scholarly journals Function suggests nano-structure: towards a unified theory for secretion rate, a statistical mechanics approach

2013 ◽  
Vol 10 (88) ◽  
pp. 20130640 ◽  
Author(s):  
Ilan Hammel ◽  
Isaac Meilijson
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Secretion Rate ◽  
Structural Investigations ◽  
Nano Structure ◽  
Fast Kinetics ◽  
Transmission Electron ◽  
Rate Transmission ◽  
Vesicle Exocytosis

The inventory of secretory granules along the plasma membrane can be viewed as maintained in two restricted compartments. The release-ready pool represents docked granules available for an initial stage of fast, immediate secretion, followed by a second stage of granule set-aside secretion pool, with significantly slower rate. Transmission electron microscopy ultra-structural investigations correlated with electrophysiological techniques and mathematical modelling have allowed the categorization of these secretory vesicle compartments, in which vesicles can be in various states of secretory competence. Using the above-mentioned approaches, the kinetics of single vesicle exocytosis can be worked out. The ultra-fast kinetics, explored in this study, represents the immediately available release-ready pool, in which granules bound to the plasma membrane are exocytosed upon Ca 2+ influx at the SNARE rosette at the base of porosomes. Formalizing Dodge and Rahamimoff findings on the effect of calcium concentration and incorporating the effect of SNARE transient rosette size, we postulate that secretion rate (rate), the number ( X ) of intracellular calcium ions available for fusion, calcium capacity (0 ≤ M ≤ 5) and the fusion nano-machine size (as measured by the SNARE rosette size K ) satisfy the parsimonious M – K relation rate ≈ C × [Ca 2+ ] min( X,M ) e − K /2 .

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 The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery

2013 ◽  
Vol 203 (2) ◽  
pp. 283-298 ◽  
Author(s):  
Sandrine Poëa-Guyon ◽  
Mohamed Raafet Ammar ◽  
Marie Erard ◽  
Muriel Amar ◽  
Alexandre W. Moreau ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Fusion Pore ◽  
Membrane Domain ◽  
Reversible Dissociation ◽  
Open Time ◽  
Vesicle Exocytosis ◽  
Kinetics Of

Several studies have suggested that the V0 domain of the vacuolar-type H+-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1–V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

scholarly journals Imaging SNAREs at work in ‘unroofed’ cells – approaches that may be of general interest for functional studies on membrane proteins

2003 ◽  
Vol 31 (4) ◽  
pp. 861-864 ◽  
Author(s):  
T. Lang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Secretory Granules ◽  
General Interest ◽  
Functional Studies ◽  
Protein Receptor ◽  
Vesicle Exocytosis

When cultured cells are subjected to a brief ultrasound pulse, their upper parts burst, but the basal plasma membranes with their embedded membrane–protein complexes remain intact. Such two-dimensional, paraformaldehyde-fixed plasma membrane sheets have been used in the past to visualize the morphology of the inner plasmalemmal leaflet by electron or light microscopy. More recently, fluorescence microscopy of unfixed native membranes has been applied to study SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) function. For instance, biochemical reactions of the plasmalemmal SNAREs with soluble fluorescent SNAREs, patching of SNARE and raft domains, and online monitoring of SNARE-mediated membrane fusion has been performed. The results obtained with the membrane sheet system have added some novel aspects to our understanding of the regulation of neuronal exocytosis. Surprisingly, SNAREs are concentrated in cholesterol-dependent microdomains that are different from membrane rafts. SNAREs in such domains are highly reactive, and define sites for vesicle exocytosis. Secretory granules that fuse on the membrane sheets are retrieved intact in a dynamin-dependent process, suggesting that the ‘kiss-and-run’ mechanism is not a reversed SNARE reaction, but is driven by a biochemically different mechanism. So far, studies of this type have focused on neuronal exocytosis; however, the method might be widely applicable. Data obtained with this system are derived from a 100% pure plasma membrane preparation that is only several seconds old, and membrane proteins are studied in their natural microenvironment that is defined by local lipid composition and putative bound proteins. Hence this approach yields results that most probably reflect the situation in a live cell.

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.

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

A tilting high temperature gas reaction chamber for the JEM 7A T.E.M.

1978 ◽  
Vol 36 (1) ◽  
pp. 70-71
Author(s):  
Brian L. Rhoades
Keyword(s):  
Cross Section ◽  
Reaction Chamber ◽  
Objective Lens ◽  
Reduced Cross Section ◽  
Cross Sectional ◽  
Structural Investigations ◽  
Transmission Electron ◽  
Dynamic Experiments ◽  
Successful Design ◽  
Thermocouple Junction

A gas reaction chamber has been designed and constructed for the JEM 7A transmission electron microscope which is based on a notably successful design by Hashimoto et. al. but which provides specimen tilting facilities of ± 15° aboutany axis in the plane of the specimen.It has been difficult to provide tilting facilities on environmental chambers for 100 kV microscopes owing to the fundamental lack of available space within the objective lens and the scope of structural investigations possible during dynamic experiments has been limited with previous specimen chambers not possessing this facility.A cross sectional diagram of the specimen chamber is shown in figure 1. The specimen is placed on a platinum ribbon which is mounted on a mica ring of the type shown in figure 2. The ribbon is heated by direct current, and a thermocouple junction spot welded to the section of the ribbon of reduced cross section enables temperature measurement at the point where localised heating occurs.

The Ultrastructure of Lymphocytic Hypophysitis

1981 ◽  
Vol 39 ◽  
pp. 466-467
Author(s):  
S.L. Asa ◽  
K. Kovacs ◽  
J. M. Bilbao ◽  
R. G. Josse ◽  
K. Kreines
Keyword(s):  
Electron Microscopy ◽  
Plasma Cells ◽  
Secretory Granules ◽  
Sella Turcica ◽  
Uranyl Acetate ◽  
Lymphocytic Hypophysitis ◽  
Pituitary Cells ◽  
Transmission Electron ◽  
Ultrathin Sections ◽  
Mass Lesions

Seven cases of lymphocytic hypophysitis in women have been reported previously in association with various degrees of hypopituitarism. We report two pregnant patients who presented with mass lesions of the sella turcica, clinically mimicking pituitary adenoma. However, pathologic examination revealed extensive infiltration of the anterior pituitary by lymphocytes and plasma cells with destruction of the gland. To our knowledge, the ultrastructural features of lymphocytic hypophysitis have not been studied so far.For transmission electron microscopy, tissue from surgical specimens was fixed in glutaraldehyde, postfixed in OsO4, dehydrated and embedded in epoxy-resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope.Electron microscopy revealed adenohypophysial cells of all types exhibiting varying degrees of injury. In the areas of most dense inflammatory cell infiltration pituitary cells contained large lysosomal bodies fusing with secretory granules (Fig. 1), as well as increased numbers of swollen mitochondria, indicating oncocytic transformation (Fig. 2).

Ultrastructural studies of the relationship between actin filaments and secretory granules

1990 ◽  
Vol 48 (3) ◽  
pp. 458-459
Author(s):  
Ellen Holm Nielsen
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Granule Membrane ◽  
Ultrathin Sections ◽  
Lowicryl K4m

In secretory cells a dense and complex network of actin filaments is seen in the subplasmalemmal space attached to the cell membrane. During exocytosis this network is undergoing a rearrangement facilitating access of granules to plasma membrane in order that fusion of the membranes can take place. A filamentous network related to secretory granules has been reported, but its structural organization and composition have not been examined, although this network may be important for exocytosis.Samples of peritoneal mast cells were frozen at -70°C and thawed at 4°C in order to rupture the cells in such a gentle way that the granule membrane is still intact. Unruptured and ruptured cells were fixed in 2% paraformaldehyde and 0.075% glutaraldehyde, dehydrated in ethanol. For TEM (transmission electron microscopy) cells were embedded in Lowicryl K4M at -35°C and for SEM (scanning electron microscopy) they were placed on copper blocks, critical point dried and coated. For immunoelectron microscopy ultrathin sections were incubated with monoclonal anti-actin and colloidal gold labelled IgM. Ruptured cells were also placed on cover glasses, prefixed, and incubated with anti-actin and colloidal gold labelled IgM.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

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