scholarly journals Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

2010 ◽  
Vol 21 (16) ◽  
pp. 2821-2831 ◽  
Author(s):  
Zhen Zhang ◽  
Enfu Hui ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Lipid Bilayers ◽  
Fusion Pore ◽  
Fusion Event ◽  
Event Frequency ◽  
Synaptic Release ◽  
Sensitive Factor ◽  
Fusion Pores ◽  
Kinetics Of ◽  
Stable Fusion

Synaptotagmin (syt) serves as a Ca2+ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca2+, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca2+ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

scholarly journals Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

2009 ◽  
Vol 20 (24) ◽  
pp. 5086-5095 ◽  
Author(s):  
Zhen Zhang ◽  
Enfu Hui ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Gradual Increase ◽  
Fusion Pore ◽  
Dependent Manner ◽  
Synaptotagmin I ◽  
Pore Opening ◽  
Kinetic Effects ◽  
Two Phases ◽  
Fusion Pores ◽  
Kinetics Of

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca2+-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca2+-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca2+-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca2+-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca2+-triggered Syt I binding is discussed along with other possible roles of PS.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

scholarly journals Characterization of the immature secretory granule, an intermediate in granule biogenesis.

1991 ◽  
Vol 115 (6) ◽  
pp. 1491-1503 ◽  
Author(s):  
S A Tooze ◽  
T Flatmark ◽  
J Tooze ◽  
W B Huttner
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Pc12 Cells ◽  
Secretory Granules ◽  
Fusion Event ◽  
Secretogranin Ii ◽  
High K ◽  
Golgi Network ◽  
Kinetics Of

The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfate-labeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell. Immature secretory granules were converted to mature secretory granules with a half-time of approximately 45 min. This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labelling of PC12 cells followed by stimulation with high K+, which causes the release of secretogranin II, showed that not only mature, but also immature secretory granules were capable of undergoing regulated exocytosis. The kinetics of secretion of secretogranin II, as well as those of a constitutively secreted heparan sulfate proteoglycan, were reduced by treatment of PC12 cells with nocodazole, suggesting that both secretory granules and constitutive secretory vesicles are transported to the plasma membrane along microtubules. Our results imply that certain membrane proteins, e.g., those involved in the fusion of post-TGN vesicles with the plasma membrane, are sorted upon exit from the TGN, whereas other membrane proteins, e.g., those involved in the interaction of post-TGN vesicles with the cytoskeleton, may not be sorted.

scholarly journals Temperature Dependence of Fusion Kinetics and Fusion Pores in Ca2+-triggered Exocytosis from PC12 Cells

2008 ◽  
Vol 131 (2) ◽  
pp. 117-124 ◽  
Author(s):  
Zhen Zhang ◽  
Meyer B. Jackson
Keyword(s):  
Temperature Dependence ◽  
Pc12 Cells ◽  
Bulk Diffusion ◽  
Fusion Pore ◽  
Spike Shape ◽  
Fusion Probability ◽  
Temperature Dependences ◽  
Membrane Components ◽  
Fusion Pores ◽  
Single Vesicle

The temperature dependence of Ca2+-triggered exocytosis was studied using carbon fiber amperometry to record the release of norepinephrine from PC12 cells. Single-vesicle fusion events were examined at temperatures varying from 12 to 28°C, and with release elicited by depolarization. Measurements were made of the initial and maximum frequencies of exocytotic events, of fusion pore lifetime, flux through the open fusion pore, kiss-and-run versus full-fusion probability, and parameters associated with the shapes of amperometric spikes. The fusion pore open-state flux, and all parameters associated with spike shape, including area, rise time, and decay time, had weak temperature dependences and activation energies in the range expected for bulk diffusion in an aqueous solution. Kiss-and-run events also varied with temperature, with lower temperatures increasing the relative probability of kiss-and-run events by ∼50%. By contrast, kinetic parameters relating to the frequency of exocytotic events and fusion pore transitions depended much more strongly on temperature, suggesting that these processes entail structural rearrangements of proteins or lipids or both. The weak temperature dependence of spike shape suggests that after the fusion pore has started to expand, structural transitions of membrane components are no longer kinetically limiting. This indicates that the content of a vesicle is expelled completely after fusion pore expansion.

scholarly journals Calcium-triggered fusion of lipid membranes is enabled by amphiphilic nanoparticles

2020 ◽  
Vol 117 (31) ◽  
pp. 18470-18476 ◽  
Author(s):  
Mukarram A. Tahir ◽  
Zekiye P. Guven ◽  
Laura R. Arriaga ◽  
Berta Tinao ◽  
Yu-Sang Sabrina Yang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Biomedical Applications ◽  
Lipid Membrane ◽  
Fusion Pore ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Kinetic Barriers

Lipid membrane fusion is an essential process for a number of critical biological functions. The overall process is thermodynamically favorable but faces multiple kinetic barriers along the way. Inspired by nature’s engineered proteins such as SNAP receptor [soluble N-ethylmale-imide-sensitive factor-attachment protein receptor (SNARE)] complexes or viral fusogenic proteins that actively promote the development of membrane proximity, nucleation of a stalk, and triggered expansion of the fusion pore, here we introduce a synthetic fusogen that can modulate membrane fusion and equivalently prime lipid membranes for calcium-triggered fusion. Our fusogen consists of a gold nanoparticle functionalized with an amphiphilic monolayer of alkanethiol ligands that had previously been shown to fuse with lipid bilayers. While previous efforts to develop synthetic fusogens have only replicated the initial steps of the fusion cascade, we use molecular simulations and complementary experimental techniques to demonstrate that these nanoparticles can induce the formation of a lipid stalk and also drive its expansion into a fusion pore upon the addition of excess calcium. These results have important implications in general understanding of stimuli-triggered fusion and the development of synthetic fusogens for biomedical applications.

scholarly journals Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion.

1993 ◽  
Vol 102 (6) ◽  
pp. 1131-1149 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
M E Peeples ◽  
F S Cohen
Keyword(s):  
Lipid Bilayers ◽  
Mdck Cells ◽  
Fusion Pore ◽  
Planar Bilayers ◽  
Influenza Hemagglutinin ◽  
Cell Membrane Capacitance ◽  
Opening And Closing ◽  
Fusion Pores ◽  
Planar Membranes ◽  
Quiescent Stage

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of-phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell-containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time-resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.

scholarly journals Nascent fusion pore opening monitored at single-SNAREpin resolution

2021 ◽  
Vol 118 (5) ◽  
pp. e2024922118
Author(s):  
Paul Heo ◽  
Jeff Coleman ◽  
Jean-Baptiste Fleury ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Energy Landscape ◽  
Three Dimensional ◽  
Fusion Pore ◽  
Fusion Event ◽  
Direct Estimate ◽  
Target Membrane ◽  
Pore Opening ◽  
Two Peaks ◽  
Fusion Pores ◽  
Second Peak

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

scholarly journals Activity-evoked and spontaneous opening of synaptic fusion pores

2019 ◽  
Vol 116 (34) ◽  
pp. 17039-17044 ◽  
Author(s):  
Dinara Bulgari ◽  
David L. Deitcher ◽  
Brigitte F. Schmidt ◽  
M. Alexandra Carpenter ◽  
Christopher Szent-Gyorgyi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Zone ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Pain Perception ◽  
Fusion Pore ◽  
Synaptic Release ◽  
Pore Opening ◽  
Active Zones ◽  
Fusion Pores

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+. SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

scholarly journals The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion

2018 ◽  
Vol 29 (7) ◽  
pp. 834-845 ◽  
Author(s):  
Mounir Bendahmane ◽  
Kevin P. Bohannon ◽  
Mazdak M. Bradberry ◽  
Tejeshwar C. Rao ◽  
Michael W. Schmidtke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Silico ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Structural Specificity ◽  
Fusion Pores ◽  
Kinetics Of ◽  
Pore Expansion

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

scholarly journals Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

2020 ◽  
Author(s):  
Sarah B. Nyenhuis ◽  
Nakul Karandikar ◽  
Volker Kiessling ◽  
Alex Kreutzberger ◽  
Anusa Thapa ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Fusion Pore ◽  
Fusion Event ◽  
Arginine Residues ◽  
Synaptotagmin 1 ◽  
Membrane Contact ◽  
Curved Membrane ◽  
Kinetics Of ◽  
Novel Model ◽  
Pore Expansion

AbstractSynaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

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