Synchronization of secretory protein traffic in populations of cells

Boncompain, Gaëlle; Divoux, Séverine; Gareil, Nelly; Forges, Hélène de; Lescure, Aurianne; Latreche, Lynda; Mercanti, Valentina; Jollivet, Florence; Raposo, Graça; Perez, Franck

doi:10.1038/nmeth.1928

Cited by 520 publications

(723 citation statements)

References 34 publications

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“…We took advantage of the RUSH assay which is based on the retention of newly synthesized proteins in the ER and their synchronized release upon addition of biotin. 29 As reporter, we used VSVG-and GPI-anchored mCherry-tagged fluorescent proteins, which are expected to segregate at the exit of the Golgi and follow different routes from the Golgi toward the PM. 30 One hour after biotin addition at 37 C, surface staining intensity of mCherry-VSVG in WT and KO cells was similar while mCherry-GPI surface signal was strongly reduced in KO compared to WT, suggesting that GPI-anchored proteins transport toward the PM was delayed in KO ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For secretion studies, cells were transfected with mCherrytagged GPI and VSVG constructs of the RUSH system generated by Boncompain et al 29 24 h after transfection, secretion was induced with 40 mM Biotin (Sigma Aldrich). After induced release cells were quickly washed at 4 C with DMEM buffered with 20 mM HEPES and then incubated in the same solution with primary mouse anti-mCherry antibody for 20 min at 4 C to allow surface epitope labeling.…”

Section: Secretion Assaysmentioning

confidence: 99%

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Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

et al. 2015

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Keywords: exocytosis, Golgi apparatus, SNARE, sphingolipids, TI-VAMP/VAMP7 Abbreviations: BFA, Brefeldin A; Cer, Ceramide; ER, Endoplasmic Reticulum; GlcCer, Glucosylceramide; GM3, ganglioside monosialic acid 3; GPI, Glycosylphosphatidylinositol; GSL, Glycosphingolipids; LC, Long Chain; PI, Phosphatidylinositide; PM, Plasma Membrane; SM, Sphingomyelin; TGN, D Trans-Golgi Network; TI-VAMP/VAMP7, Tetanus neurotoxin-insensitive vesicle-associated membrane protein / Vesicle associated membrane protein 7; VLC, very long vhain; VSVG, Vesicular Stomatitis Virus GlycoproteinBiological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI-anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.

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Section: Resultsmentioning

confidence: 99%

Section: Secretion Assaysmentioning

confidence: 99%

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

et al. 2015

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“…The question we addressed here is whether post-Golgi transport intermediates can be produced in the absence of microtubules. We monitored secretory cargos transport in normal and in microtubule-depletion conditions using the retention using selective hooks (RUSH) assay (Boncompain et al, 2012). We revealed the co-existence of distinct subpopulations of Golgi elements early after removal of microtubules and assessed their secretion capacity.…”

Section: Introductionmentioning

confidence: 99%

Microtubule-independent secretion requires functional maturation of Golgi elements

Fourrière

Divoux

Roceri

et al. 2016

Journal of Cell Science

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The Golgi complex is responsible for processing and sorting of secretory cargos. Microtubules are known to accelerate the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex and from the Golgi to the plasma membrane. However, whether postGolgi transport strictly requires microtubules is still unclear. Using the retention using selective hooks (RUSH) system to synchronize the trafficking of cargos, we show that anterograde transport of tumor necrosis factor (TNF) is strongly reduced without microtubules. We show that two populations of Golgi elements co-exist in these cells. A centrally located and giantin-positive Golgi complex that sustains trafficking, and newly formed peripheral Golgi mini-stacks that accumulate cargos in cells without microtubules. Using a genomeedited GFP-giantin cell line, we observe that the trafficking-competent Golgi population corresponds to the pre-existing population that was present before removal of microtubules. All Golgi elements support trafficking after long-term depletion of microtubules and after relocation of Golgi proteins to the ER after treatment with Brefeldin A. Our results demonstrate that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking, and that microtubule-driven post-Golgi transport is not strictly required.

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“…We focused on E-cadherin, a well-characterized anterograde transport cargo protein. Using the 'Retention Using Selective Hooks' (RUSH) system (Boncompain et al, 2012), E-cadherin was reversibly anchored in the ER through a fusion construct composed of streptavidin-binding protein (SBP) and mCherry. Upon addition of biotin, SBP-mCherry-E-cadherin was released from an ER-resident membrane-bound streptavidin-hook construct.…”

Section: Effects Of Vamp2 Vamp3 and Vamp8 Depletion On Other Trafficmentioning

confidence: 99%

“…The Retention Using Selective Hooks (RUSH) system was used to quantify anterograde transport of E-cadherin, as previously described (Boncompain et al, 2012). HeLa cells were transfected with siRNAs, and seeded after two days into 24 well plates.…”

Section: Anterograde E-cadherin Transportmentioning

confidence: 99%

Shiga toxin stimulates clathrin-independent endocytosis of VAMP2/3/8 SNARE proteins

Renard

García-Castillo

Chambon

et al. 2015

Journal of Cell Science

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Endocytosis is an essential cellular process that is often hijacked by pathogens and pathogenic products. Endocytic processes can be classified into two broad categories, those that are dependent on clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 and VAMP8 are internalized in a clathrin-dependent manner. However, the full scope of their endocytic behavior has not yet been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 are localized on plasma membrane invaginations and very early uptake structures that are induced by the bacterial Shiga toxin, which enters cells by clathrin-independent endocytosis. We show that toxin trafficking into cells and cell intoxication rely on these SNARE proteins. Of note, the cellular uptake of VAMP3 is increased in the presence of Shiga toxin, even when clathrin-dependent endocytosis is blocked. We therefore conclude that VAMP2, VAMP3 and VAMP8 are removed from the plasma membrane by non-clathrin-mediated pathways, in addition to by clathrin-dependent uptake. Moreover, our study identifies these SNARE proteins as the first transmembrane trafficking factors that functionally associate at the plasma membrane with the toxin-driven clathrin-independent invaginations during the uptake process.

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Synchronization of secretory protein traffic in populations of cells

Cited by 520 publications

References 34 publications

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

Microtubule-independent secretion requires functional maturation of Golgi elements

Shiga toxin stimulates clathrin-independent endocytosis of VAMP2/3/8 SNARE proteins

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