scholarly journals CK2 Phosphorylation Is Required for Regulation of Syntaxin 1A Activity in Ca2+-Triggered Release in Neuroendocrine Cells

2021 ◽  
Vol 22 (24) ◽  
pp. 13556
Author(s):  
Noa Barak-Broner ◽  
Dafna Singer-Lahat ◽  
Dodo Chikvashvili ◽  
Ilana Lotan
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Neuroendocrine Cells ◽  
Null Mutant ◽  
Spontaneous Release ◽  
Triggered Release ◽  
Charge Neutralization ◽  
Juxtamembrane Region ◽  
Molecular Determinant ◽  
Ck2 Inhibitor

The polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was previously shown by us to act as a fusion clamp in PC12 cells, as charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release. Using a Syx-based FRET probe (CSYS), we demonstrated that 5RK is required for a depolarization-induced Ca+2-dependent opening (close-to-open transition; CDO) of Syx, which involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the CDO requirement for 5RK and identified phosphorylation of Syx at Ser-14 (S14) by casein kinase 2 (CK2) as a crucial molecular determinant. Thus, following biochemical verification that both endogenous Syx and CSYS are constitutively S14 phosphorylated in PC12 cells, dynamic FRET analysis of phospho-null and phospho-mimetic mutants of CSYS and the use of a CK2 inhibitor revealed that the S14 phosphorylation confers the CDO requirement for 5RK. In accord, amperometric analysis of catecholamine release revealed that the phospho-null mutant does not support Ca2+-triggered release. These results identify a functionally important CK2 phosphorylation of Syx that is required for the 5RK-regulation of CDO and for concomitant Ca2+-triggered release. Further, also spontaneous release, conferred by charge neutralization of 5RK, was abolished in the phospho-null mutant.

scholarly journals CK2 Phosphorylation is required for Regulation of Syntaxin 1A activity in Ca2+-triggered release in neuroendocrine cells

2020 ◽  
Author(s):  
Noa Barak-Broner ◽  
Dafna Singer-Lahat ◽  
Dodo Chikvashvili ◽  
Ilana Lotan
Keyword(s):  
Pc12 Cells ◽  
Structural Transition ◽  
Phosphorylation Site ◽  
Neuroendocrine Cells ◽  
Triggered Release ◽  
Juxtamembrane Region ◽  
Molecular Determinant ◽  
Vesicle Exocytosis ◽  
Summary Statement ◽  
Kinase Ck2

AbstractThe polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was shown by us to act as a fusion clamp in PC12 cells, making release dependent on stimulation by Ca2+. By using a Syx-based FRET probe, we demonstrated that 5RK is absolutely required for a depolarization-induced Ca+2-dependent, close-to-open transition (CDO) of Syx that involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the 5RK requirement, and identified phosphorylation of Syx at Ser-14 (S14) by protein kinase CK2 as a crucial molecular determinant. Following biochemical verification that both endogenous Syx and CSYS are constitutively S14 phosphorylated in PC12 cells, dynamic FRET analysis of phospho-null and phospho-mimetic mutants of CSYS and the use of a CK2 inhibitor revealed that it is the S14 phosphorylation that confers the 5RK requirement. Concomitant amperometric analysis of catecholamine release revealed that the phospho-null mutants do not support release, spontaneous and evoked. Collectively, these results identify a functionally important CK2 phosphorylation site in Syx that is required for 5RK-regulation of CDO and for concomitant Ca2+-triggered release.Summary statementMany phospho-proteins participate in vesicle exocytosis. We show that a recently identified structural transition of syntaxin1A that accompanies Ca2+-regulated exocytosis in neuroendocrine cells is controlled by CK2 phosphorylation of syntaxin1A.

scholarly journals The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells

2017 ◽  
Vol 38 (1) ◽  
pp. 220-231 ◽  
Author(s):  
Dafna Singer-Lahat ◽  
Noa Barak-Broner ◽  
Anton Sheinin ◽  
Dafna Greitzer-Antes ◽  
Izhak Michaelevski ◽  
...  
Keyword(s):  
Neuroendocrine Cells ◽  
Dual Function ◽  
Spontaneous Release ◽  
Triggered Release ◽  
Syntaxin 1A ◽  
Juxtamembrane Region

scholarly journals Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

1997 ◽  
Vol 137 (2) ◽  
pp. 445-458 ◽  
Author(s):  
Anne Schmidt ◽  
Matthew J. Hannah ◽  
Wieland B. Huttner
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Membrane System ◽  
Neuroendocrine Cells ◽  
Immunogold Electron Microscopy ◽  
Cell Surface Biotinylation ◽  
Digitonin Permeabilization ◽  
Triton X

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

scholarly journals ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

2003 ◽  
Vol 162 (4) ◽  
pp. 647-659 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Dense Core Vesicle ◽  
Type I ◽  
Endosomal Membranes ◽  
Phosphatidylinositol 4 ◽  
Adp Ribosylation Factor

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.

scholarly journals Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

2016 ◽  
Vol 27 (4) ◽  
pp. 654-668 ◽  
Author(s):  
Greg Kabachinski ◽  
D. Michelle Kielar-Grevstad ◽  
Xingmin Zhang ◽  
Declan J. James ◽  
Thomas F. J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Protein Complexes ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Snare Complex ◽  
Snare Protein ◽  
Dense Core Vesicles ◽  
Vesicle Docking ◽  
Single Vesicle

The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.

scholarly journals Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells

2002 ◽  
Vol 115 (11) ◽  
pp. 2399-2412 ◽  
Author(s):  
Bethe A. Scalettar ◽  
Patrizia Rosa ◽  
Elena Taverna ◽  
Maura Francolini ◽  
Takashi Tsuboi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Growth Cones ◽  
Neuroendocrine Cells ◽  
Beta Activity ◽  
Calcium Sensor ◽  
Neuronal Calcium Sensor

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

1999 ◽  
Vol 112 (12) ◽  
pp. 1957-1965 ◽  
Author(s):  
K. Venkateswarlu ◽  
F. Gunn-Moore ◽  
J.M. Tavare ◽  
P.J. Cullen
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Head Group ◽  
Nucleotide Exchange ◽  
Ph Domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

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 Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

2004 ◽  
Vol 279 (50) ◽  
pp. 52677-52684 ◽  
Author(s):  
Mitsunori Fukuda ◽  
Eiko Kanno ◽  
Megumi Satoh ◽  
Chika Saegusa ◽  
Akitsugu Yamamoto
Keyword(s):  
Plasma Membrane ◽  
Neuropeptide Y ◽  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Small Interfering Rna ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Pc12 Cell Line ◽  
Interfering Rna

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx

2016 ◽  
Vol 124 (4) ◽  
pp. 878-884 ◽  
Author(s):  
Liping Han ◽  
Stephen Fuqua ◽  
Quanlin Li ◽  
Liyu Zhu ◽  
Xiaoyan Hao ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Calcium Influx ◽  
Sympathetic Neurons ◽  
Reduce Blood Pressure ◽  
Catecholamine Release ◽  
Unexpected Finding ◽  
Norepinephrine Release ◽  
Triggered Release ◽  
Induced Hypotension ◽  
High K

Abstract Background Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines. Methods The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K+-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca2+ ionophore, directly induces Ca2+ influx, thus bypassing the effect of ion channel modulation by propofol. Results Propofol decreased depolarization (high K+)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 μM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca2+ (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 μM; n = 6). Conclusions Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca2+ influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

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