The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been ...demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.
Autophagy breaks down nonessential cellular components to replenish macromolecular building blocks during starvation. Nevertheless, the downstream events regulating vesicle trafficking during this ...essential cellular process are not yet fully defined. Xu et al. combined approaches of crystallography, biochemistry, and cell biology to show that the guanine nucleotide exchange factor DENND3 contains an actin-binding site they call “PHenn domain” in a region previously thought to be unstructured. PHenn domain binding to microfilaments is necessary for DENND3’s participation in autophagy, providing a new link between autophagic stimulation and actin microfilaments. The findings by Xu et al. shed important new light on how membrane trafficking participates in critical steps of autophagy in relationship with actin microfilaments.
This article describes the discovery of a novel SNARE domain that might be involved in the regulation of membrane fusion. This domain is shared by a novel family of VAMPs called long VAMPs or ...longins. Members of this family are more conserved among eukaryotes than are classical VAMPs, possibly because of their underlying basic SNARE function.
Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or ...two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.
Rabbit ileal Na + -absorbing cell Na + -H + exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately
50 % of BB NHE3 was ...associated with Triton X-100-soluble fractions and the other â¼50 % with Triton X-100-insoluble fractions;
â¼33 % of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts).
The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and
clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced
increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the
total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase
in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na + -H + exchange activity.
Disrupting rafts by removal of cholesterol with methyl-β-cyclodextrin (MβCD) or destabilizing the actin cytoskeleton with
cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically,
NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated
magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MβCD treatment.
We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3;
(iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF-
and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles
in the basal endocytosis of BB NHE3.
The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is ...necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.
Lipids in biological membranes show astonishing chemical diversity, but they also show some key conserved structures in different organisms. In addition, some of their biophysical properties have ...been related to specific functions. In this review, we aim to discuss the role of sphingolipids- and cholesterol-rich micro- and nano-membrane domains (MD) and highlight their pivotal role in lipid–protein clustering processes, vesicle biogenesis and membrane fusion. We further review potential connections between human pathologies and defects in MD biosynthesis, recycling and homeostasis. Brain, which is second only to the adipose tissues in term of lipid abundance, is particularly affected by MD defects which are linked to neurodegenerative disorders. Finally we propose a potential connection between MD and several nutrient-related processes and envision how diet and autophagy could bring insights towards understanding the impact of global lipid homeostasis on human health and disease.
•Lipid chain length is important in membrane domains' function.•Lateral segregation finds a mean in membrane bending.•Membrane domains regulate SNARE-dependent membrane fusion.•Membrane domains defects are linked to brain pathologies.
SNARE proteins are key mediators of membrane fusion. Their function in ensuring compartmental specificity of membrane fusion has been suggested by in vitro studies but not demonstrated in vivo. We ...show here that ectopic expression of the plasma membrane t-SNARE heavy chain syntaxin 1 in the endoplasmic reticulum induces the redistribution of its cognate vesicular SNAREs, TI-VAMP and cellubrevin, and its light chain t-SNARE SNAP-23. These effects were prevented by co-expressing nSec1. Expression of syntaxin 1 alone impaired the cell surface expression of TI-VAMP and cellubrevin but not the recycling of transferrin receptor. TI-VAMP, cellubrevin and SNAP-23 associated in vivo with exogenous syntaxin 1. Redistribution of TI-VAMP in the ER of syntaxin-1-expressing cells was microtubule dependent and impaired the trafficking of CD63, a cargo of TI-VAMP-containing vesicles. We conclude that the destination of v-SNAREs is driven by their specific interaction with cognate t-SNAREs. Our in vivo data provide strong support for the theory that highly specific v-SNARE-t-SNARE interactions control compartmental specificity of membrane fusion.
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