Thierry Galli

A Common Exocytotic Mechanism Mediates Axonal and Dendritic Outgrowth

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.

Na+‐H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3

1 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 ∼50 % with Triton X‐100‐insoluble fractions; ∼33 % of the detergent‐insoluble NHE3 was present in cholesterol‐enriched lipid microdomains (rafts). 2 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. 3 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. 4 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.

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP‐25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP‐23, an analogue of SNAP‐25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP‐23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t‐SNAREs SNAP‐23 and syntaxin 1 and the v‐SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis. GLIA 27:181–187, 1999.

Longins: a new evolutionary conserved VAMP family sharing a novel SNARE domain

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.

The Rod cGMP Phosphodiesterase δ Subunit Dissociates the Small GTPase Rab13 from Membranes

Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase δ subunit, was named human δ-PDE. The δ-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged δ-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-δ-PDE subcellular distribution. Purified recombinant δ-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that δ-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.

D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.

Exocytosis: SNAREs drum up!

Whereas the physiology of neuronal secretion has been well documented for the past 40 years, a molecular model was lacking. A breakthrough came with the SNARE hypothesis proposed by Rothman and coll. (So ̈llneret al., 1993a,b) (Fig. 1). In this model, synaptobrevin, a protein of the synaptic vesicle forms a complex with syntaxin and SNAP25, proteins of the neuronal plasma membrane (SNARE complex or 7S complex) (So ̈llner et al., 1993a,b). Binding of the small N-ethyl maleimide sensitive fusion protein (NSF) attachment proteins (SNAPs) (McMahonet al., 1995) and of the ATPase NSF (Söllner et al., 1993a) to the 7S complex yields a 20S complex. Subsequent ATP-hydrolysis by NSF is required to disassemble the 20S complex. These steps are proposed to lead to membrane fusion. The SNARE hypothesis was greeted with enthusiasm as it provided the first molecular model for the exocytotic process. Meanwhile, a large body of evidence has accumulated that suggests a role for the SNAREs at the heart of exocytosis. We review these experiments, and we point out to what extent the original hypothesis has been modified to take into account some of the seemingly contradictory data.

Clostridial neurotoxin‐insensitive vesicular SNAREs in exocytosis and endocytosis

The SNARE model of membrane fusion was proposed by Rothman and coworkers following their discovery of a protein complex linking synaptic vesicles to the neuronal plasma membrane (Sollner et al., 1993a,b). In the original model, synaptobrevin 2 (also called Vesicle Associated Membrane Protein 2, or VAMP2), a synaptic vesicle protein, and syntaxin 1 and Synaptosomal Associated Protein of 25 kD (SNAP25), two neuronal plasma membrane proteins, were proposed to mediate membrane fusion through the formation of a complex. This hypothesis has now been largely confirmed and generalised. Indeed, SNARE proteins have been identified in all organisms from yeast and mammals, and they are involved in all intracellular membrane fusion events, thus allowing for vesicle trafficking, cell polarisation and plasma membrane expansion (for review see Jahn and Sudhof, 1999). In the most recent period, key experiments have allowed to answer important questions raised by the original SNARE model. Indeed, the elucidation of the structure of the complex showing parallel orientation of vesicle (v–) and target (t–) SNAREs (Sutton et al., 1998), the demonstration that SNAREs are not only necessary but also sufficient for lipid bilayer fusion (Weber et al., 1998), together with the recent results showing that a significant degree of specificity is encoded in SNARE proteins themselves (McNew et al., 2000; Scales et al., 2000) have helped to strengthen and refine the SNARE model. The pioneering work of the groups of H. Niemann and C. Montecucco demonstrated that the pre-synaptic SNARE proteins, syntaxin 1, SNAP25, and synaptobrevin 2, are the targets of clostridial neurotoxins. This property has been crucial to define their function in several studies (for review see Niemann et al., 1994; Schiavo et al., 1994; Humeau et al., 2000). Most importantly, the first evidence for a role of a SNARE protein in exocytosis was deduced from the association of tetanus neurotoxin-induced block of secretion with its proteolytic activity on synaptobrevin 2 (Link et al., 1992; Schiavo et al., 1992). Nevertheless, several transport pathways are either resistant or only partially affected by clostridial neurotoxins. Among those, apical transport in polarised cells (Ikonen et al., 1995), and axonal outgrowth in neurons (Osen-Sand et al., 1996) are not inhibited by tetanus neurotoxin (for review see Johannes and Galli, 1998). The recent identification of neurotoxin-resistant SNAREs allowed to reconcile the lack of effect of clostridial neurotoxins with the implication of the SNARE proteins in all known membrane fusions. The aim of this review is to briefly discuss the latest studies on the tetanus neurotoxin-insensitive VAMP (TI-VAMP, also known as VAMP7), and endobrevin (also called VAMP8), two clostridial neurotoxinresistant v–SNAREs.