Phyllis I. Hanson - US grants

The long-term goal of this project is to understand the molecular reactions that appear to be responsible for neuronal exocytosis, and more generally for membrane trafficking throughout eukaryotic cells. A number of proteins that participate in exocytosis have been identified, including the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. These three proteins are referred to collectively as SNAREs (soluble NSF-attachment protein receptors), and are representative of families of related proteins that function at different membrane trafficking steps throughout the cell. The three SNAREs bind tightly to each other to form a SNARE complex that is thought to interconnect membranes destined to fuse with each other. SNARE complexes are stable unless dissociated by the ATPase NSF (N-ethylmaleimide sensitive fusion protein), and cycle assembly and disassembly of SNARE complexes is correlated with membrane fusion in vivo. However, it remains unclear exactly how these reactions are linked to membrane fusion. In the proposed experiments, we will determine how the SNARE complex participates in membrane fusion by generating soluble SNARE complexes, delineating their structure, and characterizing their interactions with other critical complexes in solution will lead up to predictions about how such complexes affect membranes in vivo. We ill test these predications using 'model neuronal membranes' produced by heterologously expressing the synaptic SNAREs on secretory vesicles in the yeast Saccharomyces cerevisiae, isolating these vesicles, and studying their interactions using biochemical and ultrastructural approaches. With this information we will be better able to understand how intracellular membrane fusion, and in particular neurosecretion, can be modulated or abolished and how such alterations contribute to human disease and could be exploited to therapeutic advantage.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Early onset torsion dystonia is the most common and severe form of inherited dystonia. Many cases of this disease are associated with a single amino acid deletion in the protein torsinA. TorsinA is an AAA+ ATPase found in the endoplasmic reticulum, but its normal cellular function and role in the pathogenesis of dystonia are unknown. The goal of this project is to define cellular and molecular events controlled by torsinA in order to begin to understand the etiology of this elusive disease. In preliminary studies, we used torsinA mutants expressed in cultured cells and identified a likely function for torsinA in a subdomain of the ER, the nuclear envelope. Experiments in this proposal will explore how torsinA functions as an AAA+ ATPase and define its exact role in the nuclear envelope. [unreadable] Our specific aims are: [unreadable] 1. To develop and experimentally test a structural model for torsinA function, and then apply this to understanding how disease-linked mutations affect protein function. [unreadable] 2. To study torsinA dynamics and distribution in the endoplasmic reticulum and nuclear envelope to test the hypothesis that the nuclear envelope is its primary site of action. [unreadable] 3. To define the molecular targets of torsinA in the nuclear envelope. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The endosomal system internalizes membrane and protein from the plasma membrane, and then in a topologically distinct process internalizes membrane into itself to form multivesicular bodies (MVBs). Internalization of membrane and associated cargo into the MVB is essential for digestion and degradation in the lysosome, and is also used by specialized cells to generate exosomes. A set of eighteen "class E" proteins conserved from yeast to mammals is thought to regulate and probably drive the formation of intralumenal vesicles. Some or possibly all of these proteins are also used by nonlytic viruses such as HIV to bud from the plasma membrane of infected cells in a process that is topologically equivalent to budding into the lumen of the endosome. Elegant studies in yeast and more recently mammalian systems have lent insight into how these proteins associate with each other, cargo molecules, and the endosomal membrane, but essentially nothing is known about how these or other proteins drive membrane invagination and vesicle release. Because the required membrane curvature is opposite to that of traditional coated vesicle-driven endocytosis, it is likely that novel mechanisms are involved. We found using quick-freeze deep-etch electron microscopy that ESCRT-III proteins related to Snf7 (hSnf7/CHMP4 in mammalian systems) assemble into filaments on the plasma membrane that can be induced to form tight circular arrays and bend the membrane away from the cytoplasm, creating buds and eventually tubules on the cell surface. We hypothesize that similar ESCRT-III containing polymers normally create the neck of nascent endosomal vesicles, both confining the vesicle's contents and inducing the requisite deformation in the membrane. Our plan is to define the structure and dynamics of ESCRT-III polymers in vitro, on model membranes, and in the context of normal MVB biogenesis in order to determine how assembly and disassembly of ESCRT-III polymers participate in MVB biogenesis. Aim 1 will explore the structure of ESCRT-III homo- and hetero- polymers. Aim 2 will define mechanisms that regulate ESCRT-III polymer assembly. Aim 3 will study disassembly of ESCRT-III polymers by the AAA+ ATPase VPS4. Finally, Aim 4 will study endogenous ESCRT-III proteins in cells during the process of receptor downregulation before and after silencing expression of VPS4 and hSnf7 proteins. PUBLIC HEALTH RELEVANCE: These studies will illuminate cellular mechanisms involved in receptor downregulation, lipid homeostasis, and the release of many enveloped viruses including HIV from the cell. Mutations in two of the proteins to be studied are directly responsible for familial forms of frontotemporal dementia and early-onset cataracts. Insight into the pathophysiology of these processes requires the mechanistic understanding at which this work is aimed.

DESCRIPTION (provided by applicant): Early-onset (DYT1) torsion dystonia is a devastating non-degenerative neurological movement disorder caused by autosomal dominant inheritance of a glutamic acid deletion in the protein torsinA (TOR1A), frequently referred to as the ?GAG or ?E mutation because of the deleted codon or amino acid. The CNS abnormalities underlying dystonia are poorly understood, with functional imaging and clinical electrophysiology studies suggesting abnormalities in a range of structures throughout the motor circuit. More specific insight should come from understanding the responsible genetic change. TorsinA is a member of the AAA+ family of ATPases found in the lumen of the endoplasmic reticulum and nuclear envelope. It is expressed ubiquitously, and the known failure of ?E-mutant enzyme to rescue torsinA knock-out animals from perinatal lethality suggests that this mutant lacks whatever essential activity torsinA normally provides. However, the specific functions ascribed to torsinA vary widely and are not well defined despite the fact that it has been more than a decade since the protein was first described and linked to dystonia. This lack of insight is creating a major roadblock in efforts to develop targeted and effective treatments for dystonia. We propose a set of experiments aimed at clarifying the cellular function and disease-linked dysfunction of torsinA in cultured human cells. We will build on preliminary data showing that the distribution of torsinA within the endomembrane system is regulated and likely to play an important role in defining the enzyme's activity. The specific aims of the project are (1) to define the basis for association of torsinA with the endoplasmic reticulum membrane and its distribution and retention in this organelle, (2) to delineate the mechanism by which an interacting protein LULL1 (TOR1IP2) controls the distribution of torsinA between the endoplasmic reticulum and nuclear envelope, (3) to explore the effects of torsinA on known substrates using cellular and biochemical assays, and (4) to determine how disease-associated mutations affect torsinA structure and function. These studies are broadly relevant because they address potentially novel means of regulating the localization of proteins within cells, while also providing insight into the etiology of DYT1 dystonia.

? DESCRIPTION (provided by applicant): HIV and AIDS remains a persistent problem in the US and around the world. One reason for the lack of better drugs to prevent HIV infection is insufficient understanding of how the virus is released from infected host cells. HIV and other retroviruses depend on host cell factors known as endosomal sorting complex required for trafficking (ESCRT) machinery for the critical step of membrane scission necessary for budding and release. The virally encoded Gag protein assembles on plasma membranes and produces the necessary curvature to package the viral genome. Gag also has specific motifs to bind to ESCRT-I and/or Alix, which can recruit ESCRT-III proteins. ESCRT-III proteins assemble into filaments on membranes to facilitate scission and release virus. However, the molecular mechanisms responsible for ESCRT-III recruitment and activation to release assembled virions remain unclear. In this proposal, we will use deep-etch electron microscopy (EM) in combination with correlative light microscopy to study the relationship between HIV Gag and the cellular ESCRT machinery. Deep-etch EM is a powerful technique for visualizing cellular membranes and membrane surface proteins and can provide nm resolution views of the protein machinery involved in viral particle assembly. We will apply this to (i) define the molecular architecture of connections between HIV Gag and ESCRTs stabilized by the absence of Vps4 and (ii) together with correlative light microscopy extend these studies to examine transient ESCRT structures present during normal viral particle assembly. Results from these experiments will move understanding of HIV and other retrovirus budding forward, paving the way for developing new strategies to intervene in HIV infection.

The endolysosomal network is the portal by which extracellular material enters the cell. As such, the membranes of the endosomes, phagosomes, and lysosomes that comprise this network face challenges from pathogens and other internalized materials as well as from metabolic and chemical stresses. Consequences of damage vary according to the specific compartment and degree of damage, but extensive lysosomal membrane permeabilization triggers cell death while limited disruption of endosomes and phagosomes by particulate material and pathogens leads to inflammasome activation and ensuing cytokine responses. A widely deployed strategy for removing damaged organelles involves the use of selective autophagy, referred to as lysophagy. Removal is, however, unnecessary if organelles are instead repaired. We recently discovered a new role for the ESCRT (endosomal sorting complex required for transport) machinery in responding to nano- scale disruptions in endolysosomal membranes and promoting their repair. In this proposal, we will build on this discovery and test the hypothesis that ESCRTs (and in particular ESCRT-III proteins) act as a dynamic membrane stabilizing system to protect vulnerable membranes across the endolysosomal network and beyond. Two aims will exploit and explore responses to two experimentally tractable and sterile endolysosomal disruptants that potently engage the ESCRT machinery. In Aim 1, we will determine how the ESCRT machinery recognizes and counteracts lysosomal membrane stress induced by L-leucyl-L-leucine methyl ester (LLOMe). This will involve characterizing the membrane stress responsible for engaging ESCRTs, defining the molecular pathway(s) involved and identifying ?keystone? ESCRT-III proteins, delineating the molecular features required for repair, and identifying pathways that trigger this stabilizing response. In Aim 2, we will examine how ESCRTs respond to and repair silica induced membrane damage in epithelial and phagocytic cells. This will include testing a role for Fe2+-dependent lipid peroxidation in engaging ESCRTs, imaging the relative role and dynamics of ESCRT components on phagosomal membranes, and testing the hypothesis that ESCRTs limit endolysosomal damage in phagocytic cells and thereby dampen inflammation associated with the many things that transit through these pathways. The insights gained from this work will be applicable to understanding how ESCRTs sense and respond to a broad range of membrane stresses.

ABSTRACT Amyloid-beta (A?)?the principal component of amyloid plaques in Alzheimer?s disease (AD)?is generated by sequential secretase cleavages of the amyloid precursor protein (APP), a type 1 transmembrane protein. Despite three decades of study, the precise subcellular locations of A? generation have remained elusive. This is important because not only is A? central to AD pathogenesis but reducing A? levels has been a major focus in the development of potential AD therapeutics. Determing the precise cellular locations of A? generation has been a major bottleneck because current model systems are incapable of tracking A? peptide in living cells. We have recently developed a novel APP construct which incorporates an unnatural amino acid in the extracellular A? segment of APP, enabling click-chemistry to attach a small molecule fluorophore to A? at the plasma membrane. Coincident attachment of distinct fluorescent proteins to intra- and extracellular regions of APP allows the real-time visualization of APP trafficking and A? generation in living cells. In this application we will exploit this model system to determine the impact of retromer sorting on APP trafficking and A? generation in engineered neural cell lines (Aim 1) and to determine the temporal-spatial dynamics of APP trafficking and A? generation within primary neurons (Aim 2). At the completion this grant, we will have a basic understanding of the itineraries of APP that lead to A? generation. We will also be able to examine these same pathways in primary neurons (or even iPSCs), and study how these A?-generating pathways may be altered by genetic variants or neuronal activity.

ABSTRACT The property of sensing and propagating external cues that drive directional migration is a fundamental property of biological systems, and is essential to physiological and pathological processes including embryogenesis, adult tissue homeostasis, inflammation and immune responses, and metastatic invasion. This proposal aims at understanding how chemotactic signals are packaged and propagated between neighboring cells during chemotaxis. To do so, we study human neutrophils, the most abundant leukocytes in normal human blood. When exposed to primary chemoattractants like N-formyl-Met-Leu-Phe (fMLF), which is secreted by pathogens invading the body and by necrotic cells at sites of injury, neutrophils rapidly undergo polarization that allows them to efficiently migrate up the fMLF gradient. As they react to fMLF, neutrophils secrete secondary chemoattractants that serve to maintain the robustness and sensitivity to the primary chemoattractant signals. We established that the secondary chemoattractant leukotriene B4 (LTB4) is required for the massive recruitment of neutrophils to sites of injury in vitro and in vivo. In order for LTB4 to act as a bona fide signal relay molecule, it must be released in a form that enables the generation of a stable gradient during chemotaxis. In this context, we established that LTB4 is packaged in vesicles in chemotaxing neutrophils as a way to effectively disseminate gradients between neighboring cells. We found that LTB4 and its synthesizing enzymes ? 5-lipoxigenase (5-LO) and 5-LO activating protein (FLAP) - localize to intracellular multivesicular bodies (MBVs) which, upon chemoattractant stimulation, release their content as exosomes, thereby acting as a packaging mechanism to relay chemotactic signals. Further, we found that MVB biogenesis appears to be initiated at the nuclear envelope (NE) in activated neutrophils. We hypothesize that the NE is a novel site of MVB formation that enables packaging of the LTB4 synthetic machineryinto secretory MVBs that release exosomes to relay of signals during neutrophil chemotaxis. To test this hypothesis, in Aim 1 we will directly visualize 5-LO and FLAP dynamics in live cells using mCherry/GFP fusions and photoactivatable reporters under normal conditions and when endocytosis is blocked. We will also assess the role of FLAP clustering as a driving force for MVB biogenesis at the NE, by generating FLAP mutants with distinct affinities for the 5-LO substrate arachidonic acid. Since integral membrane proteins clustering is considered a hallmark of ordered membrane microdomains, in Aim 2 we will define the role of nuclear lipid micro-domains in MVB biogenesis. Finally, in Aim 3 we will establish the role of membrane remodeling complexes in the formation of the nuclear MVBs by assessing the role of ESCRTs in this process and identify accessory proteins involved in NE remodeling. This project is poised to provide much needed insight into the mechanisms regulating the genesis of chemotactic signals during neutrophil chemotaxis and will bring unprecedented knowledge into the role of the NE in the biogenesis of MVBs and in the interplay between lipid- and ESCRT-dependent pathways in their biogenesis.