Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: Identification of VAMP3 binding to PI4K2A. (A) AP-MS analysis of PI4K2A was performed in HEK293 cells stably expressing FLAG–PI4K2A (see Materials and Methods). The sizes of the nodes (blue circles) represent the frequency of the protein identified in our internal database. The thickness of the lines represents the number of unique peptides identified in the analysis (a thicker line represents more peptides identified). See supplementary material Table S1 for details. (B) COS-7 cells co-transfected with GFP–VAMP3 and either HA–PI4K2A wt or kinase dead (KD) were immunoprecipitated (IP) using anti-GFP beads. Total cell lysates and eluted immunoprecipitated samples and were resolved by SDS-PAGE and analyzed by immunoblotting (WB), using anti-GFP (top) or anti-HA antibody (bottom). (C) Live-cell imaging of COS-7 cells expressing GFP–VAMP3 and PI4K2A–mRFP. (D) Endogenous distribution of VAMP3 and PI4K2A. COS-7 cells were fixed, permeabilized and incubated with rabbit polyclonal antibody against VAMP3 and 4C5G mouse monoclonal antibody against PI4K2A. Enlargements of the boxed area are shown in the insets. The thick arrows indicate tubular structures and thin arrows indicate vesicles. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Binding Assay, Protein-Protein interactions, Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page, Western Blot, Live Cell Imaging, Incubation

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: PI4K2A localization is affected by VAMP3 inactivation. (A,B) Fluorescence micrographs of COS-7 cells co-transfected with TeNT and either GFP–VAMP3 wt (A) or the GFP–VAMP3 VW mutant (B). Fixed cells were stained with a rabbit polyclonal antibody against PI4K2A (right panels). The dotted line indicates the cell outline. (C,D) Retrograde transport of PI4K2A following photobleaching of the perinuclear and Golgi compartment in COS-7 cells expressing GFP–PI4K2A. (C) Sequential imaging during photobleaching. (D) The rate of FRAP was faster in cells transfected with GFP–PI4K2A alone (black, n = 10), than in those coexpressing GFP–PI4K2A and TeNT (gray, n = 10). Shown are mean fluorescence intensity curves +s.e.m. (PI4K2A alone) or –s.e.m. (PI4K2A + TeNT), plotted as described in the Materials and Methods. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Fluorescence, Transfection, Mutagenesis, Staining, Expressing, Imaging

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: PI4K2A regulates VAMP3 sorting. (A) COS-7 cells were treated with control siRNA or siRNA directed against PI4K2A for 2 days prior to transfection with VAMP3–HA. At 1 day after transfection, cells were fixed, permeabilized and incubated with rabbit polyclonal antibody against PI4K2A and mouse monoclonal antibody against the HA epitope. PI4K2A knockdown resulted in an accumulation of VAMP3 on enlarged vesicles. (B–D) Live-cell confocal microscopy of COS-7 cells after a 2-day treatment with control or PI4K2A siRNA duplexes and co-transfection with GFP–VAMP3 and either LAMP-1–mRFP (B,C) or mCherry–Rab5, mCherry–Rab7 or mCherry–Rab11 (D). (C) Comparison of GFP–VAMP3 and LAMP-1–mRFP pixel (Pearson's) correlation coefficients. (D) PI4K2A depletion results in missorting of VAMP3 into enlarged LAMP1- and Rab7-containing compartments (control siRNA, n = 27; PI4K2A siRNA, n = 31). Enlargements of the boxed area are shown in the insets for A and B. The boxes in D represent VAMP3-enriched vesicles; arrows indicate Rab colocalization. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Control, Transfection, Incubation, Knockdown, Confocal Microscopy, Cotransfection, Comparison

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: VAMP3 levels on target membranes depend on PI4K2A. (A–D) VAMP3–HA was expressed in control or PI4K2A siRNA-treated cells, followed by fixation and immunostaining against HA epitope under non-permeabilizing conditions. Cells were then permeabilized, stained with an antibody against PI4K2A (A–D) and DAPI (A), for analysis either by confocal microscopy (A) or flow cytometry (B–D). Cell-surface levels of VAMP3 were reduced in PI4K2A knockdown cells (red) in comparison to control knockdown (gray) (n = 10,000 cells). (C) Surface or total levels of VAMP3 in control siRNA-treated cells (black) were compared to gated cells showing >80% PI4K2A knockdown (grey). Results are mean±s.e.m. (n = 5; 10,000 cells per sample). *P<0.05 (two-tailed Student's t-test), N.S., not statistically significant (P = 0.77). (D) Reduced cell-surface levels of VAMP3 in PI4K2A knockdown cells (gray) were rescued by expression of PI4K2A–GFP (red) to levels that did not show statistical significance in comparison to the control knockdown (black) as determined by one-way ANOVA. *P<0.05; N.S., not statistically significant (n = 4; 10,000 cells per sample). (E) VAMP3 surface levels in cells transfected with VAMP3–HA and either GFP only (black), PI4K2A–GFP (gray) or the PI4K2A–GFP kinase dead (KD) mutant (red) were determined by flow cytometry. Overexpression of catalytically dead, but not the wild-type PI4K2A, induced a significant decrease in cell surface levels, based on one-way ANOVA. *P<0.05; N.S., not statistically significant (n = 4; 10,000 cells per sample). (F,G) VAMP3 fluorescence recovery was recorded in COS-7 cells transfected with GFP–VAMP3 after 2-day treatment with control siRNA (black, n = 27) or siRNA directed against PI4K2A (gray, n = 32), or 16 h incubation with 100 nM PIK93, an inhibitor of PI4KB (blue, n = 29). The outlined area in F represents the area bleached. Only PI4K2A knockdown induced a substantial delay in fluorescence recovery. FRAP measurements represent mean±s.e.m. for three independent experiments (see Materials and Methods). AU, arbitrary units. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Control, Immunostaining, Staining, Confocal Microscopy, Flow Cytometry, Knockdown, Comparison, Two Tailed Test, Expressing, Transfection, Mutagenesis, Over Expression, Fluorescence, Incubation

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: PI4K2A is required for VAMP3 interaction with cognate Q-SNARE Vti1a. (A) COS-7 cells were transfected with control siRNA or siRNA directed against PI4K2A for 2 days, fixed, then permeabilized and incubated with a rabbit polyclonal antibody directed against PI4K2A and a mouse monoclonal antibody directed against Vti1a. (B) The cartoon depicts the proposed role of PI4K2A (blue) in sorting of VAMP3 (lavender) to endosomal membranes, where it engages in SNARE complex formation with Vti1a (green) and other cognate Q-SNAREs. NEM inhibits disassembly of the complex, trapping Vti1a bound to VAMP3. (C) COS-7 cells were treated for 2 days with control or PI4K2A siRNA, transfected with GFP–VAMP3, and either preincubated with 1 mM NEM for 30 min or directly lysed. GFP–VAMP3 was immunoprecipitated from total cell lysates using anti-GFP beads. Immunoprecipitated samples were eluted and analyzed by SDS–PAGE, followed by western blotting. (D) Quantification of Vti1a present in complex with VAMP3 in immunoprecipitated samples, as shown in C. Results are mean±s.e.m. from five experiments. *P<0.05 between NEM-treated samples, as determined by one-way ANOVA analysis; N.S. not significant compared with control. (E) Assessment of PI4K2A knockdown from the experiment shown in C. After 72 h of treatment with control or PI4K2A siRNA, cells were lysed and analyzed by western blotting using antibodies against PI4K2A and actin. A.U., arbitrary units. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Transfection, Control, Incubation, Immunoprecipitation, SDS Page, Western Blot, Knockdown

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: TfR delivery to the ERC and rate of recycling are reduced upon PI4K2A depletion. After treatment of COS-7 cells with control siRNA or siRNA directed against PI4K2A, cells were serum-starved for 30 min, then incubated with Alexa-Fluor-488-conjugated transferrin for 10 min and either fixed (A,B) or chased in complete medium for 30 min prior to fixation in 4% paraformaldehyde (C,D). PI4K2A knockdown (A, right panel) impedes delivery of transferrin to the ERC in comparison to the control siRNA-treated cells (A, left panels). (B) Appearance of transferrin in the perinuclear ERC in cells treated with siRNA oligonucleotides, shown in A, or transfected with TeNT. The ratio of transferrin fluorescence in the ERC (FERC) and total fluorescence (Ftotal) was determined as described in the Materials and Methods. Shown are ratios of mean+s.e.m. fluorescence values (n = 50). Statistical significance was determined by one-way ANOVA analysis. *P<0.05 for control versus PI4K2A knockdown and control versus TeNT). (C) Total levels of internalized transferrin, as determined by flow cytometry. Following fixation, cells were incubated with a rabbit polyclonal antibody against PI4K2A. Graphs depict endogenous levels of PI4K2A and total levels of transferrin following a 30 min chase in a representative experiment (n = 3). (D) Total intracellular levels of internalized transferrin, as described in C, in cells gated for a PI4K2A knockdown efficiency of >80%. Graph represents mean±s.e.m. fluorescence intensity (n = 3 experiments, 10,000 cells per sample). AFU, arbitrary fluorescence units. Scale bars: 10 µm.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Control, Incubation, Knockdown, Comparison, Transfection, Fluorescence, Flow Cytometry

Journal: Journal of Cell Science

Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

doi: 10.1242/jcs.148809

Figure Lengend Snippet: PI4K2A and PtdIns4P regulate VAMP3 sorting. Following VAMP3 endocytosis at the PtdIns(4,5)P2-rich plasma membrane subdomains (blue), VAMP3 passes through early endosomal compartments containing PtdIns(3)P (orange), prior to delivery to sorting endosomes enriched with PtdIns(4)P (brown). Availability of PI4K2A for interaction with VAMP3, along with the catalytic activity of PI4K2A, ensures proper sorting of VAMP3 within this compartment, a process that is a prerequisite for efficient interaction of VAMP3 with cognate Q-SNAREs, such as Vti1a, on target membranes.

Article Snippet: Knockdown of PI4K2A was performed using PI4K2A siRNA duplexes as described previously ( Balla et al., 2005 ), whereas control siRNA duplexes (AllStars RNAi control) were obtained from Qiagen (Valencia, CA).

Techniques: Clinical Proteomics, Membrane, Activity Assay