James A. McNew - US grants

Nerve cells, or neurons, communicate and relay signals by the regulated release of chemicals called neurotransmitters. This extremely complex process is tightly regulated and is required for all movement as well as higher brain functions such as learning and memory. One method to study such a complicated process is to carefully dissect the problem into smaller units and gain a better understanding of the component parts. The final step in the release of neurotransmitter is the merger of the membrane bound sac that contains these chemicals with the cell membrane, the barrier that separates the nerve cell from its environment. The consequence of this membrane fusion event is the release of neurotransmitter to the space between neurons, resulting in communication between these cells. We seek to understand the structural and functional basis of this membrane fusion event. The process of membrane fusion can be further subdivided into discrete steps. Many of the molecular components that drive the fusion process have been identified, but the precise way they interact to perform their role is not well understood. The objectives of this work are to identify the specific interactions that determine the spatial and temporal coordination of neurotransmission in a defined system. We will develop a synthetic fusion system to model neurosecretion using only the minimal components, specifically pure phospholipids and fusion proteins from the fruitfly Drosophila melanogaster. Neuronal fusion proteins, called SNAREs, will be produced in bacteria and incorporated into synthetic phospholipid vesicles. A detailed molecular analysis of the protein-protein interaction affect SNARE function will be studied. In addition, the role of regulatory factors will be analyzed. This assay will provide a unique window into the way in which cells fuse biological membranes. The ability to study these proteins in their native context of a phospholipid bilayer will be a major step toward a better understanding of their critical physiological role.

0321275
Raphael
The ability to label individual molecules inside of living cells with fluorescent probes has revolutionized our understanding of basic cell biology and opened new opportunities to bioengineer living cells. Laser scanning confocal microscopy has proved to be a powerful and versatile technique for studying fluorescent molecules in cells. Rice University recognized the importance of this technique, and purchased a Zeiss LSM 410 confocal microscope in 1993. This microscope has supported numerous research projects in cell biology and bioengineering. However, there are serious limitations in the current technology, including limited ability to resolve molecules with overlapping emission spectra and the damage to cells that occurs with laser excitation. The importance and magnitude of these issues led Carl Zeiss, Inc. to expend a significant amount of time and resources to develop a user-friendly system that can overcome these problems. Zeiss recently integrated powerful new technologies, including novel algorithms for resolving overlapping emission spectra and the ability to use multi-photon laser excitation, into their new confocal microscope. The new system is called the 510 LSM META/NLO.

Membrane fusion is a fundamental process involved in diverse cellular events such as fertilization and neurosecretion. Biological membrane fusion relies on proteins to drive membrane merger and is likely facilitated by specific lipid geometries in vivo. A family of integral membrane proteins collectively known as SNAREs mediates the fusion of intracellular transport vesicles. While SNAREs pair in specific ways to provide the mechanical energy to drive fusion, the delicate interplay of regulatory elements that orchestrate this event in space and time remain elusive. We use a combination of in vitro fusion assays with purified yeast SNARE proteins and lipids, yeast genetics, and cell biology to examine mechanistic details of membrane fusion during exocytosis. SNAREs and regulatory proteins will be manipulated in vivo and their specific effects on membrane fusion can be directly analyzed in vitro. A molecular appreciation of the dynamic protein-protein interactions that execute and regulate membrane fusion during secretion could potentially lead to important therapeutic targets. We begin by examining the regulatory role of Seclp in yeast exocytosis. Seclp binds primarily to the yeast t-SNARE complex and directly stimulates membrane fusion. We will investigatehow Seclp binds to the t-SNARE complex and fully assembled ternary SNARE complex and the functional consequences of this binding. We will also determine the mechanistic basis for Seclp stimulation by comparing neuronal Seel (n-Secl) with its yeast counterpart Seclp. Next, we will dissect plasma membrane t-SNARE complex function in vitro and in vivo. The Ssolp N-terminal regulatory domain (NRD) is dispensable in vitro but required in vivo. We will determine the function of the Ssolp N-terminal regulatory domain testing the hypothesis that the Ssolp NRD serves a chaperone function for the Ssolp core H3 domain. Additionally, we examine the function of the Ssolp polybasic juxtamembrane region, a conserved sequence in all plasma membrane SNAREs. Third, we analyze membrane fusion driven by the sporulation specific t-SNARE light chain Spo20p, which requires the addition of phosphatidic acid to the bilayer for efficient fusion. We explore the mechanistic basis for the difference between the t-SNARE light chains Sec9p and Spo20p by examining lipid requirements and structural stability of each t-SNARE complex. Finally, we compare SNARE-mediated fusion with purified organelles and synthetic liposomes. Purified secretory vesicles and inverted plasma membrane vesicles will be used with existing synthetic proteoliposomes to study fusion with native membranes in an effort to reveal differences in fusion with SNARE mutants in vitro and in vivo.

DESCRIPTION (provided by applicant): Membrane fusion is vitally important for many aspects of eukaryotic cell biology including vesicular traffic within the secretory pathway as well as the biogenesis and maintenance of the entire endomembrane system. Membrane fusion also provides cytoplasmic organelles like mitochondria and the endoplasmic reticulum the ability to change shape and size to perform their required function. This proposal examines the role of the GTPase atlastin in homotypic ER fusion. Atlastin (SPG3A) is a member of a larger family of genes that are responsible for a group of inherited neurological disorders called Hereditary Spastic Paraplegias (HSP). Mutations in atlastin-1 account for ~10% of autosomal dominant forms of HSP. A fundamental understanding of atlastin's role in generating and maintaining ER function by homotypic ER fusion will significantly inform the mechanistic basis of ER-associated pathologies such as the neuronal degeneration found in HSP. Atlastin utilizes the chemical energy of GTP hydrolysis to do work on the phospholipid bilayer. This novel mechanism of membrane fusion is unlike any known fusion protein and establishes a new paradigm. Recent structural work has allowed us to develop a detailed model of atlastin function. We will test important predictions of this model using recombinant proteins, in vitro fusion reactions, measurement of GTPase activity, and determination of oligomeric state. We will probe the specific protein requirements for membrane tethering through the conserved GTPase domain, stable membrane attachment provided by a three helical bundle segment that connects the GTPase domain to transmembrane anchors, and membrane destabilization by an amphipathic helix in the C-terminal cytoplasmic tail. This work will contribute to two very important areas of research, the pathophysiology of Hereditary Spastic Paraplegia and the general mechanism of membrane fusion. Molecular genetic analysis of the most prominent forms of HSP, including atlastin, identified proteins that are generally involved in ER function. Proper functioning of the ER is critical for all cells given the crucial activities this organelle provides with respect to te secretory apparatus, lipid biogenesis, and calcium homeostasis. Recent data suggest that maintenance of a reticular morphology is necessary for ER function and atlastin, in part, provides for the ability to change shape and maintain lumen continuity. Characterization of this new way to merge membranes will be important for understanding the biophysical mechanisms of ER homotypic fusion and ER homeostasis in general.

Project Summary Degeneration of neurons or muscle are observed in several human pathologies, including Alzheimer's, Parkinson's and the hereditary spastic paraplegias (HSP) for neuronal degeneration, and disuse atrophy, cancer cachexia, and sepsis for muscle degeneration. Despite many recent advances, the molecular mechanism(s) underlying these degenerative processes remain incompletely understood. To generate such mechanistic insights, the PIs have recently established a Drosophila model for the HSPs. The specific focus is in atlastin (atl, Spastic Paraplegia Gene 3A), which encodes an ER fusion protein. Based on the previous observation that atl knockdown in neurons causes progressive, age-dependent locomotor deficits, the we asked if this knockdown also caused progressive cellular degeneration. Investigations into the adult thoracic musculature revealed that atl loss from either neuron or muscle caused progressive degeneration associated with a number of other pathologies including accumulation of aggregates containing ubiquitin, increased reactive oxygen species (ROS), and activation of the JNK/Foxo stress response pathway. Administering the drug rapamycin, which inhibits the Tor kinase, or decreasing Tor gene dosage reversed many of these pathologies at least partially, indicating that atl loss might activate muscle Tor. Muscle Tor and Foxo activation have also been observed in denervation-induced muscle atrophy. In this application, experiments are proposed to elucidate the mechanisms by which atl loss causes progressive muscle pathologies. Aim #1 will test the hypothesis that muscle Tor is activated by atl loss, determine if Tor activity is sufficient as well as necessary for atl loss phenotypes, and test the prediction that Tor activity promotes muscle degeneration by inhibiting autophagy. Aim #2 will examine the causal relationship between activated Tor and increased ROS, and between ROS and the JNK/Foxo stress pathway. In particular, we will test two non-mutually exclusive hypotheses explaining Foxo activation; first, that activated Tor increases ROS, which in turn is responsible for JNK activation, and finally Foxo activation, and second, that activated Tor activates its target S6K, which in turn down-regulates insulin signaling, thus decreasing activity of the Foxo inhibitor Akt. Aim #3 will test the hypothesis that neuronal atl loss activates muscle Tor by attenuating glutamatergic neuromuscular transmission. In particular, it will be determined if deletion of one glutamate receptor, previously shown to be sufficient to activate muscle Tor, will cause similar muscle pathologies as is observed by neuronal atl knockdown. In addition, it will be determined if neuronal atl loss confers neuronal phenotypes similar to those conferred by glutamate receptor deletion. Successful completion of these experiments will provide novel and critical mechanistic insights linking defective synaptic input conferred by atl loss to muscle degeneration. The PIs anticipate that these experiments will also provide mechanistic insights applicable to neuronal degeneration as well, which will give these experiments a broad medical relevance.

Genetic or pharmacological interventions that decrease synaptic transmission often induce a signal produced in the postsynaptic cell that acts in a retrograde manner to increase transmitter release from the presynaptic cell. At some synapses, generation of this signal requires an increased concentration of the phospholipid phosphatidic acid (PA) in the postsynaptic cell, which then activates the ?Target of rapamycin? (Tor) kinase. Tor activation, in turn, increases translation of mRNAs encoding the retrograde signal. Although increased protein synthesis is the most prominent Tor output, Tor also inhibits autophagy by phosphorylating and inhibiting several Atg proteins; this autophagy impairment might explain the association of activated Tor with a number of neurodegenerative and muscle degenerative disorders. The Hereditary Spastic Paraplegias (HSPs) represent one family of neurodegenerative disorders caused by mutations in any of over 70 different genes. We recently developed a Drosophila model for the HSP caused by mutations in atlastin (atl, SPG3A), which encodes an ER fusion GTPase. Using this model, we found that neuronal atl loss both decreased evoked transmitter release at the larval neuromuscular junction and caused progressive muscle degeneration. Because this degeneration, as well as associated locomotor and muscle pathologies, was partially suppressed by either decreasing Tor gene dosage or by administering the Tor inhibitor rapamycin, we hypothesize that neuronal atl loss activates muscle Tor. In this proposal, we will determine the role of [PA] in mediating the muscle degeneration and related pathologies caused by atl loss. In aim #1 we use mass spectrometry and an in vivo fluorescent PA reporter to test the hypothesis that neuronal atl loss increases muscle [PA]. In aim #2, we will determine the functional role of altered muscle [PA] in muscle degeneration. In particular, we will adjust muscle [PA] levels by overexpressing or introducing mutations in genes encoding PA-metabolizing enzymes including PLD, DAG Kinase and PA phosphatase. We hypothesize that increasing muscle [PA] will be sufficient to cause muscle degeneration whereas decreasing muscle [PA] will prevent muscle degeneration caused by neuronal atl loss. If we find, as expected that, increasing muscle [PA] is sufficient to cause muscle degeneration, we will then determine if this degeneration is dependent on Tor. In aim #3 we will investigate the Tor dependent retrograde signaling pathway that occurs at the Drosophila larval neuromuscular junction. This pathway is triggered by deletion of gluRIIA, one of the two alternative subunits of post-synaptic muscle glutamate receptors. We will determine if muscle PA is both necessary and sufficient to generate this retrograde signal. If successful, these studies will establish a link between impaired synaptic transmission and postsynaptic cell degeneration and elucidate the role of postsynaptic PA in this process. We anticipate that these studies will provide critical mechanistic insights into the HSPs as well as in the process of degeneration in diseases such as Alzheimer?s with great clinical importance.