Molecular Mechanisms Orchestrating the Dynamics of Secretory Vesicle Pools.

The secretion of chemical messengers via Ca2+-dependent exocytosis of vesicles is fundamental to a wide-range of physiological events. Rab GTPases and SNARE proteins govern the temporal and spatial precision of transmitter release. Yet, little is known about their role in specifying the size and filling kinetics of functionally defined vesicle pools, which impact the strength and efficiency of exocytosis. We first sought to delineate the distinct vs. overlapping roles of highly homologous Rab GTPase proteins, Rab3 and Rab27, which display high sequence homology, share protein-effectors, and may functionally compensate. To define their actions, we overexpressed Rab3GAP and/or EPI64A GTPase-activating protein in wild-type or Rab27-null cells to transit the Rab3 family or Rab27A to a GDP-bound inactive state. We found Rab27A is essential for generation of the functionally defined immediately releasable pool, Rab3 is essential for a kinetically rapid filling of the RRP, and both cooperate in populating the readily releasable granule pool (RRP). We conclude that while Rab3 and Rab27A cooperate to generate release-ready vesicles in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway. We also investigated how the SNARE Tomosyn1 (Tomo1) regulates the partitioning of synaptic vesicle (SV) pools in hippocampal neurons. Tomo1 inhibits SV priming at the plasma membrane. Yet, its localization to SVs and cytosol uniquely positions it to coordinate SV pool partitioning. We that find that Tomo1 controls SV transition between the Resting Pool and Total Recycling Pool (TRP), and modulates the RRP size. Tomo1’s regulation of SV distribution between pools is sensitive to neural activity and requires Cdk5. We provide novel evidence for an interaction between Tomo1 and Rab3A-GTP, and through this with Synapsin1 proteins, known regulators of SV recruitment. In addition, Tomo1 regulatory control over the TRP occurred independent of its C-terminal SNARE domain. Hence, Tomo1 actions on neurotransmission extend beyond its known inhibition of SV priming into the RRP and may involve other effector proteins. Altogether, our results advance the understanding of how Rab and Tomosyn proteins coordinate steps of the vesicle cycle that lead to functional heterogeneity among vesicles and thus may determine modes of transmitter release.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116755/1/vcazares_1.pd

Tomosyn-1 is a Novel Molecular Target of the Ubiquitin-Proteasome System and Underlies Synaptic Architecture

The efficacy of information transfer at synaptic contacts between excitatory central neurons undergoes continual modification in response to neuronal activity and physiological state. This plasticity in synaptic transmission may involve changes in presynaptic release probability, postsynaptic receptor number and sensitivity, and/or synaptic morphology. The molecular mechanisms influencing these distinctive targets are an investigative focus given their importance in learning, memory, and cognitive function. Much attention has focused on transcriptional and translational regulation of the synapse, but post-translational modification and directed turnover of specific protein components is also recognized as critical. Central to targeted protein degradation is the ubiquitin-proteasome system (UPS). While an increasing number of synaptic proteins are known to be susceptible to activity-dependent regulation by the UPS, relatively little has focused on the action of the UPS on known negative regulators of synaptic function. The SNARE protein Tomosyn-1 (Tomo-1) directly inhibits evoked release at central synapses, but it is also present post-synaptically, where no known function has been identified. It was recently discovered that the related Tomosyn-2 protein is subject to ubiquitination and degradation in neuroendocrine pancreatic beta cells, suggesting their secretory activity may be under control of the UPS. The general hypothesis of this dissertation is that a central mechanism underlying modulation of the synapse is the targeted degradation of Tomo-1. This dissertation made use of a series of complementary biochemical, molecular, and imaging technologies in hippocampal neuronal culture. We demonstrate that Tomo- 1 protein level, independently of its SNARE domain, positively correlates with postsynaptic dendritic spine density in vivo. The data also indicate that the UPS regulates steady-state Tomo-1 level and function. Immunoprecipitated Tomo-1 was ubiquitinated and co-precipitated the E3 ligase HRD1, and both effects dramatically increased upon proteasome inhibition. The interaction was also found in situ, via fixed- cell proximity ligation assay. In vitro reactions indicated direct, HRD1 concentration- dependent Tomo-1 ubiquitination. Furthermore, we demonstrated that neuronal HRD1 knockdown increased Tomo-1 level, and consequently, dendritic spine density. This effect was abrogated by concurrent knockdown of Tomo-1, strongly suggesting a direct HRD1/Tomo-1 effector relationship. We confirmed Tomo-1 is a UPS substrate by identifying 12 lysine residues which are ubiquitinated by HRD1 and generated a non- ubiquitinateable Tomo-1 mutant. Finally, we performed Tomo-1 isoform and homologue comparisons, protein structure modeling, and antibody-based domain targeting of Tomo-1 in neuronal lysates to identify four lysine residues which are highly likely to be ubiquitinated in vivo. In summary, the results of this dissertation indicate that the UPS participates in tuning synaptic efficacy via the precise regulation of neuronal Tomo-1 and spine density. These findings implicate Tomo-1 as a prime target of UPS mediated degradation in the implementation of morphological plasticity in central neurons.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143947/1/jsaldate_1.pd

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

A Comparative Study of Sensory Neuron Synaptic Activity and the Role of Presynaptic Diversity, Specificity, and Regulation in Caenorhabitis Elegans

Chemical synapses are complex structures that have a diversity of specific activities that shape nervous system computation. To understand how this diversity contributes to specific circuit functions, we sought to characterize synaptic release in the stereotyped and defined neural circuitry of C. elegans. Here we use pHluorin imaging (Vglut-­pH) to monitor presynaptic glutamate release from single chemosensory neurons in intact animals and characterize the dynamics of endo-­ and exocytosis from two types of glutamatergic neurons, AWCON and ASH. In Chapter 1, we describe the optimization of Vglut-­pH, we introduce a reagent for measuring synaptic calcium influx by tethering GCaMP to synaptic vesicles, and we provide our initial characterization of glutamate release in AWCON and ASH. Our results indicate that AWCON and ASH have distinct exocytosis dynamics and that AWCON exhibits synaptic release properties similar to vertebrate photoreceptors and retinal bipolar neurons, aligning with previous results from functional calcium imaging, gene expression, and circuitry. In Chapter 2, we characterize the dynamics of endocytosis in AWCON and ASH. We find that synaptic vesicle endocytosis in these neurons have kinetic features and timescales similar to that of mammalian neurons. We show that endocytosis appears to be homeostatically regulated by previous neuronal activity and is composed of at least two kinetically distinct modes, fast and slow. We show that fast retrieval is dependent on the clathrin adaptor protein AP180/CALM, suggesting that clathrin-­mediated endocytosis is important for synaptic vesicle retrieval in these sensory neurons. In Chapter 3, we compare and contrast the dependences of AWCON and ASH Vglut-­pH responses on the core synaptic vesicle release machinery and its regulators: syntaxin, synaptobrevin, SNAP-­25, unc-­13, unc-­18, RIM, complexin, and tomosyn. We find that Vglut-­pH responses are highly dependent on the core components of the SNARE complex and its regulators, but we detected significant differences in the residual responses in these mutants that suggest AWCON and ASH synapses are distinct from each other and from those of the neuromuscular junction. We find that complexin appears to act as an inhibitor of SV release in AWCON and ASH, and we find an unexpected role for tomosyn in regulating calcium influx. In Chapter 4, we describe activity-­dependent cytoplasmic pH changes in AWCON and ASH, and we conduct a series of experiments to show that these pH changes do not interfere with the measurement or interpretation Vglut-­pH signals. Our results indicate that these activity-­dependent pH changes are consistent with a depolarization-­generated acidosis and are correlated with calcium influx. We show that these pH changes in response to stimulation are not dependent on unc-­13 or unc-­18 and are thus are largely independent of synaptic release. Finally, we show that in contrast to intracellular pH, extracellular pH changes are not detected in response to sensory stimulation. In Chapter 5, we investigate the role of synaptotagmins in AWCON and ASH glutamate release using Vglut-­pH imaging. We find that AWCON basal release is highly dependent on snt-­1, whereas ASH exocytosis is intact in snt-­1 null mutants and slightly diminished in snt-­6 mutants. Our results indicate that AWC and ASH synapses have distinct requirements for snt-­1 and may use a combination of calcium sensors to mediate glutamate release. In Chapter 6, we use a combination of genetics, behavior, calcium imaging, and Vglut-­pH imaging to investigate how loss-­of-­function mutations in pkc-­1 (protein kinase C epsilon) modulate AWCON butanone olfactory preference. We find that pkc-­1 functions in AWCON downstream of presynaptic calcium influx to modulate eat-­4 dependent glutamate release and a second form of AWCON output that is important for specifying butanone olfactory preference. We identify the receptor-­type guanylate cyclase gcy-­28 and Gqα as additional important regulators of AWCON synaptic release, and identify unc-­31 (CAPS) as an additional genetic determinant of butanone olfactory preference. Finally, we suggest a model for a dual function Gqα/DAG/pkc-­1 signaling pathway that regulates synaptic vesicle release and butanone preference in AWCON. Our work in this thesis extends the characterization of C. elegans synapses from the neuromuscular junction to the presynaptic terminals of central synapses and supports a role for presynaptic diversity among distinct neuronal cell types in C. elegans. Our work emphasizes that presynaptic diversity and regulation of neurotransmitter release are important components to specifying circuit function and suggest that C. elegans will provide a deeper understanding of how presynaptic diversity, both in terms of molecular components and activity dynamics, contribute to nervous system function

Platelet Secretion and Hemostasis Require Syntaxin-binding Protein STXBP5

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion

Exploring mechanisms of insulin secretion regulators using C. elegans

Diabetes mellitus is a group of disorders characterized by disrupted glucose homeostasis. Diabetes is one of the most dangerous diseases worldwide since it affects currently more than 500 million people. The pathogenesis of the disease is associated with the insufficient production of insulin and is characterized by increased blood glucose levels. Insulin secretion takes place in pancreatic β-cells in the response to elevated glucose levels and is regulated by various factors. This thesis is aimed to understand the functions of three proteins and characterize their novel roles in the regulation of insulin signaling and secretion. The first study showed the role of ENPL-1 in the positive regulation of insulin secretion. Loss of enpl-1 resulted in reduced insulin signaling and inhibited insulin secretion. Furthermore, we identified proinsulin as a novel client protein of ENPL-1 and showed that ENPL-1 was required for its maturation. The next study was based on the previous findings showing that ASNA-1 is a positive regulator of insulin secretion. Our study showed that ASNA-1 is present in two redox states, oxidized and reduced and that the multiple functions of ASNA-1 are dependent on its redox states. Our analysis showed, that forcing ASNA-1 into the oxidized state, reduced its function of inserting tail-anchored proteins into the endoplasmic reticulum, without affecting the insulin secretion function. In the next study, we focused on the mutual role of both previously mentioned proteins. We identified the interaction of ASNA-1 and ENPL-1 and showed that proinsulin is required for this interaction to take place. Our study indicated that oxidized ASNA-1 rather than the reduced form was likely interacting with ENPL-1. In the last study, we focused on the role of a third protein, SMN-1, and its impact on the regulation of insulin secretion. Our analysis showed that loss of SMN-1 resulted in neuropeptide secretion defect and caused redistribution of insulin from its original place. In summary, we characterized the functions of three proteins and indicated their importance in the regulation of insulin secretion processes

The Nuts and Bolts of the Platelet Release Reaction

Secretion is essential to many of the roles that platelets play in the vasculature, e.g., thrombosis, angiogenesis, and inflammation, enabling platelets to modulate the microenvironment at sites of vascular lesions with a myriad of bioactive molecules stored in their granules. Past studies demonstrate that granule cargo release is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins, which are required for granule-plasma membrane fusion. Several SNARE regulators, which control when, where, and how the SNAREs interact, have been identified in platelets. Additionally, platelet SNAREs are controlled by post-translational modifications, e.g., phosphorylation and acylation. Although there have been many recent insights into the mechanisms of platelet secretion, many questions remain: have we identified all the important regulators, does calcium directly control the process, and is platelet secretion polarized. In this review, we focus on the mechanics of platelet secretion and discuss how the secretory machinery functions in the pathway leading to membrane fusion and cargo release

FUNCTIONAL ROLES FOR POST-TRANSLATIONAL MODIFICATIONS OF t-SNARES IN PLATELETS

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo

DOC2B acts as a calcium switch and enhances vesicle fusion

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PMwas supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release. Copyright © 2008 Society for Neuroscience