scholarly journals Maturation of active zone assembly by Drosophila Bruchpilot

2009 ◽  
Vol 186 (1) ◽  
pp. 129-145 ◽  
Author(s):  
Wernher Fouquet ◽  
David Owald ◽  
Carolin Wichmann ◽  
Sara Mertel ◽  
Harald Depner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
High Resolution ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Dense Projections ◽  
Ca2 Channels

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

scholarly journals Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans

2013 ◽  
Vol 203 (5) ◽  
pp. 849-863 ◽  
Author(s):  
Maike Kittelmann ◽  
Jan Hegermann ◽  
Alexandr Goncharov ◽  
Hidenori Taru ◽  
Mark H. Ellisman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
High Resolution ◽  
Active Zone ◽  
Neuromuscular Junctions ◽  
Main Role ◽  
C Elegans ◽  
Active Zones ◽  
Dense Projections

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.

scholarly journals Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix

2019 ◽  
Vol 218 (3) ◽  
pp. 1011-1026 ◽  
Author(s):  
Nicole Scholz ◽  
Nadine Ehmann ◽  
Divya Sachidanandan ◽  
Cordelia Imig ◽  
Benjamin H. Cooper ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
C Terminus ◽  
Unknown Protein ◽  
Transmission Part ◽  
Molecular Components ◽  
In Vivo Screen

Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.

scholarly journals Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release

2016 ◽  
Vol 216 (1) ◽  
pp. 231-246 ◽  
Author(s):  
Joseph J. Bruckner ◽  
Hong Zhan ◽  
Scott J. Gratz ◽  
Monica Rao ◽  
Fiona Ukken ◽  
...  
Keyword(s):  
Neural Plasticity ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Motor Behavior ◽  
Molecular Organization ◽  
Electrophysiological Studies ◽  
Circuit Function ◽  
Synaptic Vesicle Release ◽  
Ca2 Channels

The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. We previously identified Drosophila Piccolo-RIM-related Fife, which regulates neurotransmission and motor behavior through an unknown mechanism. Here, we demonstrate that Fife localizes and interacts with RIM at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, we find a decrease in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, we find that Fife is essential for the homeostatic modulation of neurotransmission. We propose that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release.

scholarly journals The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

2013 ◽  
Vol 202 (4) ◽  
pp. 667-683 ◽  
Author(s):  
Tanja Matkovic ◽  
Matthias Siebert ◽  
Elena Knoche ◽  
Harald Depner ◽  
Sara Mertel ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Circular Array ◽  
Membrane Domain ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Family Protein ◽  
Biochemical Identification ◽  
Macromolecular Architecture ◽  
Bar Formation ◽  
Ca2 Channels

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.

scholarly journals Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites

2021 ◽  
Vol 22 (11) ◽  
pp. 5709
Author(s):  
Mitzi Díaz-Hernández ◽  
Rosario Javier-Reyna ◽  
Izaid Sotto-Ortega ◽  
Guillermina García-Rivera ◽  
Sarita Montaño ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Entamoeba Histolytica ◽  
Posttranslational Modifications ◽  
Target Protein ◽  
Binding Motif ◽  
Docking Analysis ◽  
Membrane Activity ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

scholarly journals High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803

2021 ◽  
Vol 119 (1) ◽  
pp. e2116765118
Author(s):  
Christopher J. Gisriel ◽  
Jimin Wang ◽  
Jinchan Liu ◽  
David A. Flesher ◽  
Krystle M. Reiss ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Photosystem Ii ◽  
Water Oxidation ◽  
Genetic Manipulation ◽  
Water Channel ◽  
Global Scale ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
Pcc 6803

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.

scholarly journals Atrophin–Rpd3 complex represses Hedgehog signaling by acting as a corepressor of CiR

2013 ◽  
Vol 203 (4) ◽  
pp. 575-583 ◽  
Author(s):  
Zhao Zhang ◽  
Jing Feng ◽  
Chenyu Pan ◽  
Xiangdong Lv ◽  
Wenqing Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Transcription Factors ◽  
Hedgehog Signaling ◽  
Target Gene ◽  
Cubitus Interruptus ◽  
C Terminus ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Hh Signaling

The evolutionarily conserved Hedgehog (Hh) signaling pathway is transduced by the Cubitus interruptus (Ci)/Gli family of transcription factors that exist in two distinct repressor (CiR/GliR) and activator (CiA/GliA) forms. Aberrant activation of Hh signaling is associated with various human cancers, but the mechanism through which CiR/GliR properly represses target gene expression is poorly understood. Here, we used Drosophila melanogaster and zebrafish models to define a repressor function of Atrophin (Atro) in Hh signaling. Atro directly bound to Ci through its C terminus. The N terminus of Atro interacted with a histone deacetylase, Rpd3, to recruit it to a Ci-binding site at the decapentaplegic (dpp) locus and reduce dpp transcription through histone acetylation regulation. The repressor function of Atro in Hh signaling was dependent on Ci. Furthermore, Rerea, a homologue of Atro in zebrafish, repressed the expression of Hh-responsive genes. We propose that the Atro–Rpd3 complex plays a conserved role to function as a CiR corepressor.

scholarly journals Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

2015 ◽  
Vol 208 (4) ◽  
pp. 401-414 ◽  
Author(s):  
Joseph E. Klebba ◽  
Brian J. Galletta ◽  
Jonathan Nye ◽  
Karen M. Plevock ◽  
Daniel W. Buster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Binding Domain ◽  
Terminal Region ◽  
Scaffolding Protein ◽  
Binding Partner ◽  
Centriole Duplication ◽  
Binding Domains ◽  
C Terminus ◽  
N Terminus

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

scholarly journals Colocalization of synapsin and actin during synaptic vesicle recycling

2003 ◽  
Vol 161 (4) ◽  
pp. 737-747 ◽  
Author(s):  
Ona Bloom ◽  
Emma Evergren ◽  
Nikolay Tomilin ◽  
Ole Kjaerulff ◽  
Peter Löw ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
Synaptic Activity ◽  
Immunogold Electron Microscopy ◽  
Synaptic Vesicle Recycling ◽  
Vesicle Recycling ◽  
Reserve Pool ◽  
Giant Synapse

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

GSDM family genes meet autophagy

2015 ◽  
Vol 469 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Masaru Tamura ◽  
Toshihiko Shiroishi
Keyword(s):  
Protein Function ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Autophagic Activity

Shi et al. provided a new insight of Gsdm family protein function. They reported that N- and C-terminus domain of Gsdm family protein interact each other. Moreover, the N-terminus domain of Gsdm family proteins has pro-autophagic activity, which is suppressed by the C-terminus domain.

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