scholarly journals Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

2010 ◽  
Vol 188 (3) ◽  
pp. 415-428 ◽  
Author(s):  
Arun Anantharam ◽  
Bibiana Onoa ◽  
Robert H. Edwards ◽  
Ronald W. Holz ◽  
Daniel Axelrod
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Secretory Granules ◽  
Membrane Topology ◽  
Membrane Complex ◽  
Polarization Technique ◽  
Granule Membrane ◽  
Guanosine Triphosphatase ◽  
Adrenal Chromaffin ◽  
Image Simulations

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

Recycling of a secretory granule membrane protein after stimulated secretion

1993 ◽  
Vol 106 (2) ◽  
pp. 649-655 ◽  
Author(s):  
S.M. Hurtley
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Granule ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Primary Cultures ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Antibody Complex ◽  
Adrenal Chromaffin

Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.

scholarly journals Visualization of Regulated Exocytosis with a Granule-Membrane Probe Using Total Internal Reflection Microscopy

2004 ◽  
Vol 15 (10) ◽  
pp. 4658-4668 ◽  
Author(s):  
Miriam W. Allersma ◽  
Li Wang ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Evanescent Field ◽  
Internal Reflection ◽  
Spherical Granule ◽  
Granule Membrane ◽  
Green Fluorescent

Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In ∼90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of ∼200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, ∼20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, ∼10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.

scholarly journals Restriction of Secretory Granule Motion near the Plasma Membrane of Chromaffin Cells

2001 ◽  
Vol 153 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Laura M. Johns ◽  
Edwin S. Levitan ◽  
Eric A. Shelden ◽  
Ronald W. Holz ◽  
Daniel Axelrod
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Chromaffin Cells ◽  
Botulinum Toxin A ◽  
Secretory Granules ◽  
Internal Reflection ◽  
Snare Proteins ◽  
Bovine Chromaffin Cells ◽  
Heterogeneous Matrix ◽  
Granule Motion

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the ∼300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

scholarly journals Nucleotide and bivalent cation specificity of the insulin-granule proton translocase

1983 ◽  
Vol 210 (1) ◽  
pp. 235-242 ◽  
Author(s):  
J C Hutton ◽  
M Peshavaria
Keyword(s):  
Atp Hydrolysis ◽  
Secretory Granules ◽  
Bivalent Cation ◽  
Nucleotide Hydrolysis ◽  
Storage Vesicles ◽  
Carbonyl Cyanide ◽  
Granule Membrane ◽  
Insulin Granule ◽  
Intracellular Storage ◽  
Adrenal Chromaffin

1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from adenohypophyseal tissue, adrenal chromaffin granules and yeast vacuoles. The insulin-granule activity thus appears to be a type of proton translocase, which is characteristic of intracellular storage vesicles in eukaryotic cells.

scholarly journals Platelet and Megakaryocyte Dense Granules Contain Glycoproteins Ib and IIb-IIIa

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 4047-4057 ◽  
Author(s):  
Tayebeh Youssefian ◽  
Jean-Marc Massé ◽  
Francine Rendu ◽  
Josette Guichard ◽  
Elisabeth M. Cramer
Keyword(s):  
Plasma Membrane ◽  
Dense Body ◽  
Secretory Granules ◽  
Dense Granule ◽  
Immunogold Labeling ◽  
Membrane Receptors ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Plasma Membrane Receptors

Abstract Platelets contain two main types of secretory organelles, the dense granules and the α-granules. P-selectin, a specific receptor for leukocytes that is present in the α-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the α-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti–Gp IIb-IIIa or anti–Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti–Gp IIb-IIIa or anti–Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additionnal evidence of similarities between the α-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of α-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.

scholarly journals A new role for the dynamin GTPase in the regulation of fusion pore expansion

2011 ◽  
Vol 22 (11) ◽  
pp. 1907-1918 ◽  
Author(s):  
Arun Anantharam ◽  
Mary A. Bittner ◽  
Rachel L. Aikman ◽  
Edward L. Stuenkel ◽  
Sandra L. Schmid ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Membrane Topology ◽  
Fusion Pore ◽  
Gtpase Activity ◽  
Membrane Fission ◽  
Pore Widening ◽  
Granule Membrane ◽  
Membrane Deformations ◽  
Pore Expansion

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.

scholarly journals Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules

2014 ◽  
Vol 25 (16) ◽  
pp. 2416-2427 ◽  
Author(s):  
Tejeshwar C. Rao ◽  
Daniel R. Passmore ◽  
Andrew R. Peleman ◽  
Madhurima Das ◽  
Edwin R. Chapman ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Secretory Granules ◽  
Fusion Pore ◽  
Cargo Proteins ◽  
Granule Membrane ◽  
Synaptotagmin 1 ◽  
Physiological Homeostasis ◽  
Dense Core Granules ◽  
Adrenal Chromaffin ◽  
Granule Membrane Proteins

Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

scholarly journals Insulin granule biogenesis and exocytosis

2020 ◽  
Author(s):  
Muhmmad Omar-Hmeadi ◽  
Olof Idevall-Hagren
Keyword(s):  
Type 2 Diabetes ◽  
Plasma Membrane ◽  
Secretory Granules ◽  
Glucose Production ◽  
Biologically Active ◽  
Glucose Levels ◽  
Granule Membrane ◽  
Insulin Granule ◽  
Golgi Network

Abstract Insulin is produced by pancreatic β-cells, and once released to the blood, the hormone stimulates glucose uptake and suppresses glucose production. Defects in both the availability and action of insulin lead to elevated plasma glucose levels and are major hallmarks of type-2 diabetes. Insulin is stored in secretory granules that form at the trans-Golgi network. The granules undergo extensive modifications en route to their release sites at the plasma membrane, including changes in both protein and lipid composition of the granule membrane and lumen. In parallel, the insulin molecules also undergo extensive modifications that render the hormone biologically active. In this review, we summarize current understanding of insulin secretory granule biogenesis, maturation, transport, docking, priming and eventual fusion with the plasma membrane. We discuss how different pools of granules form and how these pools contribute to insulin secretion under different conditions. We also highlight the role of the β-cell in the development of type-2 diabetes and discuss how dysregulation of one or several steps in the insulin granule life cycle may contribute to disease development or progression.

scholarly journals Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge.

1980 ◽  
Vol 85 (2) ◽  
pp. 299-312 ◽  
Author(s):  
J P Caulfield ◽  
R A Lewis ◽  
A Hein ◽  
K F Austen
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Human Lung ◽  
Secretory Granules ◽  
Amorphous State ◽  
Surface Discharge ◽  
Microscope Examination ◽  
Crystalline Structures ◽  
Granule Membrane ◽  
Release Histamine

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.

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