scholarly journals ‘Full fusion’ is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

2017 ◽  
Vol 473 (2197) ◽  
pp. 20160684 ◽  
Author(s):  
Alexander Oleinick ◽  
Irina Svir ◽  
Christian Amatore
Keyword(s):  
Cell Membrane ◽  
Endocrine Cells ◽  
Fusion Pore ◽  
Spike Time ◽  
Vesicle Membrane ◽  
Analytical Formulation ◽  
Vesicular Exocytosis ◽  
Time Courses ◽  
Fusion Pores ◽  
Pore Expansion

Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (‘Kiss-and-Run’ events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane—a stage called ‘full fusion’. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of ‘full fusion’.

scholarly journals Fusion Pore Expansion and Contraction during Catecholamine Release from Endocrine Cells

2020 ◽  
Vol 119 (1) ◽  
pp. 219-231
Author(s):  
Meyer B. Jackson ◽  
Yu-Tien Hsiao ◽  
Che-Wei Chang
Keyword(s):  
Endocrine Cells ◽  
Catecholamine Release ◽  
Fusion Pore ◽  
Pore Expansion

scholarly journals Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

2001 ◽  
Vol 155 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Thomas Haller ◽  
Paul Dietl ◽  
Kristian Pfaller ◽  
Manfred Frick ◽  
Norbert Mair ◽  
...  
Keyword(s):  
Pore Diameter ◽  
Fusion Pore ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Intact Cells ◽  
Fusion Pores ◽  
Pyridinium Dibromide ◽  
Pore Expansion

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

scholarly journals Intracellular curvature-generating proteins in cell-to-cell fusion

2011 ◽  
Vol 440 (2) ◽  
pp. 185-193 ◽  
Author(s):  
Jean-Philippe Richard ◽  
Evgenia Leikina ◽  
Ralf Langen ◽  
William Mike Henne ◽  
Margarita Popova ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Cell Metabolism ◽  
Syncytium Formation ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Intracellular Membrane ◽  
Expansion Stage ◽  
Membrane Bending ◽  
Fusion Pores ◽  
Pore Expansion

Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P2, an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.

Fusion Pore: An Evolutionary Invention of Nucleated Cells

2010 ◽  
Vol 18 (3) ◽  
pp. 347-364 ◽  
Author(s):  
N. Vardjan ◽  
M. Stenovec ◽  
J. Jorgačevski ◽  
M. Kreft ◽  
R. Zorec
Keyword(s):  
Plasma Membrane ◽  
Blood Stream ◽  
Fusion Pore ◽  
The Novel ◽  
Vesicle Membrane ◽  
Signalling Molecules ◽  
Fundamental Biological Process ◽  
Chemical Synapses ◽  
Fusion Pores ◽  
Single Vesicle

This article outlines the lecture presented by Robert Zorec at the Academia Europea meeting in Liverpool on 19 September 2008, four decades after the Sherrington Lecture of Bernard Katz who, together with his colleagues, developed a number of paradigms addressing vesicles in chemical synapses. Vesicles are subcellular organelles that evolved in eukaryotic cells 1000 to 2000 million years ago. They store signalling molecules such as chemical messengers, which are essential for the function of neurons and endocrine cells in supporting the communication between tissues and organs in the human body. Upon a stimulus, the vesicle-stored signalling molecules (neurotransmitters or hormones) are released from cells. This event involves exocytosis, a fundamental biological process, consisting of the merger of the vesicle membrane with the plasma membrane. The two fusing membranes lead to the formation of an aqueous channel – the fusion pore – through which signalling molecules exit into the extracellular space or blood stream. The work of Bernard Katz and colleagues considered that vesicle cargo discharge initially requires the delivery of vesicles to the plasma membrane, where vesicles dock and get primed for fusion with the plasma membrane, and that stimulation initiates the formation of the transient fusion pore through which cargo molecules leave the vesicle lumen in an all-or-none-fashion. However, recent studies indicate that this may not be so simple. Here we highlight the novel findings which indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. At least in pituitary lactotrophs, which are the subject of research in our laboratories, single vesicle release of peptide signalling molecules involves modulation of fusion pore diameter and fusion pore kinetics.

scholarly journals Deletion of the Cytoplasmic Tail of the Fusion Protein of the Paramyxovirus Simian Virus 5 Affects Fusion Pore Enlargement

2001 ◽  
Vol 75 (11) ◽  
pp. 5363-5369 ◽  
Author(s):  
Rebecca Ellis Dutch ◽  
Robert A. Lamb
Keyword(s):  
Cytoplasmic Tail ◽  
Simian Virus ◽  
Fusion Pore ◽  
Wild Type ◽  
Direct Role ◽  
F Protein ◽  
Simian Virus 5 ◽  
Fusion Pores ◽  
Pore Enlargement ◽  
Pore Expansion

ABSTRACT The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail− or FΔ19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail− mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail− mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail− is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.

scholarly journals Roles for the SNAP25 linker domain in the fusion pore and a dynamic plasma membrane SNARE “acceptor” complex

2020 ◽  
Vol 152 (9) ◽  
Author(s):  
Ronald W. Holz ◽  
Mary A. Bittner
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Fusion Reaction ◽  
Snare Complex ◽  
Fusion Pore ◽  
Protein Protein Interactions ◽  
Vesicle Membrane ◽  
Acceptor Complex ◽  
Linker Domain ◽  
Pore Expansion

Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein–protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein–lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE “acceptor” complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion.

scholarly journals Fusion pores and their control of neurotransmitter and hormone release

2017 ◽  
Vol 149 (3) ◽  
pp. 301-322 ◽  
Author(s):  
Che-Wei Chang ◽  
Chung-Wei Chiang ◽  
Meyer B. Jackson
Keyword(s):  
Concentration Profile ◽  
Extracellular Space ◽  
Endocrine Cells ◽  
Molecular Mechanisms ◽  
Fusion Pore ◽  
Hormone Release ◽  
Cellular Regulation ◽  
Underlying Mechanisms ◽  
Multiple Signaling ◽  
Fusion Pores

Ca2+-triggered exocytosis functions broadly in the secretion of chemical signals, enabling neurons to release neurotransmitters and endocrine cells to release hormones. The biological demands on this process can vary enormously. Although synapses often release neurotransmitter in a small fraction of a millisecond, hormone release can be orders of magnitude slower. Vesicles usually contain multiple signaling molecules that can be released selectively and conditionally. Cells are able to control the speed, concentration profile, and content selectivity of release by tuning and tailoring exocytosis to meet different biological demands. Much of this regulation depends on the fusion pore—the aqueous pathway by which molecules leave a vesicle and move out into the surrounding extracellular space. Studies of fusion pores have illuminated how cells regulate secretion. Furthermore, the formation and growth of fusion pores serve as a readout for the progress of exocytosis, thus revealing key kinetic stages that provide clues about the underlying mechanisms. Herein, we review the structure, composition, and dynamics of fusion pores and discuss the implications for molecular mechanisms as well as for the cellular regulation of neurotransmitter and hormone release.

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 Roles for the SNAP25 Linker Domain in the Fusion Pore and a Dynamic Plasma Membrane SNARE Acceptor Complex

2020 ◽  
Author(s):  
Ronald Holz ◽  
Mary Bittner
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Fusion Reaction ◽  
Snare Complex ◽  
Fusion Pore ◽  
Protein Protein Interactions ◽  
Vesicle Membrane ◽  
Acceptor Complex ◽  
Linker Domain ◽  
Pore Expansion

A recent paper demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion (Shaaban et al., 2019, Elife. 8). Remarkably, lipid perturbing agents rescue a palmitoylation-deficient phenotype that includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Furthermore, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE acceptor complex for synaptobrevin2 (Jiang, et al., 2019, FASEB J. 33:7985-7994;Shaaban et al., 2019, Elife. 8). This insight, together with biophysical and optical studies from other laboratories (Wang, et al., 2008, Molecular Biology of the Cell. 19:3944-3955; Zhao, et al., 2013, Proc Natl Acad Sci U S A. 110:14249-14254) suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the resulting trans SNARE complex with the v-SNARE synaptobrevin form just milliseconds before fusion.

scholarly journals Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

2021 ◽  
Vol 9 ◽  
Author(s):  
Ryan Khounlo ◽  
Brenden J. D. Hawk ◽  
Tung-Mei Khu ◽  
Gyeongji Yoo ◽  
Nam Ki Lee ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Snare Complex ◽  
Fusion Pore ◽  
Fusion Inhibitors ◽  
Important Regulator ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

Sign in / Sign up

Export Citation Format

Share Document