Ultrastructural morphometry of the pancreatic ?-cell

Dean, Philip M.

doi:10.1007/bf01230690

Cited by 257 publications

(202 citation statements)

References 12 publications

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“…The average number of LDCVs in MIN6 cells was 2,412 Ϯ 563 granules per cell. This is in close agreement with measurements of the average diameter (311 Ϯ 45 nm) and number (3,290 granules per MIN6 cell) of immunostained insulin granules and values reported in other studies (2)(3)(4).…”

Section: Resultssupporting

confidence: 80%

“…Roughly 1,000 granules are morphologically docked to the plasma membranes, and perhaps 100 of these are in a readily releasable pool. Only a small proportion of total insulin is released even under maximal stimulatory conditions (4,5). Mechanisms that control transfer of granules to the readily releasable pool as well as details of release activation remain unresolved.…”

mentioning

confidence: 99%

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Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Bindokas

Kuznetsov

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

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Section: Resultssupporting

confidence: 80%

mentioning

confidence: 99%

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Bindokas

Kuznetsov

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…This is not much different from the pool size deduced from the transient stimulation of exocytosis elicited by cAMP at 60 nÒ [Ca¥]é (80 granules). Collectively, these results suggest that in the pancreatic B-cell the readily releasable pool under control conditions is of very limited size and contains û0·5% of the total number of granules within the cell (13100 granules; Dean, 1973). The fact that exocytosis continues for much longer in the infusion experiments when cAMP is present ( Fig.…”

Section: Discussionmentioning

confidence: 74%

Protein kinase A‐dependent and ‐independent stimulation of exocytosis by cAMP in mouse pancreatic B‐cells

Renström

Eliasson²,

Rorsman

1997

The Journal of Physiology

284

279

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The mechanisms by which cAMP stimulates Ca2+‐dependent insulin secretion were investigated by combining measurements of whole‐cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B‐cells maintained in tissue culture. Cyclic AMP stimulated exocytosis > 4‐fold in whole‐cell experiments in which secretion was evoked by intracellular dialysis with a Ca2+‐EGTA buffer with a [Ca2+]i of 1.5μm. This effect was antagonized by inhibitors of protein kinase A (PKA). Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (≥60 nm), but was without effect in the complete absence of Ca2+. Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6‐fold PKA‐dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated‐patch whole‐cell recordings. Exocytosis triggered by single depolarizations in standard whole‐cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition. When exocytosis was triggered by Ca2+ released from Ca2+‐NP‐EGTA (‘caged Ca2+), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA‐independent phase and a late PKA‐dependent component. We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.

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“…Thus, although there are no data to rule out that secretion of DCVs normally occurs from wild-type motor neuron somas, the reconstruction data suggest that motor neurons do not use somas to store a large reservoir of DCVs for secretion. This contrasts with neuroendocrine cells, such as chromaffin cells and pancreatic b-cells, in which DCVs number in the tens of thousands per cell and can occupy 31 and 12% of the cell soma volume, respectively (Dean 1973;Plattner et al 1997).Abnormal neuropeptide function contributes to the sluggish locomotion of unc-43 lf mutantsIf unc-43 mutants secrete native neuropeptides abnormally from their motor neuron somas instead of their axons, this could affect the animal's locomotion behavior. Similarly, the 90% reduction in axonal DCVs in motor neurons could also affect the animal's locomotion behavior.…”

mentioning

confidence: 87%

“…The DCVs in neuroendocrine and PC12 cells are much more abundant and accessible to biochemical and physiological experiments than those in neurons. For example, in chromaffin and pancreatic b-cells (both neuroendocrine cells), DCVs can number in the tens of thousands per cell and can occupy 31 and 12% of the cell volume, respectively (Dean 1973;Plattner et al 1997). Exploiting these advantages, studies in PC12 and neuroendocrine cells have revealed that DCVs arise from a regulated secretory pathway.…”

mentioning

confidence: 99%

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release fromCaenorhabditis elegansMotor Neurons

Hoover

Edwards

et al. 2014

Genetics

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Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by 90% and cell soma/dendrite DCV levels by 80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and . DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II. BOTH neurons and neuroendocrine cells rely on the controlled release of neuropeptides via dense core vesicle (DCV) exocytosis to evoke or modulate behaviors (Scheller and Axel 1984;Kupfermann 1991;T. Liu et al. 2007;Li and Kim 2008). The DCVs in neuroendocrine and PC12 cells are much more abundant and accessible to biochemical and physiological experiments than those in neurons. For example, in chromaffin and pancreatic b-cells (both neuroendocrine cells), DCVs can number in the tens of thousands per cell and can occupy 31 and 12% of the cell volume, respectively (Dean 1973;Plattner et al. 1997). Exploiting these advantages, studies in PC12 and neuroendocrine cells have revealed that DCVs arise from a regulated secretory pathway. The pathway begins in the trans Golgi, where various sorting mechanisms cause regulated secretory proteins, such as neuropeptides and their processing enzymes, to coalesce into vesicles that bud from the trans Golgi to form immature DCVs. Additional sorting of non-DCV cargos away from DCV cargos occurs as DCVs mature through this pathway (Arvan and Castle 1998;Tooze et al. 2001;Borgonovo et al. 2006).DCVs must selectively retain and protect several distinct cargos that have different physical states as they mature. These include the neuropeptide core, which is thought to be in an aggregated state, the neuropeptide-processing enzymes PC-2 convertase and carboxypeptidase E, possibly soluble cargos, and transmembrane cargos.While neurons and neuroendocrine cells share this core pathway for DCV production, neurons have evolved additional...

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Ultrastructural morphometry of the pancreatic ?-cell

Cited by 257 publications

References 12 publications

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Protein kinase A‐dependent and ‐independent stimulation of exocytosis by cAMP in mouse pancreatic B‐cells

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release fromCaenorhabditis elegansMotor Neurons

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