James E. Rothman - US grants

Our goal is to understand the mechanisms governing the formation, targeting, and fusion of transport vesicles. We have reconstituted these processes in cell-free extracts containing Golgi membrane fractions, and have accumulated evidence supporting the working hypothesis that protein transport between membrane compartments in this cell-free system is due to the budding of transport vesicles from one cisterna of a Golgi stack followed by fusion of the vesicle with the next cisterna. We now hope to purify two key cytoplasmic components (C and B) that have been identified as necessary for vesicle budding and fusion respectively. Furthermore, we hope to purify a recently discovered NEM-sensitive factor (NSF) that is bound to the membranes in an ATP-dependent fashion. NSF seems to be needed for membrane fusion, and the activity of NSF is greatly stimulated by long chain acyl Coenzyme A, acting as a cofactor. We hope to purify both NSF and its presumed "receptor" that binds it to Golgi membranes, as well as to elucidate the basis of the Coenzyme A requirement, which may be for an acylation-decylation cycle in which NSF participates that regulates a step leading to fusion of transport vesicles. The role of transport vesicles as intermediates will be studied by electron microscope immunocytochemistry, an important technique which will also be exploited to determine the nature of transport intermediates which accumulate when individual transport components (like NSF) are eliminated, offering clues as to the functional role of the eliminated component.

The overall goal of the research is to elucidate the molecular mechanism by which coated vesicles execute sorting decisions -- how they succeed in including select sets of membrane-associated proteins during budding, how they bud, and how they fuse with the appropriate organelle after budding. An entry point into coated vesicle metabolism may have been forged in the last grant period with the identification and purification of an enzyme that can utilize ATP hydrolysis to power the removal of clathrin coating termed uncoating ATPase. We hope now to establish the exact role of this protein in the cell and its detailed mechanism of action, and to use the products of its action as substrates with which to assay for factors that may facilitate other steps in a clathrin-coated vesicle cycle of assembly and disassembly. Specifically, these and other related topics we plan to pursue in the next five years are: 1. Mechanism of ATP-Dependent Disassembly of Clathrin Coats and Its Possible in Vivo Significance 2. Reconstitution of Steps in the Clathrin-Coated Vesicle Cycle Subsequent to Uncoating 3. Functions of the Clathrin Light Chains 4. Transport of the VSV G Protein in Coated Vesicles 5. Isolation of Proteins That Bind the Carboxyterminus of VSV G Proteins, and Their Possible Role in Facilitating Transport 6. Expression of a Drosophila Heat Shock Cognate Gene in E. Coli: Does it Encode Uncoating ATPase?

Our goal is to understand the mechanisms governing the formation, targeting, and fusion of transport vesicles. We have reconstituted these processes in cell-free extracts containing Golgi membrane fractions, and have accumulated evidence supporting the working hypothesis that protein transport between membrane compartments in this cell-free system is due to the budding of transport vesicles from one cisterna of a Golgi stack followed by fusion of the vesicle with the next cisterna. We now hope to purify two key cytoplasmic components (C and B) that have been identified as necessary for vesicle budding and fusion respectively. Furthermore, we hope to purify a recently discovered NEM-sensitive factor (NSF) that is bound to the membranes in an ATP-dependent fashion. NSF seems to be needed for membrane fusion, and the activity of NSF is greatly stimulated by long chain acyl Coenzyme A, acting as a cofactor. We hope to purify both NSF and its presumed "receptor" that binds it to Golgi membranes, as well as to elucidate the basis of the Coenzyme A requirement, which may be for an acylation-decylation cycle in which NSF participates that regulates a step leading to fusion of transport vesicles. The role of transport vesicles as intermediates will be studied by electron microscope immunocytochemistry, an important technique which will also be exploited to determine the nature of transport intermediates which accumulate when individual transport components (like NSF) are eliminated, offering clues as to the functional role of the eliminated component.

Recent evidence from genetic studies in yeast raises the strong possibility that a new family of GTP-binding proteins related in size and sequence (30-40% homology) to the 21 kD mammalian ras oncogene family catalyzes steps in protein targeting and secretion. One member of this family in yeast (y-YPT) affects transport through the ER and Golgi when mutated. A mammalian gene 85% homologous to the yeast YPT gene has recently been cloned. We propose to test whether this gene's product (termed m-YPT; 'm' = mammalian) functions in secretion in animal cells. We propose to prepare antibodies to m-YPT expressed in E. coli, and to localize the protein in animal cells by immuno-electron microscopy. We also plan to make use of neutralizing monoclonal and polyclonal antibodies in combination with established mammalian cell-free and semi-intact cell systems that reconstitute steps in the secretory pathway to probe the function of m-YPT in secretion. We hope to use the m-YPT gene and it sequence to identify a novel family of ras-related "sec" genes that may be needed for secretion in mammalian cells. Recent evidence suggests that a human ras oncogene product causes massive uncontrolled secretion in mast cells. This, together with the evidence from yeast, raises the intriguing possibility that the normal function of certain ras and ras-related proteins (currently unknown) may be in controlling secretory and other intracellular transport processes. The potential role of members of this putative ras-related "sec" gene family as oncogenes will be tested by introducing mutations of a kind that are known to activate classical ras proto-oncogenes. The phenotypes of similar mutations in a new class of ras-related "sec" genes may well reveal a whole new class of oncogenes associated with cancers. Thus work is likely to be of significance for cell biology, and may also afford novel insights into the basis of malignant transformation.

The precise localization of newly discovered proteins and messenger RNA molecules within cells is of crucial importance for assigning functions and testing mechanistic hypotheses. Such molecules can be visualized by fluorescent and electron-dense tags using standard techniques, and then need to be imaged in relation to surrounding structure by microscopy for localization. The confocal microscope provides the most effective means to do this by light microscopy, allowing complete three dimensional reconstructions with unprecedented detail. This instrument will serve the entire biological community at Princeton, whose research requires localizations of importance and molecular biology. At lest 80% of the 25 faculty in life sciences at Princeton will be user of both instruments, about 45% being major users.

The overall goal of this project is to elucidate the mechanism of transport vesicle budding and targeting. We will take advantage of the very recent purification of Golgi-derived ("non-clathrin") coated vesicles in combination with existing cell-free systems to offer molecular insights into the mechanisms of vesicular movement between membrane-bound compartments. These coated vesicles, which transport proteins between the cisternae of the Golgi stack, can be accumulated during cell-free incubations of isolated Golgi membranes, and then purified by density gradient centrifugation following salt extraction. Sufficient quantities can be isolated to enable the individual polypeptides to be characterized by microsequencing, antibodies to be produced, and the genes cloned. Many of these proteins should be needed for vesicle budding and targeting. Antibodies prepared against them will be used as reagents to probe for function in cell-free systems. Specifically, we plan to purify Golgi-derived coated vesicles in bulk as source material for microsequencing. Anti-peptide and then polyclonal antibodies will be produced based on this microsequence information. We will use these antibodies to localize the coated vesicle polypeptides in coated vesicles fractions and in cells at the electron microscope level. Probes based on the amino acid sequences will be used to clone the genes encoding the major polypeptides. We hope to elucidate the physical arrangement of the major polypeptides in membranes and in coats. A number of functional studies are planned in addition to these structural studies. These include determining which subunits of coated vesicles originate from cytosol and which from Golgi membranes, and whether any coated vesicle component are fatty acylated during budding, since fatty acyl-CoA is required for the process. Antibodies to individual polypeptide components will be employed as specific inhibitors with which to ascertain the function preformed by each component. We hope to purify cytosol-derived components from cytosol in their native, transport-competent forms, and to use these as reagents to help develop simplified assays (partial reactions) corresponding to sub-steps in vesicle budding and targeting.

DESCRIPTION (provided by applicant): Membrane fusion is central to many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes. As a result of the work supported by this grant during the current cycle of funding, the core principle of cellular membrane fusion is now well established, consisting of the assembly of cognate SNARE proteins initially residing in apposing membranes to yield a stable, bridging complex that triggers the bilayers to merge. How does the assembly of SNARE proteins between membranes drive membrane fusion? A mechanistic understanding of the fusion event, in which as many as four separate proteins fold together to fuse apposing bilayers, will require the combined power of a variety of biophysical approaches. Such an undertaking has become possible only recently, thanks to continuing advances in both SNARE fusion biochemistry and biophysical membrane technologies. We will use different complementary technologies (cellular and molecular biology, electrophysiology, surface force/adhesion, and optical imaging), to follow SNARE dynamics and function in real time. SNARE proteins will be reconstituted in both synthetic and biological membranes, thus allowing measurements in fully reconstituted membrane environments as well as in less flexible but more physiological cellular membranes. Within these systems, we will determine the energetics of SNARE-assembly, the consequences of membrane composition and membrane tension on fusion, and the dynamics of the fusion pore itself. Insights gained in the different approaches can be linked by comparing the effects of critical mutations and other perturbations. By following this program, whose value has been proven with viral fusion proteins, we expect new and important information concerning cellular membrane fusion to emerge during the next five years.

Recent evidence from genetic studies in yeast raises the strong possibility that a new family of GTP-binding proteins related in size and sequence (30-40% homology) to the 21 kD mammalian ras oncogene family catalyzes steps in protein targeting and secretion. One member of this family in yeast (y-YPT) affects transport through the ER and Golgi when mutated. A mammalian gene 85% homologous to the yeast YPT gene has recently been cloned. We propose to test whether this gene's product (termed m-YPT; 'm' = mammalian) functions in secretion in animal cells. We propose to prepare antibodies to m-YPT expressed in E. coli, and to localize the protein in animal cells by immuno-electron microscopy. We also plan to make use of neutralizing monoclonal and polyclonal antibodies in combination with established mammalian cell-free and semi-intact cell systems that reconstitute steps in the secretory pathway to probe the function of m-YPT in secretion. We hope to use the m-YPT gene and it sequence to identify a novel family of ras-related "sec" genes that may be needed for secretion in mammalian cells. Recent evidence suggests that a human ras oncogene product causes massive uncontrolled secretion in mast cells. This, together with the evidence from yeast, raises the intriguing possibility that the normal function of certain ras and ras-related proteins (currently unknown) may be in controlling secretory and other intracellular transport processes. The potential role of members of this putative ras-related "sec" gene family as oncogenes will be tested by introducing mutations of a kind that are known to activate classical ras proto-oncogenes. The phenotypes of similar mutations in a new class of ras-related "sec" genes may well reveal a whole new class of oncogenes associated with cancers. Thus work is likely to be of significance for cell biology, and may also afford novel insights into the basis of malignant transformation.

The overall goal of this project continues to be to understand the molecular mechanism of budding of transport vesicles, with special reference to the Golgi. With the support of this grant, coated transport vesicles budding from Golgi membranes (COPI vesicles) and a general principle for budding (utilizing coatomer and the GTPase ARF) were uncovered. We now seek deeper insights into budding mechanisms and the role of transport vesicles in cell physiology by addressing new, thematically-related questions concerning: 1) how the process of budding is coupled to packaging of essential membrane proteins, such as v-SNAREs and certain cargo receptors, mainly utilizing the recently achieved reconstitution of budding from synthetic lipid bilayers; 2) how distinct species of anterograde and retrograde-selective COPI vesicles can bud from the same Golgi membranes, utilizing the above approach and immuno-electron microscopy; 3) how transport signal peptides interact with their receptor, the gamma-COP subunit of coatomer, isolating domains by limited proteolysis and utilizing X-ray crystallography; and 4) how recently discovered Rim-Derived (RIDE)-vesicles bud off from Golgi stacks enclosing aggregates that are far too large for standard COPI vesicles, utilizing mainly cell-free budding reactions, protein purification, and immuno-electron microscopy. The process of vesicular transport is of basic importance to biology and medicine, underlying the compartmental organization of the cytoplasm and the pathways of exocrine, endocrine, and neurosecretion. Knowledge of the mechanism of action of gene products in these pathways, and their allelic variants, will likely be important for predictive medicine and impact upon management of common diseases such as cancer, diabetes, cardiovascular disease and diseases of the central nervous system.

structural biology; cell biology; biochemistry; biophysics; cyclin dependent kinase; tumor suppressor proteins; integrins; protein folding; biological signal transduction; cell adhesion; carcinogenesis; oligonucleotides; protein structure; molecular chaperones; cadherins;

[unreadable] DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are common traits across different neurodegenerative disorders, such as the polyglutamine expansion disorder Huntington's disease (HD). A recently emerging theme is that if mutant protein accumulation is eliminated, symptomatic progression not only halts but also recovers. For example, in an inducible model of Huntington's disease, loss of mutant protein accumulation in symptomatic animals led to complete reversion of the disease-like symptoms. Therefore, if we can accelerate the clearance of a disease-causing mutant protein, there exists the tantalizing possibility of recovery from disease. But how do cells clear these mutant proteins? And how does clearance of the mutant proteins lead to recovery from symptoms? To gain insight into these questions we have run Affymetrix gene arrays on stably transfected cell lines that carry mutant huntingtin protein. The comparison of the different genetic profiles revealed surprisingly robust changes in pathways indicating lysosome-mediated degradation and vesicular trafficking. These two areas are little explored in Huntington's disease and polyglutamine diseases in general, and is thus a rich source of questions. In this proposal we will therefore systematically test the following hypotheses: 1) Lysosome-mediated degradation has a significant impact on the degradation of mutant huntingtin proteins; and 2) Aggregation leads to reversible deficits in vesicular trafficking. Using a combination of biochemical and genetic techniques we also propose to identify regulators of protein aggregation and clearance using a functional cell-based assay: a stable cell line that conditionally expresses mutant proteins fused to variants ol GFP. In sum, during this grant period we will reveal targets that directly alter the level of mutant proteins in a cell, elucidate the basic degradation pathways crucial for handling these difficult proteins, and examine how deficits in protein degradation alters vesicular trafficking in the cell. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Recently, we established a novel membrane fusion system in which "flipped" v- and t-SNAREs needed in exocytosis are expressed on the surface of two cell populations, driving cell-cell fusion thereby demonstrating that SNAREs are sufficient to fuse biological membranes. Here, we propose to capitalize on this development to ask key mechanistic questions about SNARE-dependent fusion, especially questions concerning precisely how regulatory proteins - known to function physiologically - act alone and in concert to control excocytosis at the molecular level. Rigorous studies of these questions require a simplified system of this kind in which protein composition and topology can be controlled in a biologically-relevant environment so that the kinetic effect of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Regulatory proteins will be flipped by adding signal sequences and co-expressed with v- or t-SNAREs on the surface of cells, or added as pure recombinant proteins to the medium. Fusion kinetics and transition states will be measured using established techniques originally developed for viral fusion proteins. We will initially study a well-established group of proteins known to regulate exocytosis in whole cells and organisms: synaptotagmins, Sec/Munc proteins, complexins, and tomosyns, as well as NSF and SNAP. While their general physiologic importance is clear, the molecular mechanism of action - and functional interactions among themselves - are not clear due to the dearth of mechanistic studies in minimal functional fusion systems. The long-term vision is to work our way up - protein by protein - until we can reconstitute the basic properties and fine-tuning of regulated exocytosis. Imbalances in exocytosis and related processes underly major forms of diabetes and obesity, and are likely important in learning, mood, and inflammatory disorders. Knowledge of how the regulators work will likely identify novel targets for intervention.

[unreadable] DESCRIPTION (provided by applicant): The Columbia Center in the MLSCN will be differentiated on the basis of its strength and experience in cell biology, high content/high resolution automated cellular imaging and image analysis, and phenotypic assay design and implementation. Building on these existing strengths, our Center proposes a strategic focus on high throughput screening using phenotypic assays at the cellular and subcellular levels to identify bioactive compounds, which will enable us to meet or exceed the screening milestones mandated in the RFA within the scope of the allowed budget. Because of its strategic differentiation, the Columbia Center will perform uniquely within and to the benefit of the MLSCN network as a whole. Sister centers in the network which screen against defined molecular targets will dynamically interact with Columbia for secondary screening services. To facilitate this and to add value, the Columbia Center will draw on the assays it implements for referring investigators to create a "house repertoire" of biological assays. Profiling of hits against this repertoire of biology will provide important information on specificity at the biological level to complement information on the compound's selectivity at the protein/target level. This kind of information will be critical for the network to achieve best practice by focusing what will be limiting chemistry resources on the most promising hits. To accomplish the above goals in three years, the Columbia Center will be structured internally as a series of five functional components (assay implementation, HTS, probe development, informatics, management) each with defined goals, milestones and timelines. Each function will be led by a dedicated senior scientist, and the project will be coordinated by a dedicated project manager: The project is strongly supported by Columbia, which has already purchased a state-of-the-art highthroughput confocal cell imaging system (GE INCell3000) for the project, and will provide space (approx 5,000 nsf) adjacent to the PI's laboratory as well capital equipment needed to allow full automation of cellular screening at the Center. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are a common link across a wide array of neurodegenerative disorders. Recently, an exciting theme has emerged: if mutant protein accumulation is eliminated, symptomatic progression not only halts but also leads to recovery from disease. The first indication that neurodegenerative diseases are reversible comes from an inducible mouse model of the polyglutamine disorder Huntington's disease (HD). In the presence of expanded polyglutamine huntingtin, mice recapitulated HD-like symptoms. When mutant gene expression was abolished, not only did the aggregates disappear, the symptoms regressed. These findings signal that neurodegenerative diseases need no longer be considered a death sentence. Unfortunately, however, the pathway underlying clearance of these mutant proteins are not yet clear. We believe that screening a well-designed cell-based assay with a chemical compound library will allow us to not only further clarify which degradative pathway is important, but may also reveal new means by which these pathways can be activated. We have therefore designed a functional cell-based assay that monitors not only aggregation of mutant huntingtin protein, but also its clearance. To do so, we created a stable cell line that conditionally expresses the N-terminus of huntingtin protein with polyQ proteins of different polyQ lengths fused to variants of GFP. These cell lines permit high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells. We hypothesize that by eliminating the accumulated protein we will bring about recovery of the neurodegenerative process, as shown in several animal models of the disease. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Clearance of neurotransmitter from the synaptic cleft, by high affinity transport proteins (neurotransporters), is an important stage in the regulation of neuronal signalling. A major mechanism by which neurotransporters are regulated occurs at the level of subcellular localization. Internalization of neurotransporters away from the cell surface, into an intracellular pool reduces the number of available neurotransporter molecules at the synaptic cleft. Changes that affect the regulation of neurotransporters are important in the progression of a number of neurodegenerative diseases. Here we describe an assay that can be developed into a high throughput screen to identify small molecules (drugs) that affect neurotransporter trafficking. The assay measures changes in the subcellular localization of fluorescently tagged neurotransporters in response to stimuli such as protein kinase C (PKC) signaling. Screening chemical compounds against this assay has the potential to identify small molecule effectors that can be used as tools to pharmacologically manipulation of neurotransmitter clearance. This will aid research into pathological conditions caused by alterations of neurotransporter regulation. In addition, identification of such molecules may yield compounds that can be developed into new therapeutic drugs, or provide information about potential therapeutic targets. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Columbia Center in the MLSCN will be differentiated on the basis of its strength and experience in cell biology, high content/high resolution automated cellular imaging and image analysis, and phenotypic assay design and implementation. Building on these existing strengths, our Center proposes a strategic focus on high throughput screening using phenotypic assays at the cellular and subcellular levels to identify bioactive compounds, which will enable us to meet or exceed the screening milestones mandated in the RFA within the scope of the allowed budget. Because of its strategic differentiation, the Columbia Center will perform uniquely within and to the benefit of the MLSCN network as a whole. Sister centers in the network which screen against defined molecular targets will dynamically interact with Columbia for secondary screening services. To facilitate this and to add value, the Columbia Center will draw on the assays it implements for referring investigators to create a "house repertoire" of biological assays. Profiling of hits against this repertoire of biology will provide important information on specificity at the biological level to complement information on the compound's selectivity at the protein/target level. This kind of information will be critical for the network to achieve best practice by focusing what will be limiting chemistry resources on the most promising hits. To accomplish the above goals in three years, the Columbia Center will be structured internally as a series of five functional components (assay implementation, HTS, probe development, informatics, management) each with defined goals, milestones and timelines. Each function will be led by a dedicated senior scientist, and the project will be coordinated by a dedicated project manager: The project is strongly supported by Columbia, which has already purchased a state-of-the-art highthroughput confocal cell imaging system (GE INCell3000) for the project, and will provide space (approx 5,000 nsf) adjacent to the PI's laboratory as well capital equipment needed to allow full automation of cellular screening at the Center. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Recently, we established a novel membrane fusion system in which "flipped" v- and t-SNAREs needed in exocytosis are expressed on the surface of two cell populations, driving cell-cell fusion thereby demonstrating that SNAREs are sufficient to fuse biological membranes. Here, we propose to capitalize on this development to ask key mechanistic questions about SNARE-dependent fusion, especially questions concerning precisely how regulatory proteins - known to function physiologically - act alone and in concert to control excocytosis at the molecular level. Rigorous studies of these questions require a simplified system of this kind in which protein composition and topology can be controlled in a biologically-relevant environment so that the kinetic effect of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Regulatory proteins will be flipped by adding signal sequences and co-expressed with v- or t-SNAREs on the surface of cells, or added as pure recombinant proteins to the medium. Fusion kinetics and transition states will be measured using established techniques originally developed for viral fusion proteins. We will initially study a well-established group of proteins known to regulate exocytosis in whole cells and organisms: synaptotagmins, Sec/Munc proteins, complexins, and tomosyns, as well as NSF and SNAP. While their general physiologic importance is clear, the molecular mechanism of action - and functional interactions among themselves - are not clear due to the dearth of mechanistic studies in minimal functional fusion systems. The long-term vision is to work our way up - protein by protein - until we can reconstitute the basic properties and fine-tuning of regulated exocytosis. Imbalances in exocytosis and related processes underly major forms of diabetes and obesity, and are likely important in learning, mood, and inflammatory disorders. Knowledge of how the regulators work will likely identify novel targets for intervention.

DESCRIPTION (provided by applicant): It is the premise of this Challenge Grant that investigations of the dynamics of membrane structure and function would be tremendously facilitated by the development of robust methods for the manufacture of "suspended" bilayers that are surrounded on both sides by aqueous solutions. This application addresses Challenge Area 06-GM-104, Dynamics of membrane structure and function. Current technology consists of "supported" bilayers that are attached to a solid surface. They are inherently non-dynamic, as proteins they contain are generally immobile by nature, and an attached bilayer is not deformable. The proposed research is organized into a series of specific aims, each corresponding to different potential method(s) for generating functional supported bilayers, which we will explore in parallel, to see which one(s) meet the required criteria for a robust mezzanine platform using functional read-outs like lateral diffusion, binding reactions at the single molecule level, and membrane dynamics as exemplified by SNARE-dependent vesicle fusion. By attacking directly the single most critical technological limitation of mezzanine studies of the dynamics of membrane structure and function, and succeeding in producing planar unsupported bilayers containing functional proteins, we can meet the challenge outlined by NIGMS (Challenge Topic 06-GM-104), and jump start a great variety of insights from many laboratories in the field of membrane dynamics. This is a realistic goal within a two year frame, which will have a broad impact on membrane research, and which would not occur without the stimulus funding. PUBLIC HEALTH RELEVANCE: In the proposed project, we aim to generate freestanding (suspended) functional planar bi-layers for the study of membrane dynamics. Existing technology supported on solid surfaces, results in bi-layers where proteins are immobile and which cannot be deformed. By attacking this limitation, we can jump start a great variety of insights from many laboratories in the field of membrane dynamics.

DESCRIPTION (provided by applicant): The release of neurotransmitters at synapses is central to information processing in the nervous system. This process is altered in many psychiatric and neurological disorders, and the genes encoding the protein machinery have been repeatedly implicated in anxiety, depression, and schizophrenia in humans and in rodent models. Even though the responsible protein machinery has been known for some time, it is still not understood how the entry of calcium at nerve endings triggers release of neurotransmitters rapidly and synchronously to enable timely correlations of information in nervous systems. In the current project period [NOTE- this grant was at that time entitled Regulation of Exocytosis Studied with Flipped SNAREs and the title was changed in this application to more precisely reflect the current technical approaches.] we have obtained an X-ray crystal structure that appears to represent a pre-fusion intermediate in which the synaptic vesicle is docked and clamped by Complexin half-way thru the zippering process of the SNARE complex, based on correlative biochemical and genetic studies and functional reconstitution. These studies have suggested an innovative structural/biochemical working model that for the first time can explain in a concrete manner how synchronous release of transmitters occurs. In this competing renewal, we now propose to probe this model and its implications deeply, and as a result we expect to gain fundamental insights into synaptic transmission, whether the starting model is fully accurate or whether it needs to be modified as a result. The focus will be the exciting, and entirely unexpected, co-operative zig-zag array of half-zippered SNARE complexes revealed by the crystal structure in which a Complexin bound to one SNARE complex in a novel 'open' conformation inhibits zippering of a second SNARE complex in trans via its 'accessory' helix. We aim to establish the functional, compositional, and dynamic properties of the array as it occurs on lipid bilayers and at vesicle-bilayer junctions, and how the calcium sensor Synaptotagmin releases this clamped complex to trigger exocytosis, using mainly single event/single molecule optical techniques in completely defined systems and with genetic correlations in Drosophila to valid the physiological significance of key findings when possible. PUBLIC HEALTH RELEVANCE: The release of neurotransmitters at synapses is central to information processing and is altered in many psychiatric and neurological disorders. Even though the responsible protein machinery has been known for some time, it is still not understood how the entry of calcium at nerve endings triggers release of neurotransmitters rapidly and synchronously. We have developed and propose to test an innovative structural/biochemical working model that for the first time can explain in a concrete manner how synchronous release of transmitters occurs.

DESCRIPTION (provided by applicant): The cortical ER (cER) plays a critical role in many important physiological processes in the body, including in nerve and muscle. Until recently, cER seemed to be either present or absent, depending on the cell type. This has led to the traditional view that cER is a static specialization found in some but not most cells. Now a variety of studies, including work from our laboratory, profoundly challenge this view. They suggest that - to the contrary - many if not most animal cells have the inherent capacity to dynamically produce cER in copious quantities upon suitable physiologic demand. This implies the existence of broadly-distributed yet still unknown core machinery that triggers cER biogenesis and turnover. We have developed the first system that permits the controlled induction of cortical ER (cER) and propose here to utilize it to dissect the sub-cellular pathways and molecular mechanisms that produce this fascinating but still obscure organelle. The system entails controlled dimerization of ER membrane proteins bearing the C-terminal peptide from a cER protein, such as yeast Ist2p and mammalian STIM1, which triggers proliferation of cER containing this protein in various mammailian cells that normally have little or no cER. Discovering this machinery, the pathways it generates, and the molecular logic involved is the long-term goal of this research grant, and will have a major impact on fundamental concepts in cell biology and related areas of neurophysiology and muscle physiology, among others.

DESCRIPTION (provided by applicant): Research in the current grant period caused us to significantly revise our model of SNARE assembly, resulting in a new and much more specific hypothesis for the fusion mechanism. We now think assembly takes place in a series of well-defined discrete steps, which we call discrete zippering. The new data have come mainly from optical tweezers and biochemical experiments with the isolated proteins showing binary switch assembly behavior. Importantly, this discrete mechanism, unlike continuous zippering, creates discrete assembly intermediates which are natural pivot points for regulation. The discrete zippering concept is helpful as a guide to current research. Our main goal (Aims 1-3 and 5) is to rigorously test the new discrete zipper model to validate or modify it. We will do this by systematically studying the physical chemical, functional, and physiological effects of targeted mutations in each of the discrete portions of the SNARE complex: the N-terminal domain (NTD), C-terminal domain (CTD), linker domain (LD), and the trans-membrane domain (TMD). Many mutations in NTD and CTD are already known that affect fusion physiology in some way (less attention has been paid to TMD and LD) but it is not known how they work molecularly to affect SNARE assembly because sophisticated physical chemical assays have not in general been used before in this connection. Our comprehensive combined physical-chemical and functional mutational analysis of CTD, LD and TMD will provide essential information to advance our understanding of membrane fusion to the next level, whatever the model. Our other goal (Aim 4) is to better understand how the essential gene product Munc18 facilitates the discrete assembly of NTD, inherently a non- physiologically slow process. New data suggest that Munc18 can act as a molecular chaperone to promote the assembly of fusion-competent all-parallel SNARE helical bundles and prevent incompetent anti-parallel arrangements.

? DESCRIPTION (provided by applicant): This MIRA proposal consolidates funded research on three central problems in cell biology concerning membrane dynamics: synaptic vesicle exocytosis, dynamic membrane contacts, and synthetic biology-enabled tests of Golgi models. Synaptic vesicles fuse to release neurotransmitters in less than 1 msec after Ca++ enters, enabling effective information processing in the brain. In spite of all of the progress in our understanding of membrane fusion generally, it is remarkable that we have no clear explanation of this. Our goal for the next 5-10 years is to develop a detailed structural biochemical and biophysical understanding of how neurotransmitter release is coupled to Ca++ and how it can occur so very rapidly. Two related problems must be solved to provide an answer: how is the half-zippered SNARE complex stabilized (clamped) from completing fusion spontaneously? How can fusion be completed 100-1000 times faster than permitted by the physical chemistry of individual SNAREs? Our general hypothesis is that sub-millisecond exocytosis is achieved in some way by a supra-molecular assembly involving the Ca++ sensor synaptotagmin and the clamp/activator complexin that harnesses and synchronizes the force of many SNAREs co-operatively to enable explosive release. Membrane contacts generally are dynamic entities, forming and breaking as the result of signals and shifts in physiology. The Golgi stack is based on such membrane contacts, and despite the fact that it looks like a stable entity, our recent and surprising results reveal that the principal proteins responsible for registered stacking (GRASP proteins) cycle on and off the membrane every minute! Nanoscopy-based imaging of suggests this is due to rapid cycles of fatty acid (palmitate) addition and cleavage in specialized transien adhesive nano-domains. Over the next 5 years we hope to elucidate the mechanisms involved using real-time nanoscopy of the enzymes, substrates and their products in living cells combined with detailed biochemical, biophysical, and genetic studies. The Golgi apparatus is found in all eukaryotes, and yet has the dubious and remarkable distinction of being the last remaining membrane organelle whose basic principle of operation is still unknown! In spite of decades of intensive research we still debate how proteins traffic across the Golgi stack in animal cells, simply because we lack the ability to directly observe protein transfer reactions in living cells at sufficient spatial/temporal resolution. The new idea we are developing is to engineer the conversion of the animal Golgi stack into a more yeast-like unstacked topology in living cells, so that we can now much more easily track individual cisternae, vesicles and tubules in relation to transport cargo and machinery, especially with the help of dynamic nanoscopy now possible in our department.

Project Summary/Abstract How can virtually the same SNARE machine operate at dramatically different speeds depending on context, often far faster than a single SNAREpin? This is one of the central questions driving the field today, and the problem it embodies stands in boldest relief at the neuronal synapse, so it is here that we focus on the structures, biophysics, and physiological properties of the key protein machinery. Our overall hypothesis is that multiple SNAREpins released synchronously, each already close to the point of triggering fusion, co-operate to achieve fusion dramatically faster than any one alone. During the current period of support we discovered that the calcium sensor Synaptotagmin (normally anchored in synaptic vesicle) can self-assemble in vitro into Ca2+- sensitive, ring-like oligomers ~30 nm in diameter and have suggested that such rings forming between the synaptic vesicle (or insulin secretory vesicle) and the plasma membrane would prevent release until they are disrupted by Ca2+. Our specific hypothesis is that such ring oligomers of Synaptotagmin (Syt) are a central organizing principle for exocytosis, enabling the clamping and rapid synchronous release of multiple SNAREpins. This hypothesis is strongly supported by recent experiments in which a targeted mutation (F349A) that de-stabilizes Syt1 rings dramatically increases spontaneous and evoked release and in hippocampal neurons, and dramatically reduces the synchronicity of release with the action potential. We propose to 1) Test the hypothesis that ring-like oligomers of Synaptotagmins regulate exocytosis; 2) Test the hypothesis that Syt1 and Syt7 play distinct structural and functional roles in synchronous and asynchronous release from the same docked vesicles; 3) Elucidate the dynamics and topology of Munc13 and its proposed oligomers and the posited dual roles as vesicle tether and outer ring chaperone templating SNAREpins; and 4) Obtain by single particle cryo-EM and cryo EM tomography high resolution structures of functional release sites in vitro and in situ trapped in defined functional states. Similar machinery mediates neuroendocrine secretory physiology, including pancreatic insulin secretion, so we expect the answers will be highly relevant to the mission of NIDDK. Further, there is little doubt in the post-leptin era of the key role of the nervous system in metabolic balance and diseases.