scholarly journals Calcium Dependencies of Regulated Exocytosis in Different Endocrine Cells

2011 ◽  
pp. S29-S38
Author(s):  
J. DOLENŠEK ◽  
M. SKELIN ◽  
M. S. RUPNIK
Keyword(s):  
Pancreatic Beta Cells ◽  
Endocrine Cells ◽  
Secretory Vesicle ◽  
Pituitary Cells ◽  
Secretory Vesicles ◽  
Regulated Exocytosis ◽  
Vesicle Exocytosis ◽  
Adrenal Chromaffin ◽  
Endocrine Tissues

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+]i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity ‘classes’ of secretory vesicles have been observed: a highly Ca2+-sensitive, a medium Ca2+-sensitive and a low Ca2+-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca2+ sensor protein.

scholarly journals Spatiotemporal organization and protein dynamics involved in regulated exocytosis of MMP-9 in breast cancer cells

2019 ◽  
Vol 151 (12) ◽  
pp. 1386-1403 ◽  
Author(s):  
Dominique C. Stephens ◽  
Nicole Osunsanmi ◽  
Kem A. Sochacki ◽  
Tyrel W. Powell ◽  
Justin W. Taraska ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Secretory Vesicle ◽  
Effector Proteins ◽  
Cellular Transport ◽  
Secretory Vesicles ◽  
Rab Gtpases ◽  
Regulated Exocytosis ◽  
Endocytic Proteins

Altered regulation of exocytosis is an important mechanism controlling many diseases, including cancer. Defects in exocytosis have been implicated in many cancer cell types and are generally attributed to mutations in cellular transport, trafficking, and assembly of machinery necessary for exocytosis of secretory vesicle cargo. In these cancers, up-regulation of trafficking and secretion of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, is responsible for degrading the extracellular matrix, a necessary step in tumor progression. Using TIRF microscopy, we identified proteins associated with secretory vesicles containing MMP-9 and imaged the local dynamics of these proteins at fusion sites during regulated exocytosis of MMP-9 from MCF-7 breast cancer cells. We found that many regulators of exocytosis, including several Rab GTPases, Rab effector proteins, and SNARE/SNARE modulator proteins, are stably assembled on docked secretory vesicles before exocytosis. At the moment of fusion, many of these components are quickly lost from the vesicle, while several endocytic proteins and lipids are simultaneously recruited to exocytic sites at precisely that moment. Our findings provide insight into the dynamic behavior of key core exocytic proteins, accessory proteins, lipids, and some endocytic proteins at single sites of secretory vesicle fusion in breast cancer cells.

scholarly journals Kiss-and-Coat and Compartment Mixing: Coupling Exocytosis to Signal Generation and Local Actin Assembly

2006 ◽  
Vol 17 (4) ◽  
pp. 1495-1502 ◽  
Author(s):  
Anna M. Sokac ◽  
William M. Bement
Keyword(s):  
Cell Types ◽  
Secretory Vesicle ◽  
Signal Generation ◽  
General Mechanism ◽  
Fusion Pore ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Vesicle Membrane ◽  
Regulated Exocytosis ◽  
Broad Variety

Regulated exocytosis is thought to occur either by “full fusion,” where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by “kiss-and-run,” where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed “kiss-and-coat,” which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.

Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D

1981 ◽  
Vol 49 (1) ◽  
pp. 261-272 ◽  
Author(s):  
J.M. Picton ◽  
M.W. Steer
Keyword(s):  
Turnover Rate ◽  
Secretory Vesicle ◽  
Pollen Tubes ◽  
Cytochalasin D ◽  
Wall Material ◽  
Secretory Vesicles ◽  
Size Distributions ◽  
Visible Effect

Pollen tubes of Tradescantia were grown in vitro and exposed to 0.3 microgram/ml cytochalasin D for 5 or 10 min. Fine-structural observations revealed no visible effect of the drug on the organelles. Stereological analysis, using a method recently developed by Rose (1980) to obtain sphere size-distributions corrected for section thickness, revealed substantial increase in the number of secretory vesicles present in the cytoplasm around the dictyosomes. Equating the rate of vesicle accumulation with the rate of vesicle production, a total of 5388 vesicles per minute are formed by a growing tube. This corresponds to 2.4 vesicles per minute per dictyosome, and a turnover rate of 3.7 min for a single dictyosome cisterna, or about 15–18.5 min for a complete dictyosome. The calculated vesicle production rate agrees well with that required to sustain the observed growth rate of such tubes, based on the addition of membrane or wall material to the tube tip.

Scinderin and cortical F-actin are components of the secretory machinery

1999 ◽  
Vol 77 (9) ◽  
pp. 660-671 ◽  
Author(s):  
J -M Trifaró
Keyword(s):  
Chromaffin Cell ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Actin Network ◽  
Filamentous Actin ◽  
Actin Networks ◽  
Reserve Pool ◽  
Vesicle Exocytosis

Secretory vesicle exocytosis is the mechanism of release of neurotransmitters and neuropeptides. Secretory vesicles are localized in at least two morphologically and functionally distinct compartments: the reserve pool and the release-ready pool. Filamentous actin networks play an important role in this compartmentalization and in the trafficking of vesicles between these compartments. The cortical F-actin network constitutes a barrier (negative clamp) to the movement of secretory vesicles to release sites, and it must be locally disassembled to allow translocation of secretory vesicles in preparation for exocytosis. The disassembly of the cortical F-actin network is controlled by scinderin (a Ca2+-dependent F-actin severing protein) upon activation by Ca2+ entering the cells during stimulation. There are several factors that regulate scinderin activation (i.e., Ca2+ levels, phosphatidylinositol 4,5-bisphosphate (PIP2), etc.). The results suggest that scinderin and the cortical F-actin network are components of the secretory machinery.Key words: F-actin, scinderin, exocytosis, cytoskeleton, chromaffin cell.

scholarly journals Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

2011 ◽  
Vol 301 (2) ◽  
pp. C507-C521 ◽  
Author(s):  
Lilian Chiang ◽  
Julie Ngo ◽  
Joel E. Schechter ◽  
Serhan Karvar ◽  
Tanya Tolmachova ◽  
...  
Keyword(s):  
Lacrimal Gland ◽  
Morphological Changes ◽  
Acinar Cells ◽  
Secretory Vesicle ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Apical Surface ◽  
Tear Proteins ◽  
Vesicle Exocytosis ◽  
Constitutively Active

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b −/− and Rab27 ash/ash /Rab27b −/− mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

scholarly journals Expression of VGF mRNA in developing neuroendocrine and endocrine tissues

2003 ◽  
Vol 179 (2) ◽  
pp. 227-235 ◽  
Author(s):  
SE Snyder ◽  
B Peng ◽  
JE Pintar ◽  
SR Salton
Keyword(s):  
Pancreatic Beta Cells ◽  
Endocrine Cells ◽  
Developmental Studies ◽  
Rat Uterus ◽  
Functional Roles ◽  
Esophageal Lumen ◽  
Enteric Plexus ◽  
Developing Rat ◽  
The Brain ◽  
Endocrine Tissues

Analysis of knockout mice suggests that the neurotropin-inducible secreted polypeptide VGF (non-acronymic) plays an important role in the regulation of energy balance. VGF is synthesized by neurons in the central and peripheral nervous systems (CNS, PNS), as well as in the adult pituitary, adrenal medulla, endocrine cells of the stomach and pancreatic beta cells. Thus VGF, like cholecystokinin, leptin, ghrelin and other peptide hormones that have been shown to regulate feeding and energy expenditure, is synthesized in both the gut and the brain. Although detailed developmental studies of VGF localization in the CNS and PNS have been completed, little is known about the ontogeny of VGF expression in endocrine and neuroendocrine tIssues. Here, we report that VGF mRNA is detectable as early as embryonic day 15.5 in the developing rat gastrointestinal and esophageal lumen, pancreas, adrenal, and pituitary, and we further demonstrate that VGF mRNA is synthesized in the gravid rat uterus, together supporting possible functional roles for this polypeptide outside the nervous system and in the enteric plexus.

scholarly journals Submaximal Responses in Calcium-triggered Exocytosis Are Explained by Differences in the Calcium Sensitivity of Individual Secretory Vesicles

1998 ◽  
Vol 112 (5) ◽  
pp. 559-567 ◽  
Author(s):  
Paul S. Blank ◽  
Myoung-Soon Cho ◽  
Steven S. Vogel ◽  
Doron Kaplan ◽  
Albert Kang ◽  
...  
Keyword(s):  
Calcium Sensitivity ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Calcium Dependent ◽  
Sea Urchin Egg ◽  
Dependent Inactivation ◽  
Graded Response ◽  
Vesicle Exocytosis

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.

scholarly journals Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

1996 ◽  
Vol 135 (6) ◽  
pp. 1471-1483 ◽  
Author(s):  
C D Austin ◽  
D Shields
Keyword(s):  
Endocrine Cells ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Secretory Vesicle ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Vesicle Release ◽  
Vesicle Budding ◽  
Phosphatase Inhibitors ◽  
Gtp Binding

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

scholarly journals Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells

1996 ◽  
Vol 314 (3) ◽  
pp. 723-726 ◽  
Author(s):  
Wai Lam W. LING ◽  
Dennis SHIELDS
Keyword(s):  
Mammalian Cells ◽  
Endocrine Cells ◽  
Secretory Vesicle ◽  
Gh3 Cells ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Vesicle Budding ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Salt Extract

The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells.

scholarly journals Analytical Methods for the Determination of Neuroactive Steroids

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 553
Author(s):  
Michal Kaleta ◽  
Jana Oklestkova ◽  
Ondřej Novák ◽  
Miroslav Strnad
Keyword(s):  
Nervous System ◽  
Steroid Hormone ◽  
Neuroactive Steroids ◽  
Physiological Changes ◽  
Biological Matrices ◽  
Pathological Conditions ◽  
Reliable Monitoring ◽  
Hormonal Steroids ◽  
Endocrine Tissues

Neuroactive steroids are a family of all steroid-based compounds, of both natural and synthetic origin, which can affect the nervous system functions. Their biosynthesis occurs directly in the nervous system (so-called neurosteroids) or in peripheral endocrine tissues (hormonal steroids). Steroid hormone levels may fluctuate due to physiological changes during life and various pathological conditions affecting individuals. A deeper understanding of neuroactive steroids’ production, in addition to reliable monitoring of their levels in various biological matrices, may be useful in the prevention, diagnosis, monitoring, and treatment of some neurodegenerative and psychiatric diseases. The aim of this review is to highlight the most relevant methods currently available for analysis of neuroactive steroids, with an emphasis on immunoanalytical methods and gas, or liquid chromatography combined with mass spectrometry.

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