Journal: Nature Communications

Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors

doi: 10.1038/s41467-019-09617-9

Figure Lengend Snippet: Vps13B binds to Stx6- and Stx13-positive vesicles. a Recruitment of Vps13B and EEA1 to liposomes containing Stx6, Stx13, Stx6, and Stx13 together. For details, see legend to Fig. . α-Tubulin was used as loading control, because the protein binds to liposomes dependent on the lipid composition (ref. ). b , c Co-immunoprecipitation of Vps13B and SNARE proteins; b shows co-precipitation of Stx6 and 13 but not of any other SNARE with Vps13B. c Conversely, Vps13B co-precipitates with Stx6 and Stx13 but not with Stx16 and Stx4. The bottom panel shows that all SNAREs were efficiently immunoprecipitated. d The N-terminal domain of Stx13 is necessary for binding to Vps13B. Lysates from cells expressing Vps13B-FLAG and GFP-Stx13, GFP-ΔN-Stx13, or GFP-Stx13-Stx6 chimera were immunoprecipitated with anti-FLAG antibody and probed by immunoblotting for GFP. e Immunofluorescence for Vps13B of cells expressing mRFP-Stx6 and GFP-Stx13. Arrows in the insert indicate triple-positive vesicles. Scale bar, 10 µm. f Co-localization between Stx6, Stx13, and Vps13B in the peripheral region of the imaged HeLa cells. Vesicles were counted from nine images, obtained in three independent experiments, with the degree of overlap shown in the bar graph on the right (numbers denote total number of vesicles analyzed). g Co-localization of Vps13B with TfnR, EEA1, M6PR, and Golgin97. White arrows show co-localized vesicles. Scale bar, 10 µm. Intensity plots of exemplary line scans are shown on the right. A bar graph shows the percentage of TfnR-, EEA1-, M6PR-positive vesicles, which are also positive for Vps13B

Article Snippet: Primary antibodies used were obtained from the following companies: anti-APPL1 (3858), anti-LC3B (4599), and anti-Rab7 (9367) from Cell Signaling; anti-EEA1 (612006) and anti-GM130 (560257) from BD Biosciences; anti-M6PR (ab2733), anti-LAMP1 (ab24170), anti-mitofilin (ab110329), anti-mitofusion2 (ab101055), and anti-cathepsin D (ab6313) from Abcam; anti-Tfn receptor (sc-65882) from Santa Cruz Biotechnology; anti-LBPA (Z-PLBPA) from Echelon; anti-RFP (R10367), anti-Golgin97 (A-21270), anti-Vps52 (PA5–24408), and anti-Rab11 (71–5300) from Thermo Fischer Scientific; anti-PDI (700782) from ABfinity; anti-Vps51 (HPA061447), Vps13B (HPA043865) from Atlas antibodies; anti-Rab6 (10187–2-P) and anti-Rab35 (11329–2-AP) from Proteintech; anti-CD63 (H5C6) from Developmental Studies Hybridoma Bank; anti-Rab14 (R0656) from Sigma-Aldrich; anti-GFP (132002), anti-α-tubulin (302211; 1:10,000 dilution for Western blotting), anti-β-actin (251003; 1:10,000 dilution for Western blotting), anti-Rab5 (108011), anti-Stx6 (110062), anti-Stx13 (110132), anti-syntaxin 4 (110042), anti-syntaxin 16 (110162), and anti-VAMP4 (136002) from Synaptic Systems.

Techniques: Liposomes, Control, Immunoprecipitation, Binding Assay, Expressing, Western Blot, Immunofluorescence

Journal: Nature Communications

Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors

doi: 10.1038/s41467-019-09617-9

Figure Lengend Snippet: Vps13B specifically binds to Rab14 and Rab6 and to PtdIns(3)P. a , b Association of endosomal Rab proteins with Vps13B, revealed by co-immunoprecipitation of endogenous Vps13B with GFP (or mRFP)-tagged Rab proteins. a GFP (or mRFP)-tagged proteins were precipitated from cell lysates using a GFP-trap or anti-mRFP antibody. Bound proteins were analyzed by immunoblotting for Vps13B, GFP, and mRFP. b Cross-linked cells were lysed and immunoprecipitated with anti-Vps13B antibody. The interaction of Rab proteins was detected and indicated Rab protein antibodies. c Coimmunoprecipitation of VPS13B with mRFP-Rab14-WT, GTP-locked mRFP-Rab14Q70L (QL), or GDP-locked mRFP-Rab14S25N (SN) expressed in HeLa cells. Anti-RFP antibody was used for immunoprecipitation of mRFP-tagged Rab14 proteins. There was no significant difference in the binding of Vps13B to the Rab14 variants. d Vps13B co-localizes with Rab6 and Rab14 upon expression in HeLa cells. Top panel: cells were transfected with mRFP-Rab14 and Vps13B-GFP and analyzed for the presence of GFP and mRFP, respectively, by fluorescence microscopy. Bottom panel: cells were transfected with GFP-Rab6. In this case, the distribution of endogenous Vps13B was monitored by immunocytochemistry. White arrows in the upper inserts show vesicles positive for both Vps13B and Rab14. Bottom panels show line scans on the lines in the upper panels and the percentage of colocalization of Rab14 with Vps13B. Scale bar, 10 µm. e PIP strip-binding assay of Vps13B. Perinuclear supernatant from Vps13B-GFP overexpressing cells was incubated with a membrane strip containing the phosphoinositides as indicated and probed for bound Vps13B using an anti-GFP antibody. f Binding of Rhodamine-labeled liposomes to Vps13B-GFP that was immobilized on magnetic Dynabeads. After incubation, the beads were washed, and bound liposomes were detected by Rhodamine fluorescence. Binding was only detectable when Vps13B-GFP was bound to the beads and when the liposomes contained 5% PtdIns(3)P. Error bars indicate S.E.M., *** P < 0.001, determined by unpaired t- test

Article Snippet: Primary antibodies used were obtained from the following companies: anti-APPL1 (3858), anti-LC3B (4599), and anti-Rab7 (9367) from Cell Signaling; anti-EEA1 (612006) and anti-GM130 (560257) from BD Biosciences; anti-M6PR (ab2733), anti-LAMP1 (ab24170), anti-mitofilin (ab110329), anti-mitofusion2 (ab101055), and anti-cathepsin D (ab6313) from Abcam; anti-Tfn receptor (sc-65882) from Santa Cruz Biotechnology; anti-LBPA (Z-PLBPA) from Echelon; anti-RFP (R10367), anti-Golgin97 (A-21270), anti-Vps52 (PA5–24408), and anti-Rab11 (71–5300) from Thermo Fischer Scientific; anti-PDI (700782) from ABfinity; anti-Vps51 (HPA061447), Vps13B (HPA043865) from Atlas antibodies; anti-Rab6 (10187–2-P) and anti-Rab35 (11329–2-AP) from Proteintech; anti-CD63 (H5C6) from Developmental Studies Hybridoma Bank; anti-Rab14 (R0656) from Sigma-Aldrich; anti-GFP (132002), anti-α-tubulin (302211; 1:10,000 dilution for Western blotting), anti-β-actin (251003; 1:10,000 dilution for Western blotting), anti-Rab5 (108011), anti-Stx6 (110062), anti-Stx13 (110132), anti-syntaxin 4 (110042), anti-syntaxin 16 (110162), and anti-VAMP4 (136002) from Synaptic Systems.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Expressing, Transfection, Fluorescence, Microscopy, Immunocytochemistry, Stripping Membranes, Incubation, Membrane, Labeling, Liposomes

Journal: Nature Communications

Article Title: SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors

doi: 10.1038/s41467-019-09617-9

Figure Lengend Snippet: Vps13B functions as a tethering protein for Tfn-positive endosomes. a Expression of Vps13B-GFP on the outer mitochondrial membrane by fusing it to a monoaminoxidase fragment (see also Fig. ) results in scattering and accumulation of TfnR-containing vesicles around mitochondria (arrows in insert). GFP signal was enhanced by immunostaining with anti-GFP antibody. Right: perinuclear index of TfnR and M6PR distribution upon expression of Vps13B-GFP-MAO. Error bars indicate S.E.M., ** P < 0.01, determined by unpaired t -test. b Expression levels of Vps13B and marker proteins in a HeLa cell line stably expressing Vps13B shRNA expressing cell line. The figure shows immunoblots of cell lysates for the proteins indicated. c Knockdown of Vps13B selectively reduces targeting of liposomes containing Stx6/Stx13 to Tfn-positive endosomes. d Vps13B KD cells, and cells expressing a control construct (scramble) were allowed to internalize Alexa Fluor 568-Tfn for 10 min and chased for indicated time at 37 °C. The images show the change in the distribution of labeled organelles after counterstaining of the nuclei with DAPI (blue channel). Lower left: quantification of fluorescence intensity of Alexa Fluor 568-Tfn over time after pulse-chase of labeled Tfn. The data show mean values ± S.E.M. of three independent experiments. Lower right: quantification of the distribution of Alexa Fluor 568-Tfn-positive endosomes between the peripheral region and the perinuclear region over time. The perinuclear index decreased in Vps13B KD cells, suggesting that transport of Tfn-positive endosomes to the perinuclear recycling endosomes was impaired. Error bars indicate S.E.M., ** P < 0.01, *** P < 0.001, determined by unpaired t -test from three independent experiments. e Immunoisolation of vesicles containing TfnR reveals a loss of association with Rab11, whereas association with Rab4 and Rab35 was increased in Vps13B KD cells. Moreover, a higher level of LAMP1 was detectable. The figure shows immunoblots for the proteins indicated, with all lanes containing equal amounts of TfnR. f Immunoblots of control (mock) and Vps13B-KD cell extracts, normalized to tubulin, reveal that Vps13B-KD leads to a reduction of Tfn levels that is preventable by the inhibition of lysosomal function by 10 µM chloroquine. g Cartoon showing the recycling of transferrin in Vps13B KD cells. After delivery to early endosomes by clathrin-mediated endocytosis, it can recycle either directly (fast recycling) using a pathway governed by Rab4 and Rab35, or via recycling endosomes, which is governed by Rab11 in control cells. Vps13B KD appears to block the slow pathway, leading to an increase in the fast recycling route and to an increased degradation via the late-endosome lysosome route

Article Snippet: Primary antibodies used were obtained from the following companies: anti-APPL1 (3858), anti-LC3B (4599), and anti-Rab7 (9367) from Cell Signaling; anti-EEA1 (612006) and anti-GM130 (560257) from BD Biosciences; anti-M6PR (ab2733), anti-LAMP1 (ab24170), anti-mitofilin (ab110329), anti-mitofusion2 (ab101055), and anti-cathepsin D (ab6313) from Abcam; anti-Tfn receptor (sc-65882) from Santa Cruz Biotechnology; anti-LBPA (Z-PLBPA) from Echelon; anti-RFP (R10367), anti-Golgin97 (A-21270), anti-Vps52 (PA5–24408), and anti-Rab11 (71–5300) from Thermo Fischer Scientific; anti-PDI (700782) from ABfinity; anti-Vps51 (HPA061447), Vps13B (HPA043865) from Atlas antibodies; anti-Rab6 (10187–2-P) and anti-Rab35 (11329–2-AP) from Proteintech; anti-CD63 (H5C6) from Developmental Studies Hybridoma Bank; anti-Rab14 (R0656) from Sigma-Aldrich; anti-GFP (132002), anti-α-tubulin (302211; 1:10,000 dilution for Western blotting), anti-β-actin (251003; 1:10,000 dilution for Western blotting), anti-Rab5 (108011), anti-Stx6 (110062), anti-Stx13 (110132), anti-syntaxin 4 (110042), anti-syntaxin 16 (110162), and anti-VAMP4 (136002) from Synaptic Systems.

Techniques: Expressing, Membrane, Immunostaining, Marker, Stable Transfection, shRNA, Western Blot, Knockdown, Liposomes, Control, Construct, Labeling, Fluorescence, Pulse Chase, Inhibition, Blocking Assay