Publications by authors named "Partho Halder"

10 Publications

  • Page 1 of 1

Implications of the null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult .

J Exp Biol 2019 10 8;222(Pt 19). Epub 2019 Oct 8.

Institute of Clinical Neurobiology, University of Würzburg, 97078 Würzburg, Germany

The gene of encodes a highly abundant 47 kDa synaptic vesicle-associated protein. null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of or on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of but not reduces lifespan, whereas knockout of and , either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in and single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.
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http://dx.doi.org/10.1242/jeb.203505DOI Listing
October 2019

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs.

Sci Rep 2017 09 21;7(1):12070. Epub 2017 Sep 21.

Institute of Physical Chemistry, Georg-August-University, Göttingen, 37077, Germany.

The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m, at which the number of fusion events is increased substantially.
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http://dx.doi.org/10.1038/s41598-017-12348-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608890PMC
September 2017

Oxidized phagosomal NOX2 complex is replenished from lysosomes.

J Cell Sci 2017 04 15;130(7):1285-1298. Epub 2017 Feb 15.

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.
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http://dx.doi.org/10.1242/jcs.196931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399780PMC
April 2017

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments.

Proc Natl Acad Sci U S A 2016 11 2;113(46):13051-13056. Epub 2016 Nov 2.

Institute of Physical Chemistry, University of Göttingen, 37077 Goettingen, Germany;

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.
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http://dx.doi.org/10.1073/pnas.1615885113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135348PMC
November 2016

PtdInsP and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

Elife 2016 10 28;5. Epub 2016 Oct 28.

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

The Ca-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from and expressed in ) binds to PtdIns(4,5)P via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca neutralizes the negative charges of the Ca-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P). These Ca-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.
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http://dx.doi.org/10.7554/eLife.15886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123861PMC
October 2016

Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors.

Proc Natl Acad Sci U S A 2016 08 19;113(31):8807-12. Epub 2016 Jul 19.

Department of Microbiology, University of Chicago, Chicago, IL 60637;

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires' disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host's disassembly machinery, thus effectively trapping VAMP4 in an inactive state.
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http://dx.doi.org/10.1073/pnas.1608755113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978295PMC
August 2016

Identification and structural characterization of interneurons of the Drosophila brain by monoclonal antibodies of the würzburg hybridoma library.

PLoS One 2013 17;8(9):e75420. Epub 2013 Sep 17.

Institute of Clinical Neurobiology, University of Würzburg, Würzburg, Germany.

Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075420PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775750PMC
June 2014

Identification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila brain.

PLoS One 2011 19;6(12):e29352. Epub 2011 Dec 19.

Department of Neurobiology and Genetics, Theodor-Boveri Institute, University of Wuerzburg, Wuerzburg, Germany.

The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029352PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244249PMC
April 2012

The Wuerzburg hybridoma library against Drosophila brain.

J Neurogenet 2009 7;23(1-2):78-91. Epub 2009 Jan 7.

Institut für Zoologie, Lehrstuhl für Entwicklungsbiologie, Regensburg, Germany.

This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.
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http://dx.doi.org/10.1080/01677060802471627DOI Listing
January 2010

Adaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation.

Bioinformation 2006 Dec 26;1(8):321-6. Epub 2006 Dec 26.

Department of Biotechnology, Birla Institute of Technology, Mesra, India.

The phenomenon of host radiation is strongly influenced by the rates of mutation of their virulence genes. We have studied the molecular evolution of virulence genes (HA, NS, PB2) of the Avian Influenza Virus H5N1 from avian to human hosts. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions for the three chosen genes in parasite populations from different hosts. Analyses were made using Maximum Likelihood (ML) genealogies for the null and alternate hypothesis based on differential gamma distribution rates. The null hypothesis had a higher rate of substitution and was found to be more suitable for all the studied genes by Likelihood Ratio Test (LRT). The study showed the NS gene to be having the fastest rate of evolution.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891714PMC
http://dx.doi.org/10.6026/97320630001321DOI Listing
December 2006
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