Publications by authors named "Alexander Vasin"

9 Publications

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The Accessory Helix of Complexin Stabilizes a Partially Unzippered State of the SNARE Complex and Mediates the Complexin Clamping Function .

eNeuro 2021 Mar-Apr;8(2). Epub 2021 Apr 7.

Ophthalmology, Visual and Anatomical Sciences Department, Wayne State University School of Medicine, Detroit, MI 48202

Spontaneous synaptic transmission is regulated by the protein complexin (Cpx). Cpx binds the SNARE complex, a coil-coiled four-helical bundle that mediates the attachment of a synaptic vesicle (SV) to the presynaptic membrane (PM). Cpx is thought to clamp spontaneous fusion events by stabilizing a partially unraveled state of the SNARE bundle; however, the molecular detail of this mechanism is still debated. We combined electrophysiology, molecular modeling, and site-directed mutagenesis in to develop and validate the atomic model of the Cpx-mediated clamped state of the SNARE complex. We took advantage of botulinum neurotoxins (BoNTs) B and G, which cleave the SNARE protein synaptobrevin (Syb) at different sites. Monitoring synaptic depression on BoNT loading revealed that the clamped state of the SNARE complex has two or three unraveled helical turns of Syb. Site-directed mutagenesis showed that the Cpx clamping function is predominantly maintained by its accessory helix (AH), while molecular modeling suggested that the Cpx AH interacts with the unraveled C terminus of Syb and the SV lipid bilayer. The developed molecular model was employed to design new Cpx poor-clamp and super-clamp mutations and to tested the predictions employing molecular dynamics simulations. Subsequently, we generated lines harboring these mutations and confirmed the poor-clamp and super-clamp phenotypes Altogether, these results validate the atomic model of the Cpx-mediated fusion clamp, wherein the Cpx AH inserts between the SNARE bundle and the SV lipid bilayer, simultaneously binding the unraveled C terminus of Syb and preventing full SNARE assembly.
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http://dx.doi.org/10.1523/ENEURO.0526-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026252PMC
June 2021

Metabolic imaging and secondary ion mass spectrometry to define the structure and function of liver with acute and chronic pathology.

J Biomed Opt 2019 12;25(1):1-14

Privolzhsky Research Medical University, Institute of Experimental Oncology and Biomedical Technolog, Russia.

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic.
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http://dx.doi.org/10.1117/1.JBO.25.1.014508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008498PMC
December 2019

Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in .

eNeuro 2019 Jul/Aug;6(4). Epub 2019 Aug 22.

Neurology Department, Wayne State University, Detroit, Michigan 48201

Synapse formation can be promoted by intense activity. At the larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. We combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the NMJ. We found that the new boutons can form rapidly in intact larva in response to intense crawling activity, and we observed two different patterns of bouton formation and maturation. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. We demonstrated that intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. We also showed that this pathway depends on synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, we took advantage of the temperature-sensitive mutant to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.
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http://dx.doi.org/10.1523/ENEURO.0060-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709223PMC
March 2020

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction.

J Vis Exp 2017 09 25(127). Epub 2017 Sep 25.

Department of Neurology, School of Medicine, Wayne State University; Department of Anatomy and Cell Biology, School of Medicine, Wayne State University;

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has a diameter of several microns, and the release sites positioned outside of the electrode rim do not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.
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http://dx.doi.org/10.3791/56493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752324PMC
September 2017

Electrophysiological analysis of synaptic transmission in Drosophila.

Wiley Interdiscip Rev Dev Biol 2017 09 24;6(5). Epub 2017 May 24.

Department of Neurology, Wayne State University, Detroit, MI, USA.

Synaptic transmission is dynamic, plastic, and highly regulated. Drosophila is an advantageous model system for genetic and molecular studies of presynaptic and postsynaptic mechanisms and plasticity. Electrical recordings of synaptic responses represent a wide-spread approach to study neuronal signaling and synaptic transmission. We discuss experimental techniques that allow monitoring synaptic transmission in Drosophila neuromuscular and central systems. Recordings of synaptic potentials or currents at the larval neuromuscular junction (NMJ) are most common and provide numerous technical advantages due to robustness of the preparation, large and identifiable muscles, and synaptic boutons which can be readily visualized. In particular, focal macropatch recordings combined with the analysis of neurosecretory quanta enable rigorous quantification of the magnitude and kinetics of transmitter release. Patch-clamp recordings of synaptic transmission from the embryonic NMJ enable overcoming the problem of lethality in mutant lines. Recordings from the adult NMJ proved instrumental in the studies of temperature-sensitive paralytic mutants. Genetic studies of behavioral learning in Drosophila compel an investigation of synaptic transmission in the central nervous system (CNS), including primary cultured neurons and an intact brain. Cholinergic and GABAergic synaptic transmission has been recorded from the Drosophila CNS both in vitro and in vivo. In vivo patch-clamp recordings of synaptic transmission from the neurons in the olfactory pathway is a very powerful approach, which has a potential to elucidate how synaptic transmission is associated with behavioral learning. WIREs Dev Biol 2017, 6:e277. doi: 10.1002/wdev.277 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wdev.277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980642PMC
September 2017

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.

J Neurosci 2017 01;37(2):383-396

Department of Neurology and

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.

Significance Statement: Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation.
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http://dx.doi.org/10.1523/JNEUROSCI.1854-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242395PMC
January 2017

Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.

Biophys J 2016 Nov;111(9):1954-1964

Department of Neurology, Wayne State University, Detroit, Michigan; Department of Anatomy and Cell Biology, Wayne State University, Detroit, Michigan. Electronic address:

Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE) and vesicle-attached (v-SNARE) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. We previously proposed a model in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, we combined molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. We investigated clamping properties in the syx paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. We used our model to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. MD simulations predicted that a weakened interaction between the Cpx accessory helix and the v-SNARE would enhance Cpx flexibility and thus promote separation of SNAREs, reducing spontaneous fusion. We generated transgenic Drosophila with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.
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http://dx.doi.org/10.1016/j.bpj.2016.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102999PMC
November 2016

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction.

J Neurosci 2014 Aug;34(32):10554-63

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00960-6032, and

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(-)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(-) NMJs was significantly diminished, and that new boutons in Syn(-) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(-) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.
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http://dx.doi.org/10.1523/JNEUROSCI.5074-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200108PMC
August 2014

Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

Biophys J 2013 Aug;105(3):679-90

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico.

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx(3-69)Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.
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http://dx.doi.org/10.1016/j.bpj.2013.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736676PMC
August 2013