scholarly journals High-Speed Imaging Reveals the Bimodal Nature of Dense Core Vesicle Exocytosis and Jet Flow through the Fusion Pore

2021 ◽  
Author(s):  
Pengcheng Zhang ◽  
David Rumschitzki ◽  
Robert H Edwards
Keyword(s):  
High Speed ◽  
Secretory Vesicle ◽  
Fusion Pore ◽  
Dense Core Vesicle ◽  
Intrinsic Properties ◽  
High Speed Imaging ◽  
Stochastic Variation ◽  
Vesicle Exocytosis ◽  
Flow Through ◽  
Dual Imaging

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Osmotic forces contribute to exocytosis but release through the pore is thought to occur by diffusion. Heterogeneity of fusion pore behavior has also suggested stochastic variation in a common exocytic mechanism, implying a lack of biological control. Imaging at millisecond resolution to observe the first events in exocytosis, we find that fusion pore duration is bimodal rather than stochastic. Loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. In addition, dual imaging shows a delay in the entry of external dye relative to release that indicates discharge at high velocity rather than strictly by diffusion.

scholarly journals SCAMP2 Interacts with Arf6 and Phospholipase D1 and Links Their Function to Exocytotic Fusion Pore Formation in PC12 Cells

2005 ◽  
Vol 16 (10) ◽  
pp. 4463-4472 ◽  
Author(s):  
Lixia Liu ◽  
Haini Liao ◽  
Anna Castle ◽  
Jie Zhang ◽  
James Casanova ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Pore Formation ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Small Gtpase ◽  
Fusion Pore ◽  
Dense Core Vesicle ◽  
Nucleotide Exchange ◽  
Phospholipase D1 ◽  
Vesicle Exocytosis

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5′-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.

Nonredundant function of secretory carrier membrane protein isoforms in dense core vesicle exocytosis

2008 ◽  
Vol 294 (3) ◽  
pp. C797-C809 ◽  
Author(s):  
Haini Liao ◽  
Jie Zhang ◽  
Svetlana Shestopal ◽  
Gabor Szabo ◽  
Anna Castle ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dense Core ◽  
Protein Isoforms ◽  
Fusion Pore ◽  
Dense Core Vesicle ◽  
Vesicle Exocytosis ◽  
Pore Opening ◽  
Pore Closure ◽  
Secretory Carrier Membrane Proteins ◽  
Pore Dynamics

Five secretory carrier membrane proteins (SCAMP-1, -2, -3, -4, and -5) have been characterized in mammalian cells. Previously, SCAMP-1 and -2 have been implicated to function in exocytosis. RNA inhibitor-mediated deficiency of one or both of these SCAMPs interferes with dense core vesicle (DCV) exocytosis in neuroendocrine PC12 cells as detected by amperometry. Knockdowns of these SCAMPs each decreased the number and frequency of depolarization-induced exocytotic events. SCAMP-2 but not SCAMP-1 depletion also delayed the onset of exocytosis. Both knockdowns, however, altered fusion pore dynamics, increasing rapid pore closure and decreasing pore dilation. In contrast, knockdowns of SCAMP-3 and -5 only interfered with the frequency of fusion pore opening and did not affect the dynamics of newly opened pores. None of the knockdowns noticeably affected upstream events, including the distribution of DCVs near the plasma membrane and calcium signaling kinetics, although norepinephrine uptake/storage was moderately decreased by deficiency of SCAMP-1 and -5. Thus, SCAMP-1 and -2 are most closely linked to the final events of exocytosis. Other SCAMPs collaborate in regulating fusion sites, but the roles of individual isoforms appear at least partially distinct.

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 Flagellar synchronization through direct hydrodynamic interactions

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Douglas R Brumley ◽  
Kirsty Y Wan ◽  
Marco Polin ◽  
Raymond E Goldstein
Keyword(s):  
Time Series ◽  
Stochastic Processes ◽  
Spatial Dependence ◽  
High Speed ◽  
Hydrodynamic Interactions ◽  
Intrinsic Properties ◽  
High Speed Imaging ◽  
Volvox Carteri ◽  
Hydrodynamic Coupling ◽  
Analysis Of Time Series

Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.

Experimental Measurement of In-Plane Rolling Nonpneumatic Tire Vibrations Using High-Speed Imaging

2019 ◽  
Vol 47 (3) ◽  
pp. 196-210
Author(s):  
Meghashyam Panyam ◽  
Beshah Ayalew ◽  
Timothy Rhyne ◽  
Steve Cron ◽  
John Adcox
Keyword(s):  
High Speed ◽  
Image Correlation ◽  
Dynamic Mode Decomposition ◽  
Dynamic Mode ◽  
High Speed Imaging ◽  
Correlation Algorithm ◽  
Mode Decomposition ◽  
Series Of Experiments ◽  
Plane Deformations ◽  
Dominant Frequencies

ABSTRACT This article presents a novel experimental technique for measuring in-plane deformations and vibration modes of a rotating nonpneumatic tire subjected to obstacle impacts. The tire was mounted on a modified quarter-car test rig, which was built around one of the drums of a 500-horse power chassis dynamometer at Clemson University's International Center for Automotive Research. A series of experiments were conducted using a high-speed camera to capture the event of the rotating tire coming into contact with a cleat attached to the surface of the drum. The resulting video was processed using a two-dimensional digital image correlation algorithm to obtain in-plane radial and tangential deformation fields of the tire. The dynamic mode decomposition algorithm was implemented on the deformation fields to extract the dominant frequencies that were excited in the tire upon contact with the cleat. It was observed that the deformations and the modal frequencies estimated using this method were within a reasonable range of expected values. In general, the results indicate that the method used in this study can be a useful tool in measuring in-plane deformations of rolling tires without the need for additional sensors and wiring.

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