MICAL-L1 is required for cargo protein delivery to the cell surface.

Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons.

Alteration of axonal transport has emerged as a common precipitating factor in several neurodegenerative disorders including Human Spastic Paraplegia (HSP). Mutations of the SPAST (SPG4) gene coding for the spastin protein account for 40% of all autosomal dominant uncomplicated HSP. By cleaving microtubules, spastin regulates several cellular processes depending on microtubule dynamics including intracellular membrane trafficking. Axonal transport is fundamental for the viability of motor neurons which often have very long axons and thus require efficient communication between the cell body and its periphery. Here we found that the anterograde velocity of VAMP7 vesicles, but not that of VAMP2, two vesicular-SNARE proteins implicated in neuronal development, is enhanced in SPG4-KO neurons. We showed that this effect is associated with a slight increase of the level of acetylated tubulin in SPG4-KO neurons and correlates with an enhanced activity of kinesin-1 motors. Interestingly, we demonstrated that an artificial increase of acetylated tubulin by drugs reproduces the effect of Spastin KO on VAMP7 axonal dynamics but also increased its retrograde velocity. Finally, we investigated the effect of microtubule targeting agents which rescue axonal swellings, on VAMP7 and microtubule dynamics. Our results suggest that microtubule stabilizing agents, such as taxol, may prevent the morphological defects observed in SPG4-KO neurons not simply by restoring the altered anterograde transport to basal levels but rather by increasing the retrograde velocity of axonal cargoes.

Effects of TENS in living kidney donors submitted to open nephrectomy: a randomized placebo-controlled trial.

BACKGROUND: Pain is a negative factor in the recovery process of postoperative patients. It causes pulmonary alterations and complications, and it also affects functional capacity. Several studies have investigated the effects of transcutaneous electrical nerve stimulation (TENS) during the postoperative period. However, no studies have assessed the effects of TENS on kidney donors. Thus, the aim of the present study was to evaluate the effect of TENS on pain, walking function, respiratory muscle strength and vital capacity in kidney donors. METHODS: Seventy-four patients were randomly allocated into two groups: active TENS or placebo TENS. All patients were assessed for pain intensity, respiratory muscle strength, vital capacity and walking function before and after the TENS application on the first day of the postoperative period. RESULTS: The use of active TENS significantly reduced pain at rest (p = 0.006), during the measurement of maximal inspiratory pressure (p = 0.006), during maximal expiratory pressure (p = 0.004) and during vital capacity (p = 0.013). Active TENS also produced a significant increase in maximal expiratory pressure when compared with the placebo TENS group (p = 0.001). Maximal inspiratory pressure, vital capacity and walking function were not significantly different between the two treatment groups. CONCLUSIONS: These results suggest that TENS decreases pain intensity at rest and during respiratory manoeuvres and increases maximal expiratory pressure during the postoperative period in kidney donors after open nephrectomy.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

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 approximately 50% with Triton X-100-insoluble fractions; approximately 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-beta-cyclodextrin (MbetaCD) 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 MbetaCD 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.

Cycling of synaptic vesicles: how far? How fast!

Synaptic transmission is based on the regulated exocytotic fusion of synaptic vesicles filled with neurotransmitter. In order to sustain neurotransmitter release, these vesicles need to be recycled locally. Recent data suggest that two tracks for the cycling of synaptic vesicles coexist: a slow track in which vesicles fuse completely with the presynaptic plasma membrane, followed by clathrin-mediated recycling of the vesicular components, and a fast track that may correspond to the transient opening and closing of a fusion pore. In this review, we attempt to provide an overview of the components involved in both tracks of vesicle cycling, as well as to identify possible mechanistic links between these two pathways.

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.

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.

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network.

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery.

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.

Sentinel lymph node identification in breast cancer: feasibility study.

The aim of the study was to evaluate in our institute the technique of sentinel node (SN) identification and biopsy in the surgical treatment of early breast cancer. Between June 1998 and November 1999 54 patients (age range, 31-75 years) where studied. Inclusion criteria were age less than 75 years, indication for conservative surgery, absence of palpable axillary nodes, Karnofksy index >70. Lymphoscintigraphy was performed 16-18 hours prior to surgery, following injection of 0.1-0.2 mL of 99mTc-Nanocoll: the administered activity was 3-4 MBq in group A (44 pts) and 7-8 MBq in group B (10 pts). The colloids were administered by transdermal supralesional injection in 49 patients with palpable nodules and by intraparenchymal ultrasound-guided injection in five patients with non-palpable nodules. Planar projections were performed starting from the 5th until the 80th min (or 180th in the event of late migration). In 10 patients further projections were acquired 14-18 h following tracer administration. All nodes identified by gamma probe (MR 100 Pol.Hi.Tech) were histologically evaluated by immunohistochemistry and standard histology. Scintigraphic visualization of the SN was obtained in 49 patients: in 38 of these patients there was only one SN while in 11 patients there were two or three SNs. The delayed scan made in 10 patients did not show any further nodes. In all patients given US-guided perilesional injections migration was late (after at least 60 min). Our study confirms the validity of the scintigraphic procedure, its safety for patients and health care workers, and the feasibility of interdisciplinary collaboration.

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment.

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.

Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract.

Our laboratory has previously shown that the vacuolar H(+)-ATPase, located in a subpopulation of specialized cells establishes a luminal acidic environment in the epididymis and proximal part of the vas deferens (Breton S, Smith PJS, Lui B, and Brown D. Nat Med 2: 470-472, 1996). Low luminal pH is critical for sperm maturation and maintenance of sperm in a quiescent state during storage in these organs. In the present study we examined the regulation of proton secretion in the epididymis and vas deferens. In vivo microtubule disruption by colchicine induced an almost complete loss of H(+)-ATPase apical polarity. Endocytotic vesicles, visualized by Texas red-dextran internalization, contain H(+)-ATPase, indicating active endocytosis of the pump. Cellubrevin, an analog of the vesicle soluble N-ethyl malemide-sensitive factor attachment protein (SNAP) receptor (v-SNARE) synaptobrevin, is highly enriched in H(+)-ATPase-rich cells of the epididymis and vas deferens, and tetanus toxin treatment markedly inhibited bafilomycin-sensitive proton secretion by 64.3+/-9.0% in the proximal vas deferens. Western blotting showed effective cleavage of cellubrevin by tetanus toxin in intact vas deferens, demonstrating that the toxin gained access to cellubrevin. These results suggest that H(+)-ATPase is actively endocytosed and exocytosed in proton-secreting cells of the epididymis and vas deferens and that net proton secretion requires the participation of the v-SNARE cellubrevin.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

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.

Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells.

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.