Membrane traffic in health and disease

Thierry Galli, INSERM Research Director (Biographical sketch)

E-mail: thierry.galli@inserm.fr  

Phone: +33 1 57 27 80 39


Our research focuses on the role of SNARE proteins in exocytosis mediating epithelial and neuronal cell differentiation, with particular emphasis on the tetanus neurotoxin-sensitive routes, mediated by cellubrevin/VAMP3 and synaptobrevin/VAMP2, and the tetanus neurotoxin-insensitive routes mediated by TI-VAMP/VAMP7. We are also interested in the role of Sec22b in neurite growth and the regulation of ER-plasma membrane contact sites.


Main results

Unconventional secretion and neurite growth

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. We found that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer (Petkovic et al, 2014).

Vesicular trafficking sensitive to neurotoxins

- Role of VAMP2 in Sema3A-dependent repulsion

VAMP2 is crucial for the exocytosis of synaptic vesicles in mature neurons but its potential function during development was not known. In developing neurons, we showed that semaphorin 3A (Sema 3A)-mediated signaling and axonal repulsion require VAMP2-dependent vesicular trafficking. Indeed, the botulinum neurotoxin D, which inactivates VAMP2 and invalidation of the VAMP2 gene, have no effect on the attractive guidance by Sema 3C but block Sema3A-induced repulsion. Moreover, when treated with Sema3A, neurons deficient in VAMP2 do not collapse and do not carry the Sema3A receptor to the cell bodies (Zylbersztejn et al., 2012). These studies suggest a new function of VAMP2 in axonal guidance during brain development. They suggest that clostridial neurotoxins, already widely used to treat neurological diseases, may be beneficial to remove the sensitivity to repulsive molecules. By removing this repulsion, one could stimulate the regeneration of axons and their passage through the sites of injury in pathologies secondary to traumatic injuries (Zylbersztejn and Galli, 2012).

 

Figure 1. Role of Syb2 in receptor trafficking during axon guidance. Semaphorin-3A induces growth cone repulsion. Semaphorin-3A binds its receptor neuropilin-1/plexin- A1 which interacts with Syb2. The complex then undergoes clathrin–dependent endocytosis. The receptor is then recycled to the plasma membrane. Syb2 is necessary for the endocytosis and recycling of the receptor. Sema3A: semaphorin-3A NRP1: neuropilin-1; PlexA1: plexine-A1; Stx1: syntaxin-1; Syb2: synaptobrevin- 2 (Zylbersztejn and Galli, 2012).


Vesicular trafficking insensitive to neurotoxins

- Identification of a molecular network involved in TI-VAMP/VAMP-7 traffic

The v-SNARE TI-VAMP/VAMP-7 was previously shown to mediate an exocytic pathway involved in neurite growth and epithelial apical exocytosis, but its regulation is still largely unknown. Human immunodeficiency virus Rev-binding protein (Hrb) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. Eps15 and AP-2 are involved in endocytosis. We identified Hrb as a partner of TI-VAMP/VAMP-7 in yeast two-hybrid screens and using biochemical assays. In the cytoplasm, Hrb colocalized with clathrin-, AP-2-, Eps15-, and transferrin receptor-containing vesicles. We went on to show that endocytosis of transferrin and pHLuorin-TI-VAMP/VAMP-7 is strongly reduced in Hrb knockdown cells. Altogether these results suggest that Hrb is involved in clathrin-dependent endocytosis and recruits TI-VAMP/VAMP-7 in this process (Chaineau et al., 2008).

 

Figure 2. TI-VAMP/VAMP-7 routes. The main TI-VAMP/VAMP-7 routes are represented. TI-VAMP/VAMP-7 is involved in the transport from the Golgi to the cell surface. After mediating exocytic events via pairing with its t-SNAREs at the plasma membrane, TI-VAMP/VAMP-7 is endocytosed in a clathrin-dependent manner by interacting with Hrb, to be included in clathrin-coated structures. In early endosomes, TI-VAMP/VAMP-7 interacts with AP-3 to reach late endosomes and lysosomes. Then, TI-VAMP/VAMP-7 mediates lysosomal secretion. ER: endoplasmic reticulum, TGN: trans-Golgi Network, Lys: lysosome, EE: early endosome, LE: late endosome.

 

We further showed that TI-VAMP/VAMP-7 interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21. Varp, TI-VAMP/VAMP-7 and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in neurites. Silencing the expression of Varp by RNA interference or expressing its domain of interaction with TI-VAMP/VAMP-7 or a form of Varp deprived of its Vps9 domain impair neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP/VAMP-7 (Burgo et al., 2009). We further established that Varp interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP-7 vesicles from the cell center to the cell periphery. Therefore, the v-SNARE TI-VAMP/VAMP-7 is the starting point of a dynamic molecular network involving the exchange factor Varp, Rab21, GolginA4, a Kif5 kinesin motor and the spectraplakin MACF1. This network regulates vesicular transport from the Golgi apparatus, in the cell center, to the cell periphery where TI-VAMP/VAMP-7 mediates exocytosis (Burgo et al., 2012b).

 

Figure 3. Network of molecular interactions of TI-VAMP/VAMP-7. Interactions identified in our two-hybrid screens and / or biochemical approaches are shown in blue. The other interactions described in the literature are shown in red (Figure was prepared using the software Cytoscape ®). (Burgo et al., 2012a)

 

- Integrative characterization of the role of TI-VAMP/VAMP-7

With the Mouse Clinic Institute (Strasbourg), we generated a strain of mice invalidated for the gene coding for TI-VAMP/VAMP-7. The mutant mice (VAMP7 KO) are viable and fertile and show no major developmental defect probably due to compensation by another v-SNARE during development. Several defects were nevertheless identified in adults: decreased brain weight, lowered body temperature, increased circulating levels of glycerol, increased volume of the third ventricle and higher anxiety in the elevated plus maze (Danglot et al., 2012b). In addition, we have shown that TI-VAMP/VAMP-7 is involved in Golgi to plasma membrane transport of cargoes like VSVG or the tetraspanin CD82. CD82 is a partner of the EGF receptor, responsible for the formation of membrane microdomains. In the absence of TI-VAMP/VAMP-7, the dynamics of EGFR on the cell surface, EGFR signaling and endocytosis are altered (Danglot et al., 2010). We are now further analyzing synaptic receptors dynamics in light of data suggesting defects associated with the transport of cholesterol-rich domains in the mutant mice. In conclusion, TI-VAMP/VAMP-7 is involved in the transport of membrane microdomains that may regulate several signaling pathways. One goal is now to precisely determine the molecular and cellular mechanisms involved, particularly in the mutant mice.

 

Figure 4. TI-VAMP/VAMP-7-dependent secretion of CD82 regulates cell surface diffusion and endocytosis of EGFR. TI-VAMP/VAMP-7 mediates the transport from the Golgi to the cell surface of the marker VSV-G and potentially of the tetraspanin CD82. One possibility would be that AP3 is present on this vesicle since it has already been involved in the transport of VSV-G from the Golgi complex to the plasma membrane and since the targeting of TI-VAMP/VAMP-7 is dependent on AP3 both in HeLa cells and in neuronal cells. At the cell surface, CD82 assembles into tetraspanin-enriched microdomains constituting the tetraspanin web. EGFR strongly interacts with CD82 and is endocytosed by clathrin-dependent endocytosis. In TI-VAMP/VAMP-7-depleted cells, the transport of CD82 to the cell surface is inhibited, leading to a defect in the tretraspanin web at the cell surface. When stimulated by EGF, the lack of TI-VAMP/VAMP-7 and/or CD82 results in a diminished lateral diffusion of EGFR, increased recruitment of EGFR in AP-2-coated pits and by increased endocytosis by CME, thus impairing MAPK signaling. (Danglot et al., 2010)

 

- Role of Vezatin in dendritic spine morphogenesis and memory

Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. We showed that Vezatin localizes in spines in mature mouse hippocampal neurons. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear conditioning memory response. Vezatin knock down in cultured hippocampal neurons and Vezatin conditional knock out in mice led to reduced proportion of mature dendritic spines. Accordingly, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results identify Vezatin as a novel spine-associated protein critical for dendritic spine morphogenesis and functional synaptic maturation (Danglot et al., 2012a).

 

 

References

Burgo A, Formstecher E, Galli T (2012a) [Molecular network for the transport of intracellular vesicles from cell center to periphery]. Medecine sciences : M/S 28:1040-1041.

Burgo A, Sotirakis E, Simmler MC, Verraes A, Chamot C, Simpson JC, Lanzetti L, Proux-Gillardeaux V, Galli T (2009) Role of Varp, a Rab21 exchange factor and TI-VAMP/VAMP7 partner, in neurite growth. EMBO Rep 10:1117-1124.

Burgo A, Proux-Gillardeaux V, Sotirakis E, Bun P, Casano A, Verraes A, Liem RK, Formstecher E, Coppey-Moisan M, Galli T (2012b) A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery. Dev Cell 23:166-180.

Chaineau M, Danglot L, Proux-Gillardeaux V, Galli T (2008) Role of HRB in clathrin-dependent endocytosis. J Biol Chem 283:34365-34373.

Danglot L, Chaineau M, Dahan M, Gendron M-C, Boggetto N, Perez F, Galli T (2010) Role of TI-VAMP and CD82 in EGFR cell-surface dynamics and signaling. J Cell Sci 123:723-735.

Danglot L, Freret T, Le Roux N, Neme NN, Burgo A, Hyenne V, Roumier A, Contremoulins V, Dauphin F, Bizot JC, Vodjdani G, Gaspar P, Boulouard M, Poncer JC, Galli T, Simmler MC (2012a) Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation. J Neurosci 32:9007-9022.

Danglot L, Zylbersztejn K, Petkovic M, Gauberti M, Meziane H, Combe R, Champy MF, Birling MC, Pavlovic G, Bizot JC, Trovero F, Della Ragione F, Proux

Gillardeaux V, Sorg T, Vivien D, D'Esposito M, Galli T (2012b) Absence of TI-VAMP/Vamp7 Leads to Increased Anxiety in Mice. J Neurosci 32:1962-1968.

Petkovic M, Jemaiel A, Daste F, Specht CG, Izeddin I, Vorkel D, Verbavatz JM, Darzacq X, Triller A, Pfenninger KH, Tareste D, Jackson CL, Galli T (2014) The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion. Nat Cell Biol 16:434-444. 

Zylbersztejn K, Galli T (2012) Membrane traffic, a new actor in axon guidance. Med Sci (Paris) 28:267-269.

Zylbersztejn K, Petkovic M, Burgo A, Deck M, Garel S, Marcos S, Bloch-Gallego E, Nothias F, Serini G, Bagnard D, Binz T, Galli T (2012) The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion. J Cell Biol 196:37-46.

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