investigated. If, and how, these regulatory inputs are influenced by endocytic cargo will also be tested. Finally, how the rhomboid protease Rbd2 regulates the transition from the early to late stages of the endocytic pathway will be investigated. (2) The roles of specific plasma membrane lipids and protein-lipid interactions during each stage of the CME pathway will be elucidated. Endocytosis involves a series of membrane shape changes. A variety of endocytic proteins associate intimately with the lipid bilayer during each stage of the process, responding to and affecting lipid composition and membrane geometry. The specific roles and protein interactions of PS, PIP2 and sphingolipids will be investigated. Purified endocytic proteins and a recently developed cell extract system that reconstitutes the actin assembly burst near the end of the endocytic pathway will now be used to reconstitute endocytic steps on microbeads and lipid bilayers, establishing biochemical systems to reveal mechanism and to complement and synergize with powerful genetic and imaging approaches. A highly productive theory collaboration will continue to facilitate data synthesis and development of novel mechano-chemical concepts, and to generate experimentally testable hypotheses.

PROJECT SUMMARY Proposed are complementary studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME in budding yeast and human stem cells. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane and for selective removal of plasma membrane proteins. It is also one of the main routes for COVID-19 to enter cells. Therefore, this process is crucial for determining how cells respond to their surroundings and has heightened translational significance. Many proteins and lipids that mediate CME have been identified and their functions determined biochemically and in living cells. Imaging of fluorescently labeled CME proteins in live cells has revealed the intricate recruitment timing and order for some 60 CME proteins. However, how cargo capture is coordinated with vesicle formation, how correct protein recruitment order and timing are achieved, which events and molecules play critical roles in the pathway, and how forces curve the membrane and drive vesicle scission, are not fully understood. The following key questions will be addressed in budding yeast and human stem cells: 1) How does membrane curvature affect biochemical reaction rates? 2) How does CME become specialized for different cell types during differentiation? 3) How does a checkpoint sense cargo and regulate CME progress? and, 4) How does actin assemble at CME sites and how does its ultrastructure contribute to CME force production and adapt to increased membrane tension? Yeast studies will be empowered by a rich legacy in the lab of elucidating actin assembly and force production mechanisms. Human cell studies will be empowered by over 120 stable human tissue culture and stem cell lines generated using genome editing to express CME and actin cytoskeleton proteins as fluorescent protein fusions at native, endogenous levels. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and humans will complement and inform each other. Together, these studies will provide a comprehensive mechanistic understanding that could not be achieved by studies in only one cell type. Because the actin cytoskeleton has been adapted by evolution for diverse, essential activities including cell motility, organelle transport, adhesion, and cell polarity development, what is learned will apply broadly for many cellular processes and will join the growing armamentarium of possible defensive measures against the pandemic.

ABSTRACT ? NO CHANGES FROM ORIGINAL Studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME, and their critical importance to cell function in both budding yeast and mammalian cells, are proposed. Actin functions in countless processes including cell motility, organelle transport, adhesion, contractility, cell shape, cell polarity, and maintenance of membrane tension and cell mechanical rigidity. Significant gaps exist in knowledge of actin mechanisms and assembly regulation. Two key questions concerning actin regulation and function will be addressed in studies of budding yeast: (1) How does the cell cycle regulate actin cable assembly? (2) How do type 1 myosin and the Arp2/3 complex work together to create forces that generate membrane curvature? For the former studies, recent observation that fimbrin phosphorylation by Clb2/Cdk1 is crucial for cell cycle regulation of actin assembly will be leveraged to develop a mechanistic understanding of how actin assembly is regulated in the cell cycle. For the latter studies, in- depth biochemical, biophysical, genetic, and cell biological approaches will be combined to determine how type 1 myosins contribute to force production by Arp2/3-nucleated actin networks during CME. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane, and therefore, is crucial for determining how cells respond to their surroundings. Many proteins and lipids that mediate CME have been identified, and their functions determined biochemically and in cells. Live cell imaging of fluorescently labeled CME proteins has revealed the intricate recruitment timing and order for some 60 CME proteins. However, how cargo capture is coordinated with vesicle formation, how correct protein recruitment order and timing are achieved, which events and molecules play critical roles in the pathway, and how forces curve the membrane and drive vesicle scission, are not fully understood. The following key questions will be addressed in budding yeast and mammalian cells: How are CME site initiation and maturation regulated? What activities are essential for CME vesicle formation? Does a checkpoint monitor CME? What biophysical principles govern CME? What are actin's endocytic functions and how are they regulated? How do chemical and physical parameters affect CME dynamics and efficiency? How does CME change during cellular differentiation? Mammalian cell studies will be conducted on over 80 stable tissue culture and stem cell lines generated using genome editing to express CME proteins as fluorescent protein fusions at native, endogenous levels. Effects of cell differentiation on CME dynamics and efficiency will be conducted in the genome-edited stem cells. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and mammals will each complement and inform the other and provide a comprehensive mechanistic understanding that neither alone could generate.