Fusion between vesicles

This kind of process is interesting in several respects. It is a way to induce reactivity between the solutes entrapped in two different vesicle species. Fusion between vesicles is also a way to increase the molecular complexity of the incorporated species for example, one can bring together enzymes and nucleic acids, or more enzyme species in order to induce, in principle, a metabolic cycle, etc. 

Specificity in fusion between vesicles Involves two discrete and sequential processes. Describe the first of the two processes and its regulation by GTPase switch proteins. What effect on the size of early endosomes might result from overexpression of a mutant form of Rab5 that Is stuck in the GTP-bound state ... 

Fusion events may be homotypic or heterotypic. Homotypic fusion indicates fusion between membranes that originate from the same compartment (i.e. fusion of ER-derived vesicles to form tubular vesicular clusters, see below). Heterotypic membrane fusion indicates fusion of membranes originating from different compartments (i.e. synaptic vesicles and the plasma membrane). Triggered fusion like neurotransmitter release is typically heterotypic. 

Early endosomes antigen 1, which regulates fusion between endocytic vesicles... 

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Figure 4.8 The hypothetical prebiotic fusion between nucleotide- and peptide-containing vesicles.

The preparation of giant liposomes79) made it possible to apply electrical field-induced fusion to the fusion of vesicles made from natural and polymerizable lipids. Since in dielectrophoresis the net force pulling vesicles towards higher field intensities is proportional to the volume of the particle, only large vesicles can be aligned in parallel to the field lines between the electrodes. 

In principle, the activation of a presynaptic ionotropic receptor may either increase or decrease the amount of transmitter being released. However, the effect may depend on the situation prior to the activation of the receptor. In this context one has to mention that transmitter release occurs either due to spontaneous fusion of vesicles with the plasma membrane or as a consequence of an event that triggers increases in the intracellular Ca2+ concentration at the active zone where vesicle exocytosis takes place. The Ca2+ concentrations required to promote exocytosis towards maximal rates lie between approximately 10 and 100 pM (Augustine 2001 Schneggenburger and Neher 2005), depending on the synapse being investigated. These two types of transmitter release are usually named spontaneous and stimulated (or stimulation-evoked) release, respectively. 

Amatore C, Arbault S, Bonifas I, Bouret Y, Erard M, et al. 2005. Correlation between vesicle quantal size and fusion pore chromaffin cell exocytosis. Biophys J 88 4411-4420. 

FIGURE 6.10 The neuromuscular junction. The region of contact between one nerve and another nerve, or between a nerve and a muscle cell, is called a s)maptic cleft. The secretory vesicles are represented by circles in which acetylcholine is represented by dots. The nerve impulse provokes the entry of calcium ions (not shown) from the extracellular fluid into the nerve cell. Calcium ions act as a signal that stimulates the fusion of vesicles with the plcisma membrane, releasing acetylcholine into the extracellular fluid. Acetylcholine binds to membrane-bound proteins (acetylcholine receptors) on the plasma membrane of the muscle cell, resulting in stimulation of the muscle cell. Acetylcholinesterase of the neuromuscular junction catalyzes the destruction of acetylcholine in the moments after transmission of the nerve impulse. The enzyme is extracellular and is bound to proteoglycan, a molecule of extracellular matrix. 

When a DC pulse is applied to a couple of fluid-phase vesicles, which are in contact and oriented in the direction of the field, electrofusion can be observed. Vesicle orientation (and even alignment into pearl chains) can be achieved by application of an AC field to a vesicle suspension. This phenomenon is also observed with cells [164, 165] and is due to dielectric screening of the field. When the suspension is dilute, two vesicles can be brought together via the AC field and aligned. A subsequent application of a DC pulse to such a vesicle couple can lead to fusion. The necessary condition is that poration is induced in the contact area between the two vesicles. The possible steps of the electrofusion of two membranes are schematically illustrated in Figure 7.8a. In Sections 7.5.2.1 and 7.5.2.2, consideration will be given to the fusion of vesicles with different membrane composition or different composition of the enclosed solutions. 

Rab proteins exist in all cells and form the largest branch of the Ras superfamily. This family performs a central function in vesicular transport. Rab proteins influence and regulate the budding, targeting, docking and fusion of vesicles as well as processes of exocytosis and endocytosis involving clathrin-coated vesicles. During these functions, Rab proteins cycle between the cytosol and the cell membrane, and this cyle is superimposed on a GDP/GTP cycle. The cytosolic pool of the Rab pro-... 

The regions of the presynaptic membrane where fusion of vesicles and the plasmalemma occur are limited to what has been termed the active zones. Closely associated with these active zones are what electron microscopists believe may be clusters of calcium ionophores necessarv for the entry of Ca + for the initiation of exocytotic release. Other morphological entities at the active zone have also been identified, but their physiological role in transmitter release has not been elucidated. It should be noted that numerous freeze-fracture micrographs taken of active synapses reveal many more vesicle fusions than would be predicted for one or two release events. These observations are not consistent with the one vesicle-one quantum hypothesis that was briefly discussed earlier. To date, no explanation for the discrepancy between the number of vesicles and the number of quanta released has been proposed except to suggest that the vesicle, in fact, only releases a fraction of the quantum, which has been termed a microquantum. 

Using the in vitro liposome fusion assay, researchers have tested the ability of various combinations of individual v-SNARE and t-SNARE proteins to mediate fusion of donor and target membranes. Of the very large number of different combinations tested, only a small number mediated membrane fusion. To a remarkable degree the functional combinations of v-SNAREs and t-SNAREs revealed in these in vitro experiments correspond to the actual SNARE protein interactions that mediate known membrane-fusion events in the yeast cell. Thus the specificity of the interaction between SNARE proteins can account for the specificity of fusion between a particular vesicle and its target membranes. 

Lumina at the nuclear face of the Golgi apparatus (Figure 4.33) are termed cis cisternae those at the apical face trans cisternae. Proteins appear to enter the complex at the cis face and progress, undergoing post-translational modification, towards the trans face. Transfer between adjacent Golgi cisternae is thought to be achieved by budding and subsequent fusion of vesicles. 

Figure 17.5 Experimental methods to delivery a second reactant Y inside a X-containing vesicle. (1) Free (passive) diffusion ofY from outside to vesicle inside. (2) Fusion between two or more vesicles. (3) Microinjection of Y inside a giant vesicle. (4) Keeping the vesicles at the phase transition temperature (or by thermal cycles around T ). The permeability of lipid membranes is generally maximal at T - (5) Adding detergents at sublytic concentration, so that the membrane permeability is increased (especially for small solutes) without dramatic changes of membrane integrity. (6) Incorporation of pore-forming compounds in liposomes (a-hemolysin, OmpF porin,. ..)...

K. Tsumoto, K. Kamiya, T. Yoshimura, Membrane fusion between a giant vesicle and small enveloped particles possibilities for the application to construct model cells. Micro-NanoMechatronics and Human Science, 2006, International Symposium on 2006, pp. 1-6. 

Orsel, J.G. Bartoldus, I. Stegmann. T. Kinetics of fusion between endoplasmic recticulum vesicles in vitro. J. Biol. Chem. 1997, 272 (6). 3369 - 3375. 

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