Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A) Schematic representation of Rab11a-regulated recycling in mock- and IAV-infected cells. In mock-infected cells, Rab11a endosomes are involved in recycling material from several organelles to the plasma membrane. Upon IAV infection, progeny vRNPs bind to Rab11a endosomes and start concentrating at ERES (steps 1–2) to form liquid viral inclusions by a mechanism ill-defined (step 3). The ER likely facilitates formation of viral inclusions to promote assembly of the 8-vRNP genome (step 3). How assembled genomes reach the plasma membrane is unknown (step 4). (B) Cells (GFP-Rab11a WT low and DN low ) were infected or mock-infected with PR8 virus for 12 h at an MOI of 3. The levels of Tf-Alexa647-fluorescent conjugates were quantified inside cells and at the cell surface by flow cytometry upon 5, 10, and 15 min of incubation at 37°C. Results were plotted as the percentage (%) of recycled Tf as a function of time of incubation. Values were normalized to the mock condition at 15 min of incubation. Three pooled independent experiments are shown. Statistical analysis was done by two-way ANOVA, followed by a Sidak’s multiple comparisons test (** p < 0.01). (C) The levels of Tf were quantified inside cells and at the cell surface by flow cytometry at 15 min of incubation at 37°C. Results were plotted as the percentage (%) of recycled Tf as a function of cell type. Four pooled independent experiments are shown. Statistical analysis was done by one-way ANOVA, followed by a Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001). (D) Cells (GFP-Rab11a WT low , green) were simultaneously transfected with a plasmid encoding mCherry tagged to the ER (magenta) and infected or mock-infected with PR8 virus for 12 h at an MOI of 10. Cells were imaged under time-lapse conditions at 12 h postinfection. Representative cells are shown on the left. The respective individual frames with single moving particles are shown in the small panels on the right. The yellow arrowheads highlight fusion/fission events of viral inclusions (green), as well as their interaction with the ER (magenta). Bar = 10 μm. Images were extracted from and Videos. (E) A linescan was drawn as indicated to assess Rab11a dynamics associated with the ER. The fluorescence intensity of ER tubules (magenta) and Rab11a endosomes or viral inclusions (green) at indicated times was plotted against the distance (in μm). Representative analysis was performed using images from ( D ). Experiments were performed twice. For each condition, at least 10 cells were analyzed. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; ERES, ER exit site; IAV, influenza A virus; MOI, multiplicity of infection; Tf, transferrin; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Clinical Proteomics, Membrane, Virus, Flow Cytometry, Incubation, Transfection, Plasmid Preparation, Fluorescence

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A) Schematic representation of a liquid viral inclusion according to 2D light and electron microscopy analysis. (B) Schematic representation of how 4 sequential tomograms (of 120 nm each) were acquired and stitched together (approximately 480 nm total thickness). (C, D) Cells (GFP-Rab11a WT low ) were infected or mock-infected with PR8 virus for 12 h at an MOI of 3. Cells were processed by high-pressure freezing/freeze substitution and imaged by ET-TEM. Representative cells are shown with 3 individual sections (including section height in nm) and the 3D cumulative model. Bar = 500 nm. Images were extracted from , , , and . Abbreviations: pm, plasma membrane (gray); er, endoplasmic reticulum (blue); v, budding virions (pink); m, mitochondria (purple); smv, single-membrane vesicle (light green); dmv, double-membrane vesicle (yellow); *, ER dilation (dark green). (E) Photomontages of single sections covering an entire plane of the cell were acquired by TEM at several times postinfection (4–16 h), as exemplified here at 16 h postinfection. The photomontages were used to score the number of single (smv) and double (dmv) membrane vesicles in ( F ) and ( G ). Bar = 2 μm. (F, G) The number of single (smv) and double (dmv) membrane vesicles was manually scored and plotted as a function of time of infection. Statistical analysis was done by Kruskal–Wallis test (* p < 0.05). On average, 10 cells were analyzed per condition ( C - G ). Experiments were performed twice. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; ET, electron tomography; MOI, multiplicity of infection; TEM, transmission electron microscopy; 2D, 2-dimensional; 3D, 3-dimensional.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Electron Microscopy, Infection, Virus, Clinical Proteomics, Membrane, Tomography, Transmission Assay

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: Cells (A549) were treated with siRNA non-targeting (siNT) or targeting ULK1 (siULK1), ULK2 (siULK2), TBC1D14 (siTBC1D14), ATG2A (siATG2A), or ATG9A (siATG9A) for 48 h and then infected or mock-infected (M) with PR8 virus for 8 h, at an MOI of 3. (A) Viral production was determined at 8 h postinfection by plaque assay and plotted as PFU per mL ± SEM. Data were pooled from 3–6 independent experiments. Statistical analysis was done by one-way ANOVA, followed by a Dunnett’s multiple comparisons test (*** p < 0.001). (B) The mRNA level of ULK1/2 and TBC1D14 before infection was quantified by real-time RT-qPCR and plotted as the relative expression to GAPDH mRNA level ± SEM. Expression was normalized to siNT from mock-infected cells. Data are a pool from 3 independent experiments. Statistical analysis was done by Student t test (*** p < 0.01). (C) Protein levels of ATG2A and ATG9A before infection were determined by western blotting and plotted as the relative expression to actin protein levels ± SEM. Expression was normalized to siNT from mock-infected cells. Data are a pool from 3–6 independent experiments. Statistical analysis was done by Student t test (*** p < 0.01). The original uncropped blots can be found in Images. (D) Localisation of Rab11a (gray), tubulin (green), and viral NP (magenta) proteins at 8 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/vRNPs/Rab11a are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Bar = 10 μm. The figure panels for the corresponding mock-infected cells can be found in . (E) A schematic representation of shape classification based on circularity versus roundness is shown. (F) The roundness and circularity of viral inclusions/vRNPs, marked by NP staining, were determined at 8 h postinfection using the Shape Descriptor tool (Image J, NIH) and plotted against each other for siNT and siATG9A-treated cells. The maximum value of roundness and circularity (1) corresponds to a circular structure, whereas the minimum value represents a linear structure (0). More than 80 cells, pooled from 3 independent experiments, were analyzed per condition. Statistical analysis was done by Mann–Whitney test (*** p < 0.001). A similar analysis done for the other autophagy factors is shown in . The frequency distribution of roundness and circularity of viral inclusions/vRNPs is shown in . (G-I) Protein levels of LC3-I and LC3-II were quantified by western blotting and plotted as the relative expression to actin protein levels ± SEM. Expression was normalized to siNT from mock-infected (M) cells. Data are a pool from 6 independent experiments. Statistical analysis was done by one-way ANOVA, followed by a Tukey’s multiple comparisons test (no statistical significance detected). All the values of individual and pooled experiments are provided in File, and the original uncropped blots can be found in Images. IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; RT-qPCR, quantitative reverse transcription PCR; SEM, standard error of the mean; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Plaque Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, MANN-WHITNEY, Reverse Transcription

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–C) Cells (A549) were infected or mock-infected with PR8 virus, at an MOI of 3, for the indicated times. (A) The localization of host proteins ATG9A (green) and GM130 (gray) and viral protein NP (magenta) was determined by immunofluorescence using antibodies against these proteins. Mock-infected cells were collected at the same time as the 14 h-infected cells. Nuclei (blue, Hoechst staining) and cell periphery are delimited by white and yellow dashed lines, respectively. Bar = 10 μm. (B) Colocalization between ATG9A and GM130 in the images acquired in ( A ) was determined using the Colocalization Threshold analysis tool (FIJI/Image J, NIH) and plotted as the Pearson R value. Approximately 30 cells, from a single experiment, were analyzed per experimental condition. Red bar represents the median of values. Statistical analysis was done by Kruskal–Wallis test (** p > 0.01; *** p > 0.001). (C) The levels of ATG9A, actin, and viral NP protein in cell lysates at the indicated time points were determined by western blotting. ATG9A band intensity was quantified using FIJI (ImageJ, NIH) and normalized to actin levels. Original blots can be found in Images. Experiments ( A – C ) were performed twice. (D–F) Cells (A549) were transfected with a plasmid encoding GFP-ATG9A for 24 h and then infected or mock-infected with PR8 virus, at an MOI of 10, for 8 h. The localization of endogenous host proteins (GM130 –Golgi, Calnexin–ER, or Rab11a –recycling endosome) and viral protein NP was determined by immunofluorescence using antibodies against these proteins. Nuclei (blue or gray, Hoechst staining) and cell periphery are delimited by white and yellow dashed lines, respectively. Yellow arrowheads highlight areas of contact between viral inclusions and overexpressed GFP-ATG9A protein. Red arrowheads highlight areas of colocalization between GFP-ATG9A and the Golgi marker GM130. Bar = 10 μm. Control cells expressing GFP alone can be found in . Experiments ( D – F ) were performed twice. All the values of individual and pooled experiments are provided in File. ER, endoplasmic reticulum; GFP, green fluorescent protein; IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; TGN, trans -Golgi network.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Immunofluorescence, Staining, Western Blot, Transfection, Plasmid Preparation, Marker, Control, Expressing

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–C) Cells (GFP-Rab11a WT low or GFP-Rab11a DN low ) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 10 h, at an MOI of 3. (A) Viral production was determined by plaque assay and plotted as PFU per milliliter (mL) ± SEM. Data represent 6 replicates from a single experiment. Two independent experiments were performed. Statistical analysis was done by one-way ANOVA, followed by a Kruskal–Wallis test (* p < 0.05; *** p < 0.001). (B) The protein level of ATG9A, lamin B, GFP, and Rab11a before infection were quantified by western blotting. The levels of ATG9A were plotted as the relative expression to lamin B level ± SEM. Expression was normalized to siNT from mock-infected cells. The data are a pool from 3 independent experiments. Statistical analysis was done by unpaired t test between siNT vs. siATG9A conditions of each condition (Rab11a WT vs. DN mock; *** p < 0.01). (C) Localisation of Rab11a (magenta) and PDI (gray) at 10 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/Rab11a are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Mock-infected cells can be found in . Bar = 10 μm. (D–F) Cells (A549) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 8 h, at an MOI of 3. (D) The localisation of host Rab11a (green) and viral NP (magenta) proteins at 8 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/vRNPs are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Bar = 10 μm. Experiments were performed twice. (E) Colocalization between Rab11a and NP in the images acquired in ( D ) was determined using the Colocalization Threshold analysis tool (Image J, NIH) and plotted as the Pearson R value. At least 20 cells, pooled from 2 independent experiments, were analyzed per experimental condition. Red bar represents the median of values. Statistical analysis was done by Mann–Whitney test (n.s., not significant). (F) The roundness and circularity of Rab11a structures in the images acquired in ( D ) were determined using the Shape Descriptor tool (Image J, NIH) and plotted against each other. The maximum value of roundness and circularity (1) corresponds to a circular structure, whereas the minimum value represents a linear structure (0). Approximately 30 cells, from 2 independent experiments, were analyzed per condition. Statistical analysis was done by Mann–Whitney test (*** p < 0.001). The frequency distribution of roundness and circularity of structures marked by Rab11a is shown in . All the values of individual and pooled experiments are provided in File. GFP, green fluorescent protein; IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; SEM, standard error of the mean; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Plaque Assay, Western Blot, Expressing, Immunofluorescence, Staining, MANN-WHITNEY

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: (A–D) Cells (GFP-Rab11a WT low , magenta) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h. Upon this period, cells were infected or mock-infected with PR8 virus for 8 h, at an MOI of 3, and simultaneously treated with 200 nM Sir-Tubulin dye to stain the microtubules (green) in live cells. Cells were imaged for 10 min (2 s/frame) under time-lapse conditions at 8 h postinfection. White boxes show viral inclusions/Rab11a. Individual frames with single moving particles highlighted with yellow arrows are shown in the small panels. Bar = 10 μm. Images from selected infected cells were extracted from and Videos. Images from mock-infected cells were extracted from and Videos. For each case, a linescan was drawn as indicated to assess the dynamics of Rab11a and tubulin. The fluorescence intensity of Rab11a endosomes or viral inclusions (magenta) and tubulin (green) at indicated times was plotted against the distance (in μm). Representative analysis was performed using images from ( A - D ). (E, F) Cells (GFP-Rab11a WT low ) were treated as explained above (in A–D). At 8 h postinfection, cells were treated with DMSO or 10 μg/mL of nocodazole for 2 h. Cells were imaged at 10 h postinfection. White boxes show viral inclusions/Rab11a. Bar = 10 μm. (G) Scheme illustrates how viral inclusion/Rab11a endosome deviation from a reference position (in X and Y direction) was tracked by live cell imaging. The formula used to quantify the mean squared displacement (MSD, μm 2 ) is also shown. (H) Each viral inclusion/Rab11a endosome in a cell was tracked using the TrackMate plugin (FIJI, NIH) and displacement was quantified as explained in ( G ). Data were plotted as the MSD (μm 2 ) per treatment. The red dot indicates the median in the boxplots. Statistical analysis was done by a Kruskal–Wallis test (*** p < 0.001). (I) Colocalization between microtubules (tubulin) and viral inclusions (Rab11a) in live cells was determined as the Manders’ Overlap Coefficient tM1 (thresholded; explained in Methods section). Only infected conditions are shown. Given that very small Rab11a endosomes were scattered throughout the cytosol in mock-infected controls, we could not obtain reliable correlation coefficients. This result is, however, corroborated by quantifying colocalization between microtubules (tubulin) and viral inclusions using a fluorescent virus (PA-mNeonGreen PR8), as shown in . Between 6 and 10 cells per condition were analyzed. Statistical analysis was done by a Student t test (*** p < 0.001). Experiments were performed twice. All the values of individual and pooled experiments are provided in File.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Virus, Staining, Fluorescence, Live Cell Imaging

Journal: PLOS Biology

Article Title: ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

doi: 10.1371/journal.pbio.3002290

Figure Lengend Snippet: We currently view liquid viral inclusions, composed of Rab11a endosomes and vRNPs, as sites dedicated to the assembly of the IAV genome [ , , ]. We have previously shown that liquid viral inclusions develop in close contact with the ERES . Here, we describe the initial events on the left panel that may lead to the formation of liquid viral inclusions on the right to facilitate the formation of IAV genomic complex. In this study, we demonstrate that IAV infection reduces the Rab11a-regulated recycling capacity of the host cell (step 1). This effect is likely a consequence of vRNP binding to Rab11a endosomes, which are then rerouted to the ERES to form viral inclusions. Such trafficking of Rab11a endosomes carrying the vRNPs to the ER is regulated by the host factor ATG9A. We identified that ATG9A is mobilized from the Golgi during IAV infection (step 2) and leads to the removal of Rab11a-vRNP complexes from microtubules when at the ER (step 3). It is thus possible that ATG9A moves to the ER to promote the linkage of viral inclusions to microtubules. In this location, vRNPs-Rab11a units may establish multiple and dynamic contacts forming liquid percolation-driven condensates. We also show (although with overexpression experiments) that ATG9A engages in multiple contacts with viral inclusions (step 4). We propose that the liquid properties of viral inclusions favor the formation of the 8-segmented IAV genome that is transported to the plasma membrane (step 5). ATG9A, autophagy related gene 9A; ER, endoplasmic reticulum; ERES, ER exit site; IAV, influenza A virus; vRNP, viral ribonucleoprotein.

Article Snippet: Antibodies used were as follows: rabbit polyclonal against Rab11a (1:200; Proteintech, 15903-1-AP), ATG9A (1:200, Abcam, 108338), and viral NP (1:1,000; gift from Prof Paul Digard, Roslin Institute, UK); mouse monoclonal against viral NP (1:1,000; Abcam, 20343), PDI (1:500, Life Technologies, MA3-019), GM130 (1:500, BD Transduction Laboratories, 610823); mouse monoclonal against alpha-tubulin (1:1,000; b5-1-2, Sigma, T6074).

Techniques: Infection, Binding Assay, Over Expression, Clinical Proteomics, Membrane, Virus

Journal: Biochimica et biophysica acta

Article Title: SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover.

doi: 10.1016/j.bbamcr.2010.09.008

Figure Lengend Snippet: Fig. 5. Inhibition of SNARE-mediated membrane traffic inhibits Src trafficking from a Rab11 compartment. CHO-E329Q cells were mock treated (Wild-type NSF; A–C, G–I) or induced with doxycycline (E329Q-NSF; D–F, J–L) for 9 h and serum-starved for the last hour, before being lifted, and plated on FN for the indicated times. Cells were fixed in ice-cold 0.1% Triton X-100, 2% paraformaldehyde and stained with anti-Src (red; A, D, G, and J) and anti-Rab11 (green; B, E, H, and K). Arrowheads point to Src-Rab11-containing compartment in WT-NSF cells (I) and E329Q-NSF cells (L). Note the increased amount of Src in Rab11-containing compartment in L. Scale bar=10 μm. (M) Cells from the above experiments, as well as untreated cells, and cells where membrane traffic was inhibited with truncated SNAP23 (SN23CΔ9) and dominant-negative Rab11 (Rab11S25N) were visually scored as having primarily perinuclear Src staining (light grey bars), as seen in panel A, or peripheral Src staining (dark grey bars), as seen in panel G. Results are the means±SEM of at least 3 separate experiments in which at least 50 cells were scored.

Article Snippet: Antibodies were obtained against NSF from Stressgen Biotechnologies (Victoria, BC), Rab11, phosphoTyr397-FAK and mouse anti-FAK (F15020) from BD Biosciences (Mississauga, ON), rabbit anti-FAK from Abcam (ab40794; Cambridge, MA), phosphoTyr576-FAK from Novus Biologicals (Littleton, CO), phosphoTyr925-FAK and phosphoSer473Akt from Cell Signaling Technology, Inc. (Boston, MA), phosphoTyr418Src, phosphoTyr861-FAK and Src from Applied Biological Materials, Inc. (Richmond, BC), α-tubulin from Invitrogen, Co. (A11126; Mississauga, ON), and Rac from Cytoskeleton, Inc. (Denver, CO).

Techniques: Inhibition, Membrane, Staining, Dominant Negative Mutation

Journal: Traffic (Copenhagen, Denmark)

Article Title: Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization.

doi: 10.1111/j.1600-0854.2007.00691.x

Figure Lengend Snippet: Figure 7: Colocalization of NPY activated EGFP-tagged Y1 receptors with transferrin–Texas Red, mCherry-tagged Rab5a and endogenous Rab4 and Rab11. HEK293 cells stably expressing EGFP Y1 receptors were coincubated with transferrin–Texas Red (5 mg/mL) and 100 nM NPY for 30 min before fixation and visualization by confocal microscopy. EGFP-Y1-expressing cells were transiently transfected with mCherry–Rab5a and activated with 100 nM NPY for 30 min before fixation and visualization by confocal microscopy. For Rab4 and Rab11 localization, the same activation protocol was used on EGFP Y1-expressing cells, but after fixation, cells were permeabilized and immunostained as described in Materials and Methods. Colocalized EGFP signals with transferrin–Texas Red, mCherry– Rab5a, Rab4 or Rab11 appear yellow in the merge picture. White boxes identify selected areas for which an enlarged section is shown. Individual confocal sections are shown. Scale bar represents 10 mm.

Article Snippet: Staining with primary antibodies (1/100 dilution) was of 40 min in PBS–1% BSA–0.5% saponin with purified mouse anti-Rab4 monoclonal antibody (BD Bioscience) or rabbit polyclonal IgG anti-Rab11 antibody (Santa Cruz).

Techniques: Stable Transfection, Expressing, Confocal Microscopy, Transfection, Activation Assay

Journal: Nature Communications

Article Title: PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis

doi: 10.1038/ncomms13546

Figure Lengend Snippet: ( a ) Time-course analysis of recycled active α5β1 integrin in siCTL ECs versus siRAB11A ECs or siRAB11B ECs. RAB11B, but not RAB11A, silencing significantly impairs active α5β1 integrin recycling. Data are mean±s.e.m., n =3 independent experiments (two technical replicates for each experiment). ( b ) Confocal microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). SNAKA51 + active α5β1 integrin localizes in fibrillar adhesion in siCTL and in siRAB11A, but not in siRAB11B ECs in which it accumulates in perinuclear punctae. The number of active α5β1 integrin-containing adhesions per 100 μm 2 of cell area was quantified in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( c ) Confocal xz sectioning microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). Quantitative analysis of apico-basal mean intensity ratio reveals that SNAKA51 + active α5β1 integrin localizes on the basolateral surface of siCTL and siRAB11A, but not of siRAB11B ECs, where it redistributes all around the cell surface. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( d ) Confocal microscopy analysis of IST9 mAb + endogenous cellular ED-A FN (green) in confluent ECs. ED-A FN polymerizes into a fibrillar network in siCTL and siRAB11A, but not in siRAB11B ECs, where it accumulates in a perinuclear compartment. Relative amount of fibrillary ED-A FN area was calculated in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from 2 independent experiments. ( e ) Western blot analysis of soluble ED-A FN released by confluent ECs seeded on Transwell inserts. An equal percentage of apical and basolateral volumes of medium were collected after 72 h of culture from different wells of siCTL or siRAB11B ECs. Equal amounts of rabbit IgG were exogenously added to samples (spike normalization) for loading control purposes. Quantification of the ratio between apical or basolateral amount of ED-A FN released by siCTL over siRAB11B ECs. RAB11B silencing much more severely impairs basolateral than apical ED-A FN secretion. Data are mean±s.e.m., n =6 wells per condition pooled from three independent experiments. Scale bar, 50 μm ( d ), 20 μm ( b ), 5μm ( c ). ** P <0.01; *** P <0.001; Student's t -test.

Article Snippet: Rabbit polyclonal anti-RAB11A (TA324158) and RAB11B (TA346586) were from Origene, Rockville, MD, USA.

Techniques: Confocal Microscopy, Incubation, Microscopy, Western Blot, Control

Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 by preventing its ubiquitination and degradation. A Heat-map showing 99 circRTN4-interacting proteins in PDAC. Biotin-labelled circRTN4, RTN4 mRNA, circGFP were used to pull down circRTN4-interacting proteins. Mass spectrometry analysis was performed to identify the interacting proteins. B CircRTN4 interacted with RAB11FIP1 in PDAC cells, as revealed by RIP assay. C Bioinformatics analysis by PRIdictor revealed the RAB11FIP1-binding site (The seed region of interaction was in red) on circRTN4. Mutating RAB11FIP1-binding site on circRTN4 (The mutated seed region of interaction was in green) inhibited circRTN4-RAB11FIP1 interaction in PANC-1 cells, as revealed by circRNA pulldown assay. D CircRTN4 knockdown inhibited RAB11FIP1 expression in mice xenograft. E CircRTN4 knockdown did not affect RAB11FIP1 mRNA level in PDAC cells. F CircRTN4 knockdown decreased the stability of RAB11FIP1 after inhibition of protein synthesis by cycloheximide in PANC-1 cells. G 3-Dimensional structure of the circRTN4-RAB11FIP1 interaction revealed that circRTN4 blocked the ubiquitination site Lys578 of RAB11FIP1. H Immunoprecipitation with anti-RAB11FIP1 antibody in PANC-1 cells after circRTN4 knockdown, followed by immunoblotting analysis with anti-ubiquitin or anti-RAB11FIP1 antibody. CircRTN4 knockdown increased ubiquitination of RAB11FIP1. I RAB11FIP1 expression were upregulated in PDAC primary tumors and was positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Binding Assay, Knockdown, Expressing, Inhibition, Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 to promote EMT in PDAC. A N-cadherin expression was inhibited after circRTN4 knockdown in PANC-1 cells. B CircRTN4 knockdown in mice subcutaneous tumors inhibited N-cadherin expression. C Expression of EMT markers Slug, Snai1, Twist and Zeb1 were reduced after circRTN4 knockdown in PDAC cells. D Expression of Slug, Snai1, Twist and Zeb1 in circRTN4-overexpressing HPDE cells were rescued after RAB11FIP1 knockdown. E Slug, Snai1, Twist and Zeb1 expression were upregulated in PDAC primary tumors and were positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Expressing, Knockdown

Journal: Neuroscience Bulletin

Article Title: Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons

doi: 10.1007/s12264-023-01174-y

Figure Lengend Snippet: The interaction of MyoVa with FIP3 is required for NMDAR transport. A Schematic of MyoVa and its association with cargo proteins via Rab11 and FIPs. B Co-IP between FIP3 and associated proteins with antibodies to FIP3 in control or under PMA conditions. Non-immune IgG was used as control. C Statistical analysis of binding between FIP3 and associated proteins. Signals were divided by FIP3 signals and normalized to control. MyoVa: 1.33 ± 0.06, P <0.01; GluN1: 1.44 ± 0.06, P <0.01; GluN2A: 1.45 ± 0.05, P <0.01; Rab11: 1.38 ± 0.08, P <0.01; n = 9; unpaired Student’s t -test vs control. D PMA enhances the interaction of FIP3 and GluN1, revealed by co-IP between FIP3 and GluN1 with antibodies to GluN1 in control or under PMA conditions. Non-immune IgG was used as control. E Statistical analysis of binding between FIP3 and GluN1. FIP3 signals were divided by GluN1 signals and normalized to control. FIP3: 1.37 ± 0.12, P <0.05, n = 8; unpaired Student’s t -test vs control. F Representative images display increased colocalization of FIP3 and GluN1 in primary hippocampal neurons stained with GluN1 (green) and FIP3 (red) antibodies in control or under PMA conditions. Scale bar, 5 μm. G Quantification of the colocalization between GluN1 and FIP3 by Pearson’s coefficient. Control: n = 20 neurons, 0.62 ± 0.01; PMA: n = 26 neurons, 0.71 ± 0.01, P <0.01; data are from at least three independent cultures; unpaired Student’s t -test vs control. H Illustration of the locations used for unilateral viral injections into the hippocampal CA1 region. The virus expresses FIP3 KD or scrambled shRNA and is reported by EGFP (green). I–K FIP3 KD blocks the PMA-induced LTP of NMDA fEPSPs. The overlaid traces display changes in the average response selected at the times shown (marked by 1 and 2). J Cumulative probability of potentiation magnitude of NMDA fEPSPs. K Summary graphs of LTP magnitude from experiments shown in I . Control: n = 7, 1.69 ± 0.07; FIP3 KD: n = 8, 1.01 ± 0.01, P <0.01; scrambled: n = 7, 1.79 ± 0.09; P >0.05; one-way repeated-measures ANOVA vs control. Scale bars, 0.5 mV, 100 ms in I . L–N As in I–K , with the exception that LTP of NMDA fEPSPs was elicited by TBS. Control: n = 7, 1.69 ± 0.06; FIP3 KD: n = 7, 0.98 ± 0.02, P <0.01; scrambled: n = 7, 1.75 ± 0.08, P >0.05; one-way repeated-measures ANOVA vs control. Scale bars, 0.5 mV, 100 ms in L . The data are represented as the mean ± SEM, * P <0.05; ** P <0.01. ns, no significant difference.

Article Snippet: The major primary antibodies were: MyoVa (Sigma, St. Louis, USA, HPA001356), GluN2A (Millipore, Burlington, USA, AB1555P), GluN1 (rabbit: Abclonal, Wuhan, China, A11699; mouse: Abcam, Cambridge, UK, ab134308), GluA1 (Millipore, MAB2263), GluN2B (Cell Signaling Technology, Boston, USA, 14544), Rab11 (Invitrogen, Carlsbad, USA, 71-5300), FIP3 (Rockland, Philadelphia, USA, 600-401-994), PSD95 (Millipore, MABN68), MyoVb (Sigma, HPA040902), CaMKII (Santa Cruz Biotechnology, Inc., Dallas, USA, sc-13141), GST (glutathione-S-transferase, Genscript, Nanjing, China, A00866-100).

Techniques: Co-Immunoprecipitation Assay, Control, Binding Assay, Staining, Virus, shRNA

Journal: Neuroscience Bulletin

Article Title: Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons

doi: 10.1007/s12264-023-01174-y

Figure Lengend Snippet: FIP3 KD impairs hippocampal memory. A Schematic showing the locations used for bilateral viral injections into the CA1 region of the hippocampus. B Representative images showing EGFP expression in the CA1 region of the dorsal hippocampus prepared from FIP3 KD rats. Scale bars, 250 μm and 75 μm (enlarged images). C, D FIP3 KD rats show deficits in contextual ( C ) and trace fear memory ( D ) tested at 24 h, whereas the scrambled animals showed freezing comparable to control rats. FD, fear conditioning. C : control, n = 14 rats, 62.46% ± 3.52%; FIP3 KD, n = 9 rats, 32.33% ± 5.13%, P <0.01; scrambled, n = 8 rats, 62.23% ± 5.56%; P >0.05; one-way repeated-measures ANOVA vs control. D : Control: n = 14 rats, tone, 58.82% ± 6.40%; trace 68.73% ± 6.40%; ITI, 56.60% ± 5.45%; FIP3 KD: n = 8 rats, tone, 32.76% ± 7.21%, P <0.05; trace, 38.96% ± 7.94%, P <0.05; ITI, 33.39% ± 7.72%, P <0.05; scrambled: n = 8 rats, tone, 59.90% ± 8.03%, P >0.05; trace, 71.62% ± 6.31%, P >0.05; ITI, 65.72% ± 6.51%, P >0.05; two-way repeated-measures ANOVA vs control. E Schematic of the novel object preference (NOR) task. F, G FIP3 KD rats show deficits in the NOR task. In the acquisition phase, the FIP3 KD, scrambled, and control rats spend similar amounts of time exploring the two objects. In the test phase tested at 24 h, control and scrambled rats prefer the novel objects, whereas the FIP3 KD rats exhibit a lower preference for the novel objects. Acquisition: control, n = 10 rats, 49.58% ± 1.43%; FIP3 KD, n = 12 rats, 49.34% ± 2.87%, P >0.05; scrambled, n = 10 rats, 51.09% ± 2.21%, P >0.05. Test: control, n = 10 rats, 65.62% ± 1.67%; FIP3 KD, n = 12 rats, 52.87% ± 3.92%, P <0.05; scrambled, n = 10 rats, 67.65% ± 3.70%, P >0.05; one-way repeated-measures ANOVA vs control. H Schematic of novel place preference test (NPP). I, J FIP3 KD rats show deficits in the NPP task. In the acquisition phase of the object location task, FIP3 KD, scrambled, and control rats spend similar time exploring the two objects. In the test phase at 24 h, control and scrambled rats prefer the relocated object, whereas the FIP3 KD rats have a lower preference for the relocated object. Acquisition: control, n = 13 rats, 50.66% ± 2.88%; FIP3 KD, n = 14 rats, 49.44% ± 2.48%, P >0.05; scrambled, n = 12 rats, 51.70% ± 2.90%, P >0.05. Test: control, n = 13 rats, 68.02% ± 3.12%; FIP3 KD, n = 14 rats, 50.74% ± 3.01%, P <0.01; scrambled, n = 12 rats, 64.38% ± 3.92%, P >0.05; one-way repeated-measures ANOVA vs control. K Schematic of the temporal order memory task. L FIP3 KD rats show deficits in the temporal order memory task. In the temporal order memory task, control and scrambled rats prefer the object explored at the early stage to that explored at the end, whereas the FIP3 KD rats have a comparable preference for the two objects. Control, n = 11 rats, 68.31% ± 3.20%; FIP3 KD, n = 15 rats, 52.14% ± 3.45%, P <0.01; scrambled, n = 11 rats, 68.07% ± 3.43%, P >0.05; one-way repeated-measures ANOVA vs control. The data are represented as the mean ± SEM, * P <0.05; ** P <0.01.

Article Snippet: The major primary antibodies were: MyoVa (Sigma, St. Louis, USA, HPA001356), GluN2A (Millipore, Burlington, USA, AB1555P), GluN1 (rabbit: Abclonal, Wuhan, China, A11699; mouse: Abcam, Cambridge, UK, ab134308), GluA1 (Millipore, MAB2263), GluN2B (Cell Signaling Technology, Boston, USA, 14544), Rab11 (Invitrogen, Carlsbad, USA, 71-5300), FIP3 (Rockland, Philadelphia, USA, 600-401-994), PSD95 (Millipore, MABN68), MyoVb (Sigma, HPA040902), CaMKII (Santa Cruz Biotechnology, Inc., Dallas, USA, sc-13141), GST (glutathione-S-transferase, Genscript, Nanjing, China, A00866-100).

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

doi: 10.1038/s41467-023-36398-z

Figure Lengend Snippet: (1) Binding of GII.4 with its glycan receptor (HBGAs and possibly with a still unidentified co-receptor) on the cell surface (2) induces plasma membrane wounding (3) triggering signaling responses that direct multiple membrane repair cellular components to the injury site. (4) ASM translocation to the plasma membrane surface results in conversion of sphingomyelin (SM) to ceramide. (5) Ceramide formation along with other membrane repair processes involving gal-3 (glycan damage sensor), ALIX (Ca 2+ sensor) and membrane recycling processes regulated by Rab11 and Rab14 result in membrane reorganization and receptor clustering leading to (6) tubular carrier formation due to multiple interactions of virus with host factors causing membrane bending and (7) endocytosis regulated by Cdc42 and cholesterol. (8) GII.4 entry into the cell results in V-ATPase regulated endosomal acidification causing (9) conformational changes in the virus capsid and release of the viral genome from the endosomal compartment.

Article Snippet: Gal-3, ALIX, TSG101, LAMP-1, Rab11, Rab14, Flotilin-1, CD44 and glycosylphosphatidylinositol-anchored protein (GPI-AP) were detected using 1:200 dilution of rat anti-gal-3 (#125402, Biolegend), rabbit anti-ALIX (#12422-1-AP, Proteintech), anti-TSG101 (#14497-1-AP, Proteintech), rabbit anti-LAMP-1 (#9091, Cell Signaling Technologies), rabbit Rab11 (#5589, Cell Signaling Technologies) and rabbit Rab14 (#A12752, ABclonal), mouse anti-Flotilin-1 (# 610821, BD Biosciences), rabbit anti-CD44 (#15675-1-AP, Proteintech), and rabbit anti-GPI-AP (#10104-1-AP, Proteintech) antibodies, respectively.

Techniques: Binding Assay, Membrane, Translocation Assay, Virus