scholarly journals Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on BDNF discharge following fusion

2017 ◽  
Author(s):  
Mary A. Bittner ◽  
Kevin P. Bohannon ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Granules ◽  
Normal Function ◽  
Fusion Pore ◽  
Cellular Processes ◽  
Amphipathic Helices ◽  
Curved Structures ◽  
Amino Acid Repeats ◽  
Bovine Chromaffin Cells ◽  
Control Protein

AbstractSynuclein is strongly implicated in the pathogenesis of Parkinson’s disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, β-, and γ-synuclein are comprised of seven 11-amino acid repeats that are predicted to form amphipathic helices. α-Synuclein binds to negatively charged lipids, especially small vesicles and tubulates and vesiculates lipids. The membrane-binding and membrane-curving abilities raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous β-synuclein associates with secretory granules. Endogenous α-synuclein only rarely is found on secretory granules. Overexpression of α-synuclein but not β-synuclein quickened the median duration of the post-fusion discharge of BDNF-pHluorin by 30%, consistent with α-synuclein speeding fusion pore expansion.

scholarly journals Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on post-fusion BDNF discharge and fusion pore expansion

2019 ◽  
Vol 699 ◽  
pp. 134-139 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Kevin P. Bohannon ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Granules ◽  
Fusion Pore ◽  
Pore Expansion

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.

Rab3 is present on endosomes from bovine chromaffin cells in primary culture

1999 ◽  
Vol 112 (5) ◽  
pp. 641-649
Author(s):  
D. Slembrouck ◽  
W.G. Annaert ◽  
J.M. Wang ◽  
W.P. Potter
Keyword(s):  
Primary Culture ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Regulatory Function ◽  
Secretory Vesicles ◽  
Granule Formation ◽  
Subcellular Compartment ◽  
Starting Point ◽  
Bovine Chromaffin Cells ◽  
Vesicle Cycle

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent exocytosis. Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granules in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several groups that Rab3a dissociates from the secretory vesicle membrane during stimulated exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This ‘light’ membraneous structure could be the starting point for reassociation of Rab3a with membranes involved in granule formation, or it could be a structure unrelated to granules. The present study used several subcellular fractionation techniques and immunomicroscopy to unravel the nature of the ‘light’ Rab3a-containing structures from bovine chromaffin cells in primary culture. After stimulation, amounts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (DbetaH) increase transiently in sucrose gradient fractions enriched in endosomal markers. A diaminobenzidine-induced density shift of endosomes alters the distribution of DbetaH and Rab3a-d. At the ultrastructural level, subplasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold labelling. Taken together our data provide for the first time evidence that internalised secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates that the endosome is an important trafficking route in the biogenesis/recycling of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alternative recycling pathways for the chromaffin granule membrane.

The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells

1995 ◽  
Vol 108 (4) ◽  
pp. 1639-1649 ◽  
Author(s):  
F. Darchen ◽  
J. Senyshyn ◽  
W.H. Brondyk ◽  
D.J. Taatjes ◽  
R.W. Holz ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Pc12 Cells ◽  
Immunoelectron Microscopy ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Bovine Chromaffin Cells ◽  
Specific Association

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.

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 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.

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