scholarly journals The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bijeta Prasai ◽  
Gideon J. Haber ◽  
Marie-Paule Strub ◽  
Regina Ahn ◽  
John A. Ciemniecki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Metal Binding ◽  
Electron Tomography ◽  
Super Resolution ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Rab Proteins ◽  
Rab Gtpases

AbstractRab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we apply a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the 3D axial location of these exocytic proteins and two SNARE proteins (Syntaxin1A and SNAP25) using electron tomography. Rab proteins are distributed across the entire surface and t-SNARE proteins at the base of docked vesicles. We propose that the circumferential distribution of Rabs and Rab-effectors could aid in the efficient transport, capture, docking, and rapid fusion of calcium-triggered exocytic vesicles in excitable cells.

scholarly journals The nanoscale anatomy of exocytic dense-core vesicles in neuroendocrine cells

2020 ◽  
Author(s):  
Bijeta Prasai ◽  
Gideon J. Haber ◽  
Marie-Paule Strub ◽  
John A. Ciemniecki ◽  
Kem A. Sochacki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Metal Binding ◽  
Electron Tomography ◽  
Molecular Model ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Rab Gtpases ◽  
Dense Core Vesicles

AbstractRab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we develop a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the axial location of exocytic proteins using electron tomography. Our data show that Rab-GTPases and their effectors are distributed across the entire surface of individual docked vesicles. This circumferential distribution likely aids in the efficient transport, capture, docking, and rapid fusion of vesicles in excitable cells. The nanoscale molecular model of dense core vesicles generated from our methods reveals how key proteins assemble at the plasma membrane to regulate membrane trafficking and exocytosis.

scholarly journals A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

2006 ◽  
Vol 17 (5) ◽  
pp. 2113-2124 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Kara L. Lynch ◽  
Kristin L. Boswell ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Snare Proteins ◽  
Recycling Endosome ◽  
Protein Receptor ◽  
Interfering Rna

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

scholarly journals SNAP25, but Not Syntaxin 1A, Recycles via an ARF6-regulated Pathway in Neuroendocrine Cells

2006 ◽  
Vol 17 (2) ◽  
pp. 711-722 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Xiaofeng Xia ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Cellular Membrane ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Syntaxin 1A ◽  
Recycling Endosome ◽  
Protein Receptor ◽  
Vesicle Exocytosis ◽  
Donor Acceptor

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor–acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in ∼3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome–trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

scholarly journals Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale

2021 ◽  
Vol 72 (1) ◽  
Author(s):  
Peter D. Dahlberg ◽  
W.E. Moerner
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Nanometer Scale ◽  
Annual Review ◽  
Publication Date ◽  
Fluorescence And Electron Microscopy ◽  
Cellular Context

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis

2020 ◽  
Vol 117 (40) ◽  
pp. 25150-25158
Author(s):  
Masaru Fujimoto ◽  
Kazuo Ebine ◽  
Kohji Nishimura ◽  
Nobuhiro Tsutsumi ◽  
Takashi Ueda
Keyword(s):  
Plasma Membrane ◽  
Adaptor Proteins ◽  
Secretory Vesicle ◽  
Snare Proteins ◽  
Loss Of Function ◽  
Homologous Pair ◽  
Vegetative Development ◽  
Specific Manner ◽  
Clathrin Adaptor ◽  
Mammalian System

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

The proteins of exocytosis: lessons from the sperm model

2015 ◽  
Vol 465 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Claudia Nora Tomes
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Human Sperm ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Calcium Signal ◽  
Rab Gtpases ◽  
Attachment Protein ◽  
Protein Receptors ◽  
Signalling Cascade

Exocytosis is a highly regulated process that consists of multiple functionally, kinetically and/or morphologically definable stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13 and -18, complexins and synaptotagmins. In the present article, the molecules of exocytosis are reviewed, using human sperm as a model system. Sperm exocytosis is driven by isoforms of the same proteinaceous fusion machinery mentioned above, with their functions orchestrated in a hierarchically organized and unidirectional signalling cascade. In addition to the universal exocytosis regulator calcium, this cascade includes other second messengers such as diacylglycerol, inositol 1,4,5-trisphosphate and cAMP, as well as the enzymes that synthesize them and their target proteins. Of special interest is the cAMP-binding protein Epac (exchange protein directly activated by cAMP) due in part to its enzymatic activity towards Rap. The activation of Epac and Rap leads to a highly localized calcium signal which, together with assembly of the SNARE complex, governs the final stages of exocytosis. The source of this releasable calcium is the secretory granule itself.

scholarly journals Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

2015 ◽  
Vol 112 (52) ◽  
pp. 15922-15927 ◽  
Author(s):  
Thomas Burgoyne ◽  
Ingrid P. Meschede ◽  
Jemima J. Burden ◽  
Maryse Bailly ◽  
Miguel C. Seabra ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Fusion Proteins ◽  
Outer Segment ◽  
Actin Polymerization ◽  
Electron Tomography ◽  
Rod Photoreceptors ◽  
Immuno Electron Microscopy ◽  
Leading Edges ◽  
Membrane Fusion Proteins

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that “zipper” up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

scholarly journals From Sorting Endosomes to Exocytosis: Association of Rab4 and Rab11 GTPases with the Fc Receptor, FcRn, during Recycling

2005 ◽  
Vol 16 (4) ◽  
pp. 2028-2038 ◽  
Author(s):  
E. Sally Ward ◽  
Cruz Martinez ◽  
Carlos Vaccaro ◽  
Jinchun Zhou ◽  
Qing Tang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Fc Receptor ◽  
Live Cells ◽  
Rab Proteins ◽  
Wide Field ◽  
Rab Gtpases ◽  
Intracellular Organelles ◽  
Recycling Pathway ◽  
Discrete Domains

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcγ receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn+) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4+Rab11+ or Rab11+ compartments. For Rab4+Rab11+ compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11+FcRn+ vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.

scholarly journals Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Pascal Weber ◽  
Helena Batoulis ◽  
Kerstin M Rink ◽  
Stefan Dahlhoff ◽  
Kerstin Pinkwart ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Basic Amino Acid ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Primary Role ◽  
Amino Acid Residues ◽  
Initial Plasma ◽  
Target Membrane ◽  
Membrane Attachment ◽  
Membrane Contact

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document