David A. Agard - US grants

Proposed is a plan of research designed to determine the three-dimensional structure of polytene puffs, bands and inter-bands. The spatial arrangement of chromosomes within the the cell nucleus and its alteration as a function of cell cycle stage and development state will also be examined. A direct structural analysis is proposed using polytene interphase and diploid interphase, prophase, and metaphase chromosomes from Drosophila melanogaster. The structural analysis will rely extensively on recently developed computer methods for three-dimensional image reconstruction and enhancement. These methods are particularly powerful and require neither crystalline specimens nor internal symmetry. High-resolution three-dimensional data (100-500A) on polytene chromosomes will be collected by a HVEM tilt series on embedded, stained, thick sections. Low-resolution 3-D topology data will be collected by optical fluorescence microscopy using a computer-controlled Zeiss Axiomat microscope. Interpretation shall proceed by model building and comparison of structures from different biologically-defined chromosome states. Current research on problems of eukaryotic gene regulation has focused primarily on aspects related to DNA sequence organization and DNA modification. Nuclease sensitivity experiments have begun to hint at the importance of chromatin structural changes during gene activation. A more direct approach to understanding chromosome structure, its arrangement within the nucleus, and its degree of plasticity as a function of cell cycle stage, developmental state, and transcriptional activity is required. The proposed research is a first step in understanding these complex problems. As an outgrowth of this work development will continue on cellular tomographic techniques and computer modeling methods that should have a wide application to other areas of cell biology.

Proposed is a plan of research designed to determine the three-dimensional structure of Drosophila melanogaster chromosomes in a variety of biologically well defined functional states. The long range goal is to understand the structural complexities that underly DNA condensation and its organization into structures that can allow as well as modulate transcriptional activity. Of central interest is determining the structure of diploid mitotic chromosomes and polytene puffs, bands and inter-bands. These structures will form the starting point for examining the structural transitions accompanying chromosomal condensation during the mitotic cell cycle as well as those changes accompanying transcriptional activation. A direct structural analysis is proposed using early embryonic diploid interphase, prophase, and metaphase chromosomes and larval Malpigian tubule polytene interphase chromosomes. In collaboration with Dr. J. W. Sedat, we plan to study the 3-D arrangement of diploid chromosomes within the nuclei of intact cells in embryos, both as a function of cell cycle and developmental state of the tissue. Chromosome pairing and topological arrangement will be studied. This work will complement Sedat's studies on polytenized tissues. High resolution (100-200 Angstrom) three-dimensional data will be collected by HVEM tilt series from either isolated critical point dried chromosomes (mitotic chromosomes) or embedded and stained thick plastic sections (both diploid and polytene chromosomes). Low resolution (1500-2000 Angstrom) 3-D in situ data within intact nuclei will be collected using our recently developed 3-D optical section microscopy. The structural analyses will rely extensively on recently developed computer methods for three-dimensional image reconstruction and enhancement. These methods are particularly powerful and require neither crystalline specimens nor internal symmetry. As an outgrowth of this work, development will continue on both EM and optical cellular tomographic techniques and computer modeling methods that have a wide application to other areas of cell biology.

Proposed is a plan of research designed to determine the three-dimensional fine structure of mammalian and Drosophila melanogaster diploid mitotic chromosomes in a variety of biologically well-defined functional states. The long-range goal is to understand the structural complexities that underlie DNA condensation and its organization into higher-order structures that can support as well as modulate transcriptional activity, and that change throughout the cell cycle. This is a continuation of a well established research program that has made dramatic progress in the area of three-dimensional data collection methods for both the light and electron microscopes. The technological research has been driven by the need for tools suitable for examination of diploid chromosome structure and organization. Initial studies focus on understanding the structure of the 30nm fiber and how this fiber is packaged into the 130nm higher-order structure. Previous work had shown this 130nm fiber to be a dominant higher-order structural motif that persists throughout the cell cycle, but that is most clearly visualized in telophase. Intermediate Voltage Electron Microscope Tomography (IVEM-T) is being used to provide high-resolution (50-75 Angstroms) three-dimensional reconstructions from thick sections of embedded and stained chromosomes. The structural analyses rely extensively on recently developed computer methods for three-dimensional image reconstruction, enhancement, display and interpretation. These methods are particularly powerful and require neither crystalline specimens nor internal symmetry. Our current studies on HeLa telophase chromosomes will be extended to Drosophila embryonic and imaginal disk chromosomes and mouse lymphocyte chromosomes with experiments designed to target reconstructions to a uniquely defined region on a particular chromosome. Furthermore, these studies will be enlarged to consider a variety of cell cycle stages. Work will continue on the development of general methods designed to make IVEM-T considerably easier to accomplish. Our goal is to significantly broaden the accessibility of this powerful method for general use in cell biology.

ABSTRACT Acquisition of an X-ray Area Detector P.l. Robert J. Fletterick An area sensitive X-ray detector system is requested for X- ray crystallographic data collection for high resolution structure analyses of proteins. The new instrument is to augment the UCSF structural capability which is currently vastly oversubscribed. At present, persons typically wait in turn for one month to six weeks for time on one of the two existing area detectors. The new instrument requested is to overcome this rate limiting step in the structural science. The new phosphor storage technology used in the proposed detector system makes data collection more efficient and accurate. As structural analysis moves us to ask scientific questions using many mutations, or using modified inhibitors aimed at designing improved therapeutics, study of a single protein system can occupy as much as 50% of the time on one data collection instrument. Understanding the properties of mutant proteins or altered inhibitory compounds will require an analysis of structure as thoroughly as for the native proteins. The speed with which site-directed mutagenesis is accomplished requires rapid structural analysis for the scientist to remain close to the important scientific questions. In the following narrative, we list the projects of the three principal investigators and demonstrate the need for the new communal equipment. The laboratory to house the new machines will be renovated: a wall is to be added, plumbing changed, electrical installation and a safety enclosure will be built. The projects, most supported by NIH grants to the three P.I.s, are briefly described in narrative form, with references to published work where possible, and followed by a table. This lists the individual projects by title, the principal scientists working on the project and the status of the project. The status provides a brief summary of the current needs for data collection.

9317845 O'Shea This proposal seeks funding for circular dicroism (CD) and UV VIS spectrophotometers. These instruments will be used in a series of biochemical and biophysical studies concerned with how proteins fold and how they interact with one another and with other macromolecules. The proposed research includes studies in the following broad research areas: (1) protein structure, structure prediction and protein folding, (2) protein protein and protein nucleic acid interactions. (1) Studies on protein structure, prediction and folding will involve both model peptides and a bacterial protease, a lytic protease. Computational predictions of protein structure will be tested using synthetic polypeptides. The structure of designed peptides will be studied to investigate fundamental aspects of protein stability and structure. Structural studies of a stable intermediate in the folding of a lytic protease will be carried out to understand the forces which stabilize this intermediate. Conversion of the intermediate to the native enzyme is catalyzed by an additional polypeptide sequence. Thus, a further goal of these studies is to understand the mechanism by which folding to the native state is catalyzed. (2) Studies on protein protein interactions will involve a group of proteins that play important roles in transcriptional regulation. These include the glucocorticoid receptor from mammalian cells and the SYMBOL 97 \f "Symbol" 2, al, MCM1, PHO2, PHO4, and PHO80 proteins from yeast. A goal of these studies is to identify domains that comprise autonomous folding units, as well as domains that are responsible for inte ractions between proteins of multisubunit transcription complexes. An additional goal is to investigate changes in conformation of these molecules upon interaction with one another. These studies will provide an essential first step in obtaining three dimensional information on multi subunit protein complexes. The CD and UV VIS spectrophotometers are essential instruments for carrying out these studies. CD provides a simple and informative measure of secondary structure. It also provides a way to assess conformational changes in mixtures of macromolecules, such as two different proteins (or domains) or a protein and DNA. The UV VIS spectrophotometer provides a sensitive assay for measuring protein concentrations, a necessary component of a CD experiment. Additionally, UV VIS spectroscopy provides a method for measuring the stability of a molecule by monitoring absorbance. For proteins, the information derived from such a study is often complementary to that obtained from CD because changes in the UV spectrum are typically reflections of tertiary structure while CD is most sensitive to secondary structure. Finally, UV VIS spectroscopy provides a very sensitive way to monitor changes in nucleic acid stability and structure. These studies shall be carried out as part of ongoing, active research programs in which training of graduate students and postdoctoral fellows is a strong component. u ~ 9317845 O'Shea This proposal seeks funding for circular dicroism (CD) and UV VIS spectrophotometers. These instruments will be . / 0 x k m I K ' ) v x ! ! ! ! ! ! F x N x ; CG Times Symbol & Arial 1 Courier 0 MS LineDraw 9 N h = ABSTRACT Deseree King, BIR Deseree King, BIR

DESCRIPTION: This revised proposal requests continuing support for a research effort to determine the three dimensional fine structure of mitotic chromosomes focussing primarily on the use of an in vitro chromosome condensation system developed recently by the PI s collaborators at UCSF. This study, now in its 13th year, represents a continuation and expansion of a well established research program to use both and improve tools for high resolution structural determination using intermediate voltage transmission electron microscopy, with a specific focus on three dimensional reconstructions from thick sections of embedded and stained chromosomes. Three major goals are set forth. First, with the further use of state of the art image reconstruction methods and electron microscopy tomography, the PI will focus on use of a Xenopus in vitro DNA condensation system in which chromosomes can be moved from interphase to mitosis and studied at various stages of condensation. The key focus here will be to use immunoelectron microscopic localizations and/or immunodepletion, to identify specific roles played by individual proteins. Beyond this, higher order chromatin structure will be further investigated focusing on the 130-nm fiber already seen both in telophase and interphase cells. The hope is that with an expansion and refinement of current imaging methods, the path of the three dimensional chromatin fiber within these higher order structures may be mapped. A second goal is to use the same set of tools to identify the structural organization of centrosomes, the structures that nucleate microtubule assembly in animal cells. The initial efforts will focus on gamma tubulin, as well as two centrosomal proteins previously discovered in Drosophila centrosomes. Here again, by focussing on in vitro methods, the consequences of the removal of specific components by immunodepletion will be assessed. Lastly, both the chromatin condensation and centrosomal efforts will be the focus of additional microscopic tool development with a goal of further improvements in immuno- tomography methodology.

As an integral part of our structural studies of proteins we routinely need to oxidatively fold polypeptides. Both synthesized peptides and proteins cytoplasmically expressed in vivo do not form disulfide bonds. In a number of cases our lab is intensively working out the in vitro conditions necessary for formation of the native disulfide bonding pattern. Analysis of these efforts would be greatly accelerated by the ability to quickly and accurately determine the weight of the products of such reactions. This information will allow us to watch the removal of blocking groups (on synthesized polypeptides), check for unwanted labeling of labile residues withoxidative agents (such as iodine), and, if possible, determine the relative amounts of SH and S-S groups.

proteins; enzymes; biomedical resource; biomaterials; biological products;

The 90 kilodalton heat shock protein (hsp90) is a highly abundant, highly conserved protein in both prokaryotes and eukaryotes. In certain mammalian cell types, the isozymes of hsp90 can comprise as much as 2% of total cellular protein under nonstess conditions. At elevated temperatures, both the transcription and translation of hsp90 increase dramatically suggesting that it plays a major role in the heat shock response. In fact, like several other heat shock proteins, hsp90 has been shown to chaperone protein folding in vitro; that is, addition of hsp90 prevents nonproductive aggregation of protein molecules during refolding reactions. In addition, hsp90 has been shown to modulate the activities of a variety of signal transduction molecules including steroid hormone receptors (such as the glucocorticoid and estrogen receptors) as well as nonreceptor tyrosine kinases (such as v-src). Finally, hsp90 has been found to be associated with molecules such as calmodulin, actin, tubulin and serine/threonine kinases such as casein kinase II and eIF-2a kinase. Overall, the studies of the interactions between hsp90 and these various signal transduction molecules hint that the mechanism through which hsp90 modulates the activities of these molecules and its role as a chaperone may overlap; these signaling molecules may have co-opted the ability of hsp90 to stabilize folding intermediates into regulating conformational changes necessary for signaling. Therefore, to begin probing these mechanisms, we have initiated a structural study of htpG (high temperature production protein G), the Escherichia coli member of the hsp90 family.

DESCRIPTION (provided by applicant): The Molecular Biophysics Graduate Program provides UCSF students with a unique opportunity to bring methodologies and approaches from physics, mathematics, chemistry and engineering to bear on the most sophisticated problems in quantitative biology in a stellar biomedical environment. The program emphasizes interdisciplinary training, and a hallmark of the program is the use of experimental and computational approaches to address fundamental questions in molecular function and cellular processes. We feel it is imperative to train the next generation of scientists not only to understand the biology and structural biology but also to be able to synthesize vast amounts of information into quantitative and testable models. Reflecting these scientific opportunities and needs, we have expanded our efforts in computational biophysics while maintaining a solid emphasis on the physical basis for complex biological behavior. As a consequence we have changed the title of this application from "Structural Biology" to "Molecular Biophysics" to acknowledge the importance of training students in a broad array of approaches ranging from structural biology to computational chemistry to bioinformatics to systems biology. To provide a more integrated inter-disciplinary training experience and specifically encourage disciplinary cross-over, our unique training program has evolved to focus on very rapidly bringing all students to a common understanding, breaking down sociological barriers to interdisciplinary research, building confidence and enhancing bonding within the entering class. This is coupled with our traditional strong emphasis on critical thinking, careful mentoring, building communication skills, and the benefits of collaborative research. Relevance: The extraordinary complexity of biological systems ultimately derives from the properties of individual molecules and how they interact to form supramolecular machines and networks. To provide new insight into this complexity, the UCSF Molecular biophysics training grant will train a new generation of scientists to simultaneously understand the physical basis for molecular behavior and to have the tools required for integrating the vast amounts of available information into testable quantitative models of overall function.

A state-of-the-art Philips Tecnai F30 Helium Electron Microscope with an attached Gatan Imaging Energy Filter (GIF) and 2Kx2K CCD detector will be used for high-resolution biological imaging at both the atomic and cellular levels. Increasingly, the frontiers of cell biology focus on elucidating the control of cellular shape and organization, the nature and function of cellular organelles, and the role of complex protein machines. Such project areas also define a new frontier for structural biology where the goals are to determine not the structures of individual molecules, but structures of supramolecular complexes as large as entire cellular organelles. Embodied in this goal is a switch from the largely reductionist approach of the past to one that is, at its heart, integrative. Electron microscopy (EM) is uniquely poised to meet this challenge. Single particle reconstruction methods (SPR) hold the promise of near atomic resolution structures of very large complexes while Intermediate Voltage EM Tomography (EMT) provides the unique ability to integrate this information into the context of the whole cell.

The combination of Intermediate voltage (300kV) and Field Emission Gun provide optimal imaging for both thin and thick samples. Liquid helium cooling of the sample significantly reduces the effects of beam damage as well as the completely redesigned tilting stage provides unprecedented sample stability and freedom from drift. Addition of an imaging energy filter will revolutionize thick-section imaging and improve cryo imaging by removing inelastic electron scatter. The large area CCD will provide optimal on-line digital image recording. The result will be a facility unique in the US. Goals are to reach 25 angstrom resolution on EMT reconstruction of samples > 200nm thick and to reach 4-7 angstrom resolution from SPR of particles larger than 500 KDa. The combination of the capabilities of this microscope with automated data collection methods being developed, should enable near atomic resolution reconstruction of single macromolecular complexes to become a reality.

This instrumentation will empower major advances in cell biology and polymer science. A core user group of scientists from UCSF, Stanford, Berkeley, as well as the more distant Harvard and U. North Carolina has substantial tomography expertise and focuses on fundamentally important problems in biology and polymer science. Projects range from the understanding mechanisms controlling actin and microtubule cytoskeletal organization, the structure of chromosomes and mitotic spindle, the structure of the transcriptional initiation complex, the organization of the neuromuscular junction, and the guiding principles of organization in complex plastics.

The equipment will be in the existing Electron Microscope Laboratory in the UCSF Biochemistry Department and in August 2002 will be moved to the new Mission Bay campus. This facility is available to the entire campus as well as to outside users. A center for electron microscope tomography will act as a magnet to attract and train top students and postdocs. The UCSF programs in Biophysics and Cell Biology provide excellent opportunities for training graduate students and actively seek to attract minority students to campus.

Proposed is a plan of research designed to determine the mechanism of microtubule nucleation by the centrosome in molecular detail. A combination of hierarchical structural approaches (EM Tomography, EM single particle reconstruction, and x-ray crystallography) and biochemical dissection and reconstitution methods will be utilized to determine the structures of gamma-tubulin and its complexes in vivo and in situ. Spanning size scales from the atomic to the entire organelle, our goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between tubulin, gamma-tubulin complexes, and the centrosomal matrix. This is a continuation and expansion of a well-established research program that has made dramatic progress in the analysis of large, complex, supramolecular assemblies through a combination of light and electron microscopies. In addition, we plan to continue our efforts to determine the three-dimensional fine structure of mitotic chromosomes in a variety of biologically well-defined functional states. The long range goal is to understand the structural complexities that underlie DNA condensation and its organization into higher-order structures that can support as well as modulate transcriptional activity, and that change throughout the cell cycle. Current efforts focus on the structural analysis of Xenopus sperm chromatic condensed in vitro using Xenopus extracts as pioneered by Professor T. Mitchison (Harvard). By providing a reproducible and well controlled sampling of haploid chromosome condensation states, this will allow us to understand the condensation process as well as to determine the 3D organization of key protein components being discovered by other laboratories using this system to understand function. Furthermore, the structural consequences of immuno-depletion of such key protein components will be determined. In addition, we continue to pursue structural studies on HeLa telophase chromosomes. While ultimately a less powerful system than the Xenopus vitro condensation approach, for now it offers several important technical advances for structural analysis that makes it worthwhile. Key will be the use of High Pressure Freezing, cyro embedding, DNA-specific staining and cryo, low dose automated Intermediate Voltage Electron Microscope Tomography (IVEM-T) to allow tracing the 3D chromatin paths.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The proposed conference "Protein Folding and Assembly in the Cell" is the sixth in a series of FASEB (Federation of American Society of Experimental Biologists) Conferences that began in 1990. This meeting is unique in its efforts to bring together cell biologists/geneticists and biophysicists/biochemists to tackle the complex problems of protein folding and misfolding and understand the implications for human disease. This was the concept behind the first conference in 1990, and it remains a unique opportunity for scientists from very diverse backgrounds in 2000. Most protein folding conferences consider either the theoretical and in vitro aspects of protein folding or the role of molecular chaperones and protein folding catalysts in the cell. However, the need for interdisciplinary approaches is stronger than ever. The cell biology has progressed to where reaching a mechanistic understanding is crucial, and biological constraints play a critical role in formulating theory and interpreting in vitro data. In addition to examining recent advances in in vitro folding and molecular chaperone mechanism, this meeting will focus on protein translocation across membranes, protein misfolding to form amyloids, catalyzed disaggregation and protein degradation. Formation of protein aggregates and their dissolution are of central importance in human disease. Our goal is to bring together physical and biological scientists in an interactive environment, to promote education and understanding of the approaches, experimental limitations and to explore common interests. The conference's isolated setting with all participants residing and taking their meals in close proximity will promote scientific exchange. The conference format will provide arenas for formal presentations in the context of extensive discussions, as well as extended information discussions amongst participants. Poster sessions will allow all participants to interact scientifically with other investigators. Young investigators and women will be encouraged to attend and fully participate in the conference.

Increasingly, the frontiers of cell biology focus on elucidating the control of cellular shape and organization, the nature and function of cellular organelles, and the role of complex protein machines. Such project areas also define a new frontier for structural biology where the goals are to elucidate not the structure of individual molecules, but structures of molecular and supramolecular complexes as well as organelles. EM methods such as single particle reconstruction and EM Tomography (EMT) are ideally suited to the analysis of such large structures. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. While great strides in the development of EMT have been made at UCSF, our instrumentation is inadequate to meet the demands of current problems in structural cell biology. Our goals are to acquire a new microscope (Phillips 300kV FEG He stage) equipped with an energy filter and a 2Kx2K CCD camera. As a first step in this expensive process we request the purchase of a Gatan Imaging Energy Filter (GIF) and a Gatan 2K x2K pixel CCD camera. This equipment will be immediately useful on our existing microscope and readily transferable to the new microscope. Acquisition of the energy filter and 2Kx2K CCD will create a facility for the collection of high resolution EM Tomography data that is absolutely unique in the US and perhaps the world. This new equipment will allow us to do 1) higher resolution tomography on thick biological samples, 2) image larger samples, 3) collect high resolution image data for single particle reconstruction using the CCD and 4) collect high quality diffraction data from 2D crystals. These benefits result from the use of the GIF to remove inelastic and multiple inelastic scattered electrons and having a larger format CCD that is significantly better suited for imaging at 200-300keV. The equipment will be attached to our pre-existing Philips 300keV electron microscope in the Electron Microscope Laboratory of the Department of Biochemistry at UC San Francisco. This facility is available to the entire campus as well as to outside users. All of the core users except two are currently funded by the NIH, and all have extensive experience in electron microscope. The core users plus the supervisor of the EM lab will form an advisory committee to oversee use of the instrument and to set policy. Use by other regional and national users will be encouraged in the time remaining after core investigator use. UC San Francisco will provide space and staff support (including software) for operating the equipment.

ABSTRACT NOT PROVIDED

molecular assembly /self assembly; tubulin; protein structure; intermolecular interaction; biomedical resource;

DESCRIPTION (provided by applicant): Proposed is a plan of research designed to determine the mechanism of microtubule (MT) nucleation by the centrosome in molecular detail. A combination of hierarchical structural approaches (EM Tomography, EM single particle reconstruction, and x-ray crystallography) will be utilized to determine the structures of gamma-tubulin and its complexes in vitro and in situ and the structure of intact centrosomes. Detailed analysis of MT nucleation kinetics will reveal how gamma-tubulin and its larger assemblies alter the mechanism and rate of MT-nucleus formation. The role of guanine nucleotide binding and hydrolysis in the nucleation process will also be determined. We will extend these studies to assay effects of a broad array of kinases including known regulators of centrosome function. Cellular and embryonic extracts will be screened for novel factors that regulate MT nucleation by the purified gamma-tubulin ring complex. Through mass spectrometry, we will determine the protein components of intact and extracted centrosomes, proteins that interact with the gamma-tubulin ring complex, and seek to discover their functions. siRNA knockdowns in Drosophila S2 cells will be used as a first screen for phenotypes. This will be complemented by in vivo and in situ localization using 3D fluorescence microscopy and 3D electron microscopic tomography. The results from structural, proteomics, and siRNA approaches will be integrated with our in vitro functional studies and in vivo analyses to build an understanding of the mechanisms by which MT nucleation is regulated within the cell. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between tubulin, gamma-tubulin complexes, regulatory proteins and the centrosomal matrix. This is a continuation and expansion of a well-established research program that has made dramatic progress in the analysis of large, complex supramolecular assemblies through a combination of biochemical analyses and light and electron microscopic studies. To facilitate these scientific directions, we will continue to pioneer the development of new software technologies for the collection, processing, reconstructing and understanding of EM data. In addition, we continue our efforts to build a new class of light microscopes that provide dramatically improved resolution.

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 small gamma tubulin complex is part of the microtubule-nucleating unit. The complex consists of two Tub4p and one each of Spc97p and Spc98p. Tub4p, gamma tubulin, binds the alpha-beta tubulin heterodimer, the building blocks of microtubules. Two-hybrid experiments have shown that Spc110 interacts with Spc98 and Spc97 and weakly with Tub4. Spc110 is able to directly bind Spc98 in IP experiments from baclovirus extracts. Spc110 provides the link between the gamma tubulin complex and the inner plaque of the spindle pole body (SPB) through its interaction with Spc97 and Spc98. Our goal is to use FRET to refine the structure of the Tub4p complex in vivo.

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 ubiquitous molecular chaperone Hsp90 is required in the maturation and maintanence of signalling and regulatory molecules including serine/threonine kinases. Hsp90 function requires ATP hydrolysis and this hydrolysis cycle is coupled to large conformational changes in the protein. Hsp90 is further regulated by the interaction of other proteins known as cochaperones. For the activation of kinase clients, Hsp90 forms a complex with the cochaperone p50. It is now well understood how the Hsp90 complex functions to activate its client proteins. Using SAXS, we propose to gain a structural insight into the mechanism of Hsp90's interaction with p50 and how this complex then interacts with the client protein Cdk4. We also propose to use SAXS to dissect the role of Hsp90's ATPase in regulating its interaction with both p50 and Cdk4. The structural information obtained from SAXS will be included with ongoing biochemical studies, and the combination of techniques will provide a detailed mechanistic insight into the activation of kinases by Hsp90.

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 Leginon software has been extended to support automated collection of tilt series for electron tomography using the UCSFTomo predictive tracking algorithm.

DESCRIPTION (provided by applicant): The centrosome is of fundamental importance to animal cells in that it is the principal nucleator of the microtubule (MT) cytoskeleton and harbors the centrioles, specialized structures that are required for cilia formation and contribute to spindle-pole positioning. Through these principal roles, the centrosome is important in embryonic development and human disease. During interphase, MTs are required for vesicle trafficking and cell polarity. In most cell types, many of the MTs that form the mitotic spindle originate at the centrosome. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. The primary aims of this proposal are to determine the molecular mechanism of MT nucleation, and to understand how the nucleating machinery is assembled and regulated. Moreover we will begin to explore the mechanism by which microtubule doublets and triplets are formed at the centriole by focusing on the newly discovered ?- and ?-tubulins and their as yet unknown, binding partners. Our laboratory is uniquely poised to utilize a hierarchical array of structural approaches (x-ray crystallography, electron microscopic (EM) single particle reconstruction, and EM Tomography, small-angle x-ray scattering (SAXS)) to determine the structures of ?-, ?- and ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. By combining structural studies, MT assembly experiments and kinetic modeling, our previous work is leading to a paradigm shift in understanding the underlying principles of MT formation, suggesting a new role for GTP, a different model for MT assembly and an unexpected mode of regulation of nucleation in the ?-tubulin complexes. The proposed experiments will continue and expand upon these results, providing a detailed understanding of the physical origins of MT assembly, and the cellular machinery that dictates MT formation. In vitro predictions of modes of action or regulation will be assayed in vivo using mutagenesis and siRNA and live imaging. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between 12-tubulin, ?-, ?- and ?-tubulin complexes, regulatory proteins and the centrosomal matrix. Public Health Relevance: The normal function of centrosomes and centrioles is directly relevant to human disease, and the proposed work is aimed at understanding the molecular mechanisms of these cellular components. Abnormal centrosome number and behavior is a common hallmark of cancer cells, and centriole defects are involved in many human ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel syndrome, and Oral-Facial-Digital syndrome.

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 centrosome plays a fundamental role in organizing the microtubule cytoskeleton. Defects in centrosome function lead to errors in chromosome segregation, a major factor in both the initiation and progression of cancer. We aim to understand the mechanism and regulation of microtubule nucleation at the centrosome by focusing on the structure of a key 300 kDa heterotetrameric complex that underlies nucleation in all eukaryotes: the [unreadable]-tubulin small complex ([unreadable]-TuSC). We have determined a negative stain EM structure by the random conical tilt method, and have preliminary images under cryo conditions. From a higher resolution cryo-EM structure we hope to determine the overall organization of the complex and particularly which surfaces of [unreadable]-tubulin are exposed for interaction with [unreadable][unreadable]-tubulin at the microtubule minus ends, and to map the binding sites of proteins which link [unreadable]-TuSC to microtubule organizing centers.

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. Tech R&D Core Support for AIDS Research

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

This Major Research Instrumentation-Recovery and Reinvestment (MRI-R2) award funds the development of an 8kx8k pixel direct detection CMOS camera with single electron quantized detection for high-resolution single particle cryo electron microscopy (cryoEM) at the University of California-San Francisco (UCSF). The new camera system will enable high frame readout rates, with extreme sensitivity and high resolution. Because it uses so little material and does not require crystals, single particle cryoEM has become an indispensable tool for studying the three-dimensional (3D) structures of complex biological assemblies, providing critical information not obtainable by more traditional methods such as x-ray crystallography or NMR spectroscopy. The camera development project will be coordinated through collaborations among UCSF, the Lawrence Berkeley National Laboratory (LBNL), and Gatan, Inc., a company specializing in EM cameras and peripherals. The camera will benefit a large number of research projects and improve the training prospects for 15-25 students and postdoctoral fellows in advanced cryoEM technology. UCSF PIs are also active participants in summer research programs and have been hosting undergraduates in their labs to provide research opportunities with advanced technologies. Performance and availability of the camera will be widely disseminated through a web site, in publications, and through presentations at local and international meetings. Commercialization of the new technology may lead to even wider dissemination of similar devices.

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 molecular chaperone Heat Shock Protein 90 (Hsp90) is the central player in a multi-component complex that is required for the folding and activation of numerous essential proteins including nuclear receptors and cell-cycle kinases. Very little is known about 'client'substrate protein and cochaperone interactions on Hsp90 or the structural rearrangements involved in the initial stages of chaperone activity. Previous biochemical work has shown that nuclear receptors and other clients are delivered to an Hsp90:Hop (Hsp90 organizing protein) complex by the Hsp70 chaperone.

DESCRIPTION (provided by applicant): The integrity of the cellular proteome is critically dependent on an elaborate network of protein quality control machines that both aid in the folding of newly made proteins and allow for the recognition and disposal of terminally misfolded forms. Many diverse human diseases, including familial protein folding diseases, neurodegenerative diseases, diabetes, and cancer, as well as normal aging have been linked to the failure to maintain proper protein homeostasis. Thus defining the mechanism of action of the protein quality control machinery is a major goal in the quest for understanding of health and pathology in all living cells. A common theme to this machinery is the ability to recognize portions of unfolded polypeptide chains, either to facilitate their subsequent folding/refolding or degradation, or to signal in adaptive responses aimed at restoring the balance between supply and demand of the protein folding capacity. Most molecular events in protein quality control work on many diverse substrates and hence possess considerable plasticity in substrate binding. While much progress has been made in structural and functional analysis of individual components of these machines, there are few examples where substrate-bound structures have been determined or where a substrate recognition code has been defined and validated. As such, we are lacking in our understanding of core principles that govern workings of these protein machines. We propose to bridge this gap by focusing on a core set of physiologically critical systems that cover a range of molecular features but share the common requirement of having to balance specificity and plasticity in molecular recognition events. In particular, we wil focus on cytosolic chaperone substrate recognition (using examples of the hsp70, hsp90, and TRIC families of chaperones) and the recognition of unfolded proteins in the lumen of the endoplasmic reticulum (ER) for degradation via the ER-associated degradation pathway (ERAD) and for signaling via the unfolded protein response (UPR). PUBLIC HEALTH RELEVANCE: The proper folding of newly made proteins is very important for every cell. Numerous human diseases, including neurodegenerative diseases, diabetes, and cancer, as well as normal aging are linked to the failure to fold proteins properly. We will determine the structure of cellular machines that recognize misfolded proteins to help them fold or target them for degration. A detailed structural understanding will contribute to the development of new therapies.

DESCRIPTION (provided by applicant): The centrosome is of fundamental importance to animal cells in that it is the principal nucleator of the microtubule (MT) cytoskeleton and harbors the centrioles, specialized structures that are required for cilia formation and contribute to spindle-pole positioning. Through these principal roles, the centrosome is important in embryonic development and human disease. During interphase, MTs are required for vesicle trafficking and cell polarity. In most cell types, many of the MTs that form the mitotic spindle originate at the centrosome. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. The primary aims of this proposal are to determine the molecular mechanism of MT nucleation, and to understand how the nucleating machinery is assembled and regulated. Moreover we will begin to explore the mechanism by which microtubule doublets and triplets are formed at the centriole by focusing on the newly discovered ?- and ?-tubulins and their as yet unknown, binding partners. Our laboratory is uniquely poised to utilize a hierarchical array of structural approaches (x-ray crystallography, electron microscopic (EM) single particle reconstruction, and EM Tomography, small-angle x-ray scattering (SAXS)) to determine the structures of ?-, ?- and ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. By combining structural studies, MT assembly experiments and kinetic modeling, our previous work is leading to a paradigm shift in understanding the underlying principles of MT formation, suggesting a new role for GTP, a different model for MT assembly and an unexpected mode of regulation of nucleation in the ?-tubulin complexes. The proposed experiments will continue and expand upon these results, providing a detailed understanding of the physical origins of MT assembly, and the cellular machinery that dictates MT formation. In vitro predictions of modes of action or regulation will be assayed in vivo using mutagenesis and siRNA and live imaging. Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between 12-tubulin, ?-, ?- and ?-tubulin complexes, regulatory proteins and the centrosomal matrix. Public Health Relevance: The normal function of centrosomes and centrioles is directly relevant to human disease, and the proposed work is aimed at understanding the molecular mechanisms of these cellular components. Abnormal centrosome number and behavior is a common hallmark of cancer cells, and centriole defects are involved in many human ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel syndrome, and Oral-Facial-Digital syndrome.

DESCRIPTION (provided by applicant): Spanning size scales from the atomic to the entire organelle, our long-term goal is to synthesize an atomic resolution picture of all the relevant structural and functional interactions between ¿¿- and ?-tubulin complexes, regulatory proteins, and the centrosomal matrix. Here we focus on determining the molecular mechanism of microtubule (MT) nucleation, and understanding the assembly and regulation of the nucleating machinery. Our laboratory is uniquely poised to use a hierarchy of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of ?-tubulin complexes in vitro and in situ, and to understand their mechanism of action through functional studies and innovative kinetic modeling. Previously we discovered that yeast ?-tubulin small complex (?TuSC ) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering an unexpected mode of regulation and assembly. The proposed experiments expand upon these results, providing a detailed understanding of the physical origins of MT nucleation, and the cellular machinery that dictates it. Specifically we address the following questions: 1) Determine the structure of yeast and Drosophila ring complexes and their interactions with MTs: We will improve the resolution of our yeast ?TuSC rings and will generate a complete pseudo-atomic structure. We will extend the resolution (goal ~ 1nm) of our preliminary of cryoEM structure (3.5nm) of isolated Drosophila ?TuRCs (2.2MDa ?-tubulin ring complex) and identify how the different GCPs assemble around the ring. Structures of ?TuSC rings ?TuRCs bound to MTs or 1 layer of non-polymerizing yeast ¿¿-tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography. 2) Mechanism of Spc110/72 facilitated assembly of ?TuSC rings and regulation by phosphorylation: We know that Spc110 stabilizes formation of yeast ?TuSC rings. We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spc110 and Spc72 are required for assembly and the role of Spc110/72 phosphorylation. 3) Activation of nucleation by yeast ?TuSC rings: While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both ?TuSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined. 4) Structural organization of centrosomes - role of non ?-tubulin components: Our cryoEM tomography of basal bodies revealed new non-tubulin structures decorating the triplets. We propose a combination of SIM and STORM microscopy and cryoTomography to determine the molecular identity of these novel structures. Our SIM/STORM imaging has revealed unexpected structural domains within the centrosomal pericentriolar material. We continue these efforts assessing interactions and assembly including a structural analysis of Plp.

DESCRIPTION (provided by applicant): Cytoskeletal proteins are of ancient origin, predating the divergence of prokaryotes and eukaryotes. Although these proteins play key roles in a variety of cellular processes, the proteins that make up the prokaryotic cytoskeleton are still poorly defined. In bacteria, only a few distinct families of tubulin have been characterized: FtsZ, a widely distributed protein critical for cell division, TubZ, involved in plasmid segregation and BtubA/BtubB, whose functions are still unknown. We recently discovered a divergent tubulin-like cytoskeletal protein, PhuZ, encoded by the very large (317 kb) Pseudomonas chlororaphis bacteriophage, 201?2-1. By expressing a GFP-tagged PhuZ at low levels in Pseudomonas, we could observe filament formation during lytic phage infection. We solved the structure of PhuZ to 1.67A resolution, and found a conserved tubulin fold with a novel, extended C-terminus that we showed to be critical for polymerization both in vitro and in vivo. Surprisingly, we found that PhuZ assembles a dynamic spindle that positions a single large complex of phage DNA at the center of the cell during lytic growth. Moreover, using PhuZ mutants designed from our structure, we could show that the dynamic nature of PhuZ filaments is required for phage centering. Bacterial viral particles appear to assemble around the periphery of this central DNA mass, creating a corona-like structure similar to the replication factories of herpes viruses, whic are distantly related to dsDNA bacteriophage. This is the first example of a prokaryotic spindle that performs a genome centering function analogous to the role of microtubule-based spindles of eukaryotes. Here, we propose to elucidate the biochemical, structural, and genetic basis of the ability of PhuZ to center DNA and the underlying mechanisms by which the polymer participates in viral lytic growth. Plausible roles for the polymer and centering include: defininga site to coordinate replication and packaging, facilitating phage head and or tail assembly, and facilitating cell lysis. Not only will we seek to answer these questions, but our work will also provide new insights into how tubulin family polymers can participate in such divergent functions as cell division, separation of plasmid DNA and organizing DNA into replication factories. Specifically, we propose the following research aims: 1. Examine the role of PhuZ in viral lytic growth. 2. Examine the possible connections between PhuZ assembly and DNA replication and movement, phage assembly and cell lysis in vivo. 3. Structurally and functionally characterize the mechanism and properties of PhuZ filaments assembled in vitro. 4. Identify phage and host proteins that interact with PhuZ and determine if they affect PhuZ polymerization, localization or other aspects of function. 5. Perform electron tomography and cryoTomography at various stages of infection to gain high resolution insights into the structural organization of PhuZ and viral capsids assembled in vivo during lytic growth.

The centrosome is the principal nucleator of the microtubule (MT) cytoskeleton, which is required for cell polarity, vesicle trafficking, and spindle formation and function. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morphologically distinct, a conserved set of Y-tubulin complexes is used to nucleate MT assembly. In this Project we focus on the assembly and regulation of the nucleating machinery using a broad combination of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of Y-tubulin complexes in vitro and in situ, and to understand their mechanism of action through quantitative in vitro functional studies and innovative kinetic modeling. Previously we discovered that yeast Y-tubulin small complex (YTUSC) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering unexpected modes of regulation and assembly. Based on our previous results we propose that there are three phases of regulation: Y-TUSC ring assembly restricted to the spindle pole body by requiring interactions with Spcl 10 or Spc72, ring closure to fully match MT symmetry and, activation of the Y-tubulins for efficient nucleation. The proposed experiments expand upon our previous results with the long-term goal of synthesizing an atomic resolution picture of all the relevant structural and functional interactions between aP- and Y-tubulin complexes, regulatory proteins, and how these complexes are linked to the spindle pole body or centrosome matrix. Specifically we will (i) improve the resolution of our cryoEM reconstruction of yeast YTUSC rings and, collaborate with the Bioinformatics Core to generate a complete pseudo-atomic structure. Structures of yTuSC rings bound to MTs or 1 layer of non-polymerizing yeast ap-tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography of yeast SPBs. (ii) We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spcl 10 and Spc72 are required for assembly and the role of Spcl 10/72 phosphorylation, (iii) While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both YTUSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined.

? DESCRIPTION (provided by applicant): This MIRA application is to consolidate my effort on three separate GM grants: our long standing centrosome structure and microtubule nucleation grant (GM031627-30), yeast spindle pole body program project grant (Winey 1P01GM105537) and a MPI grant on the newly discovered phage tubulin PhuZ (Joe Pogliano, GM104556). This work is exciting and has been well appreciated. Most importantly, the collaborative efforts have been invaluable, resulting in remarkable synergies and a great expansion of our reach and understanding (genetics, in vivo function, computational modeling, single molecule biophysics). Similarly, our unique combination of biophysics and structure has significantly enhanced what our collaborators could accomplish. Unfortunately under the new rules, I cannot continue my funded participation in these efforts. Tubulins play important and remarkably varied roles throughout eukaryotic and prokaryotic biology. In most eukaryotes, the microtubule (MT) cytoskeleton is responsible for chromosome segregation, nuclear positioning, vesicle trafficking and cell polarity. As the principle nucleator of MTs, the centrosome is of fundamental importance and harbors the centrioles, specialized structures required for cilia formation and contribute to spindle-pole positioning. The centrosome also has important roles beyond MT nucleation, including functions in cytokinesis, progression through the cell cycle, and in sequestering or positioning some proteins to control when and where they are active. Thus, the centrosome is important in embryonic development and human disease. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morphologically distinct, a conserved set of ?-tubulin complexes is used in both systems to nucleate MT assembly. Our long-term goals are to synthesize an atomic resolution picture of the relevant structural and functional interactions between ??- and ?-tubulin complexes, and to elucidate the principles underlying assembly and regulation. We have determined the structure of the yeast ?TuSC ring to 6.5Å resolution, revealing principles of MT nucleation and protofilament number determination, and have identified at least two unexpected levels of regulation: via assembly mediated by oligomerization of the Spc110p attachment factor, and a closure event, lead- ing to activated nucleation. Proposed, we seek to expand the structural studies to atomic resolution by cryoEM and discover the role of PTMs and other factors in regulation. We will place in context via structures of the SPB and centrosome determined by cryoTomography, and better understand how the pericentrioloar material forms and uncover attachment factors that connect ?TuRCs to the Golgi. Our work on MT nucleation by ?-tubulin complexes has been complemented by our progress on the structure and biochemistry of prokaryotic tubulin homologs, including a ~3.3Å structure of PhuZ:GMPCPP and ~4.Å of PhuZ:GDP, providing new insights into filament structure and general regulation by GTP hydrolysis. Here we focus on the novel PhuZ spindle and associated cell biology.

Not Applicable ? no change

ABSTRACT My previous MIRA period focused on mechanisms of microtubule nucleation, centrosome structure and the phage-encoded cytoskeleton and ?nucleus?. Now that my HHMI has ended, our strong efforts on protein ho- meostasis are included in this MIRA proposal. Throughout, our work seeks to understand fundamental molecu- lar mechanisms that underly cellular function. Where possible, complex systems are reconstituted in vitro and analyzed in atomic detail with the implications explored at a cellular level. The research has three parts. I. Birth, life and destruction: mechanisms of Hsp90/Hsp70-driven proteostasis: Maintenance of the cellular proteome is one of the most fundamental aspects of all organisms. Molecular chaperones facilitate folding and activation, sequester or recover aggregated proteins, participate in the removal of irreversibly mis- folded proteins, and help regulate folding capacity according to cellular need. While critical players have been identified, the molecular mechanisms by which most of these tasks are accomplished remain unknown. We focus on the cytosolic Hsp90 chaperones that facilitate the folding and activation of ~10% of the proteome. Hsp90's ?clients? are enriched in proteins important for cellular signaling, proliferation, and survival making Hsp90 a valuable therapeutic target for multiple diseases. Despite the biological importance, the underlying mechanism of client remodeling is unknown, as is how the chaperones facilitate folding vs degradation triage decisions by presenting clients to E3 ligases. Through in vitro reconstitution, extensive biochemical, biophysi- cal and cryoEM structural analyses our goal is to elucidate the molecular mechanisms of these processes. II. Structure of the basal body transition zone, tomography technology: In non-dividing cells, centrioles mature into basal bodies that dock at the membrane leading to the formation of a primary cilium which serves as a sensory organelle on virtually all animal cells, or motile cilia to move fluid. These structures are important in numerous human diseases, including cancer and a broad array of ciliopathies. Unfortunately, there is only limited understanding of centriole or basal body structure, how the basal body docks at the membrane, transi- tions to an axoneme, or provides a distinct cellular compartment. We will use cultured mouse tracheal epithelial cells which can be grown and differentiated on grids to produce arrays of motile cilia. Cells will be high pres- sure frozen and FIB-milled to create thin lamella for high-resolution in situ cryoEM. Importantly, key proteins can be knocked out by CRISPR or tagged with Ferri-tag for simultaneous like/cryoEM visualization. Phage ?nucleus? and host immunity evasion: The cell biology being revealed by Phi-KZ jumbo phages is simply extraordinary (collaboration Pogliano, UCSD), demonstrating what appears to be an entirely new concept in compartment formation. Upon infection, these phage form a ?nucleus? from a self-assembling pro- tein monolayer shell that is centered by a dynamically unstable tubulin cytoskeleton. The shell grows as the phage DNA replicates, selectively imports DNA replication and transcription machinery, yet excludes cytosolic proteins and GFP. Collaborating with (Bondy-Denomy, UCSF) has shown that the shell confers resistance to all known host immunity factors (CRISPRs, restriction endoncleases). The molecular basis for these processes is unknown. We focus on the determining shell assembly principles and the mechanism of selective transport.

PROJECT SUMMARY Pathological tau deposition occurs in a subset of neurodegenerative disorders including frontotemporal lobar degeneration with tau inclusions (FTLD-tau). Much of what is known about FTLD-tau is derived from mutant tau models or overexpression. However, a unified view of overall tau metabolism?from its initial production, interactions with molecular chaperones, post-translational modifications, targeting to lysosomes/autophagy, and resultant degradation, to our knowledge has not been generated The long- term goal of this proposed FTD Center without Walls (CWOW) is to improve our understanding of the pathobiological mechanisms underlying FTD-tau. Its overall objective is to elucidate the genes, molecules and pathways that regulate tau metabolism and to determine the impact of disease- associated mutations and variants. Our central hypothesis is that proper tau metabolism requires the precise, coordinated action of molecular chaperones, co-chaperones, PTMs and degradation machinery that each represent regulatory nodes. Genetic mutations in tau and other pathway members can disrupt tau metabolism, leading to tau accumulation, secretion and neurodegeneration. The Center will be led by Dr. Aimee Kao, who will also lead Core A: Administration and Data Core (with Co-lead Dr. Yokoyama) and Project 1: Tau Molecular Chaperones, targeting and proteolysis (with Co-I Dr. Agard). Dr. David Agard will oversee Core B: Macromolecular and Cellular Structure Core. Dr. Jennifer Yokoyama will lead Core C: Genomics and Transcriptomics. Finally, Dr. Celeste Karch will lead Project 2: Tau Half Life and Secretion. We will achieve these objectives through four Specific Aims. Aim 1: Understand the normal process of tau metabolism as a series of decisions that are made at regulatory nodes. Aim 2: Identify and test the functional relevance of genetic variants in MAPT and other tau metabolism genes, in in vitro, cell and iNeuron models, on each of the tau metabolism regulatory nodes. Aim 3: Integrate findings from Projects and Cores to produce a Tau Metabolism and Variant Database (TMVdb), that will serve as a reference point for the field. Aim 4: Integrate findings from Projects and Cores to produce a Tau Polygenic Risk Score (TPRS), which will stratify genetic risk for tauopathy. Upon successful completion of these Aims, the proposed FTD CWOW will have provided fundamental information about tau metabolism, defined mechanistic nodes predisposing to tauopathy and generated the TMVdb and TPRS, new resources for the fields of tauopathy and neurodegeneration research. It will generate critically important information about tau homeostasis and a foundational basis from which to build and frame subsequent investigations into tau pathobiology and toxicity.