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.

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 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 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 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.

scholarly journals Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion

2021 ◽  
Vol 8 ◽  
Author(s):  
Josh Pierson ◽  
Yeon-Kyun Shin
Keyword(s):  
Neurotransmitter Release ◽  
Fluorescence Probe ◽  
Mechanistic Model ◽  
Reciprocal Relationship ◽  
Fusion Pore ◽  
Vesicle Recycling ◽  
Binding Core ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

Temperature Dependences in the O + OH → O2 + H Reaction. Quasiclassical Trajectory Calculation

1993 ◽  
Vol 58 (2) ◽  
pp. 234-243 ◽  
Author(s):  
Viliam Klimo ◽  
Martina Bittererová ◽  
Stanislav Biskupič ◽  
Ján Urban ◽  
Miroslav Micov
Keyword(s):  
Temperature Dependence ◽  
Energy Surface ◽  
Analytical Form ◽  
Mean Values ◽  
Quantum Numbers ◽  
Mean Lifetime ◽  
Temperature Dependences ◽  
Quasiclassical Trajectory Calculation ◽  
The Mean ◽  
Combustion Processes

The reaction O + OH → O2 + H in conditions of combustion of hydrocarbons and polymers was modelled by using the method of quasiclassical trajectories. The potential energy surface was determined by the multiconfiguration interaction method and fitted with the analytical form of the extended LEPS function. Attention was paid to the mean values of the vibrational and rotational quantum numbers of O2 molecules and their temperature dependence. The temperature dependence of the mean lifetime of the OOH collision complex was also examined. The calculated rate constants were analyzed and compared with the experimental data over the temperature region of the combustion processes.

The Urbach Rule for the Sodium- and Potassium-Halides

1974 ◽  
Vol 29 (1) ◽  
pp. 145-157 ◽  
Author(s):  
Tetsuhiko Tomiki ◽  
Takeo Miyata ◽  
Hirokazu Tsukamoto
Keyword(s):  
Temperature Dependence ◽  
Acoustic Phonon ◽  
Frequency Factor ◽  
Host Lattice ◽  
Exciton Peak ◽  
Urbach Rule ◽  
Temperature Dependences ◽  
Hole Band ◽  
Sodium And Potassium ◽  
Potassium Halides

Phenomenological and physical aspects of the intrinsic tail spectra of the alkalihalides are studied referring to the new results on the intrinsic tail spectra of KBr and KI and to the temperature dependences of the lowest-energy Γ-exciton peak of the sodium- and potassium-halides. Systematically analysing the temperature dependence of the steepness parameter σs (T) of the Urbach rule for these halides, it is found that the frequency factor has the value nearly equal to the acoustic phonon energy at X or L of each host lattice and the steepness constant σs0 becomes larger in passing from fluoride to iodide. This halogen dependence of σs0 is discussed in terms of the hole band-mass of the Γ8-level.

scholarly journals Synaptotagmin-Ca2+triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation

2006 ◽  
Vol 570 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Chih-Tien Wang ◽  
Jihong Bai ◽  
Payne Y. Chang ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Fusion Pore ◽  
Regulated Exocytosis ◽  
Pore Opening ◽  
Pore Dilation

scholarly journals The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Radhika C. Desai ◽  
Bimal Vyas ◽  
Cynthia A. Earles ◽  
J. Troy Littleton ◽  
Judith A. Kowalchyck ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Essential Step ◽  
Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

Sign in / Sign up

Export Citation Format

Share Document