Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: Representative confocal fluorescence micrograph of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and GFP‐BspF and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐MICAL‐L1, GFP‐STX16, GFP‐STX6, or GFP‐VAMP3 and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of GFP‐MICAL‐L1, GFP‐STX16, GFP‐STX6, or GFP‐VAMP3 to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab11a or VAMP4‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of GFP‐Rab11a or VAMP4‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

Article Snippet: pEGFP‐ VAMP4 Homo sapiens , Addgene , Cat#42313.

Techniques: Fluorescence, Transfection, Labeling

Journal: Nature cell biology

Article Title: EARP, a multisubunit tethering complex involved in endocytic recycling

doi: 10.1038/ncb3129

Figure Lengend Snippet: Schematic representation of TfR recycling, and the roles of EARP and GARP. GARP participates in retrograde transport by promoting the assembly of the Stx6-Stx16-Vti1a-VAMP4 SNARE complex at the TGN , while EARP and, to a lesser extent, GARP, promote transport through recycling endosomes by acting on Stx6 in conjunction with endosomal SNAREs.

Article Snippet: Antibodies to Stx2 (Cat# 110022) (1:1000 for IB), Stx6 (Cat# 110062) (1:1000 for IB), Stx16 (Cat# 110162) (1:1000 for IB), and VAMP4 (Cat# 136002) (1:500 for IB) were from Synaptic Systems (Göttingen, Germany).

Techniques:

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: Distribution and known functions of host SNAREs used in this study.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques:

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, Localization of endogenous SNAREs in HeLa cells infected for 24 h with C. trachomatis serovar D was assessed using specific antibodies. Vamp3 and Vamp7 encircle the inclusion (arrows), while Sec22 does not (arrowheads). SNAREs are shown on the left, host and bacterial DNA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. B, Cells were transfected with different GFP-SNARE constructs 8 h before infection. The inclusion was labelled with anti-IncA antibodies followed with TRITC-coupled secondary antibodies. GFP-Vamp8 or GFP-Vamp7 encircle the inclusion membrane (arrows), while GFP, GFP-Vamp4 or GFP-Vamp7 deleted from its SNARE domain (GFP-Longin) do not (arrowheads). GFP-tagged SNAREs are shown on the left, IncA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. C, Quantification of SNARE recruitment to the inclusion. Cells were scored as positive when the entire circumference of the inclusion was surrounded by the SNARE protein. Means and standard deviations of at least two independent experiments are shown.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Infection, Transfection, Construct, Membrane

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, Distribution of GFP was observed in fields with an inclusion, in cells transfected with GFP-Vamp4 (top) or GFP-Vamp8 (bottom), and infected. Two representative panels are shown for each construct. Arrowheads point to gold particles less than 50 nm from the inclusion membrane. I, inclusion, B, bacteria. B, Equivalent numbers of gold particles were counted in both sets of images and their localization relative to the inclusion membrane was assessed. Levels of significance are indicated by p-values comparing the distribution of GFP-Vamp4 and GFP-Vamp8 for each category.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Transfection, Infection, Construct, Membrane, Bacteria

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing IncA or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA co-immunoprecipitated with Vamp3, Vamp7 and Vamp8, and very little with Vamp4 (top right panel, anti-IncA blot). B, Purified IncA-His and purified GST-Vamp3, GST-Vamp4 or GST-Vamp8 were inserted in two populations of liposomes, mixed, and GST-SNAREs were pulled-down using glutathione agarose beads. Pull-down complexes were resolved on SDS-PAGE gels and analyzed after staining with Coomassie.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Purification, Liposomes, SDS Page, Staining

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing CT813-His* or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of CT813-His* in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. CT813-His* co-immunoprecipitated with Vamp7 and Vamp8 (top right panel, anti-His blot), and not with Vamp3, Vamp4, GFP or GFP-Longin. Absence of immunoprecipitation with Vamp3 might be due to insufficient expression level of both CT813-His* and GFP-Vamp3, which were consistently low in these experiments.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, GFP-SNARE proteins were immunoprecipitated from HeLa cells co-expressing IncA wild-type, mutated in motif Nter (T126R) or in both SNARE-like motifs (TRQR), or empty vector (pQE) as control. The top left panel (anti-IncA blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA wild-type and T126R immunopecipitated with the SNAREs, and the double mutant did not (anti-IncA blot,top right panel). B, Cells transfected for 18 hrs with the indicated His-tagged IncA constructs were infected for 20 h with C. trachomatis serovar D before fixation. Coverslips were stained with anti-His antibodies and anti-EfTu antibodies to label the bacteria. Histograms show the percentage of cells (n>100) with a normal inclusion for each population of transfected cells, in one experiment representative of three (means and standard deviations of two independent counts are shown). Only inclusions that looked intact were scored; in cells transfected with IncA wild-type and IncAT126R, and, to a lesser extent, IncAQ244R and IncAT126RQ244R (TRQR), many disrupted inclusions were also observed.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Control, Mutagenesis, Transfection, Construct, Infection, Staining, Bacteria