Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Munc13-4 KD strongly impairs exosome release. (A) SDS-PAGE Western blot of indicated proteins in MDA-MB-231 cells after stable expression of shRNA for Munc13-4 or Rab27a or a scrambled control (Ctrl). (B) Culture medium from MDA-MB-231 cells either untreated or stimulated with 1.25 µM ionomycin for 30 min was centrifuged at 1,000 g to remove cellular debris and 10,000 g to remove large extracellular vesicles. (C) The resulting 10,000- g supernatant was filtered onto a nitrocellulose membrane and analyzed for CD63, CD9, ALIX, and GM130 content by antibody blotting. (D) Quantification of CD63, CD9, and ALIX blots in C are shown as exosome release as a percentage of total cellular material with mean values ± standard error (SE) for n ≥ 3. *, P < 0.05 for comparison with corresponding control samples. (E) Panc-1 or A549 cells were left untreated or were treated with TGFβ-1 for 24 h. Indicated proteins were detected by SDS-PAGE Western blot. (F) Panc-1 cells were left untreated or were treated with TGFβ-1 for 24 h, and Munc13-4 levels were determined by immunofluorescence. TGFβ-1–treated cells exhibited a mesenchymal morphology. Bars, 5 μm. (G) A549 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA were left untreated (Untr) or were treated with TGFβ-1 for 24 h, and SDS-PAGE Western blotting for indicated proteins was conducted. (H) Culture media supernatants (as in B) from A549 cells that were either untreated or were stimulated with 1.25 µM ionomycin for 30 min were filtered onto nitrocellulose membrane and analyzed for CD63 and GM130. (I) Quantification of CD63 + exosome release shown as a percentage of total cellular material with mean values ± SE for n = 5. *, P < 0.05; **, P < 0.01 for comparison with corresponding control samples.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: SDS Page, Western Blot, Expressing, shRNA, Membrane, Comparison, Immunofluorescence, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Munc13-4 translocation to membrane is Ca 2+ dependent. (A) Live-cell epifluorescence imaging of GFP-Munc13-4 in MDA-MB-231 cells at indicated times after ionomycin stimulation. See Video 1. (B) MDA-MB-231 cells expressing wild-type GFP-Munc13-4, GFP-Munc13-4 C2A*, or GFP-Munc13-4 C2B* either left untreated or stimulated with 1.25 µM ionomycin for 5 min were fixed and imaged by confocal microscopy. (C) Indicated proteins were detected by SDS-PAGE and Western blotting of lysates with Munc13-4 antibody from MDA-MB-231 cells stably expressing control shRNA (Ctrl) or shRNA targeting Munc13-4 (KD), or Munc13-4 KD cells rescued with shRNA-resistant wild-type Munc13-4, Munc13-4 C2A*, or Munc13-4 C2B*. (D) Culture media supernatants (as in ) from MDA-MB-231 cells as in C either untreated or stimulated with 1.25 µM ionomycin for 30 min were filtered onto membrane and analyzed for CD63 or GM130. (E) Quantification of CD63 + exosome release (from ) shown as mean values ± SE for n = 3. *, P < 0.05 for comparison between ionomycin-treated and basal. Bars, 5 μm.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Translocation Assay, Membrane, Imaging, Expressing, Confocal Microscopy, SDS Page, Western Blot, Stable Transfection, shRNA, Comparison

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Munc13-4 recruitment to recycling endosomes is dependent on Rab11a. (A) MDA-MB-231 cells immunolabeled for endogenous CD63 (red) and Munc13-4 (green) by confocal microscopy. (B) MDA-MB-231 cells expressing GFP-Munc13-4 and mCherry-Rab27a, mCherry-Rab27b, or mApple-Rab11a imaged by confocal microscopy. (C) Percentage of cells with Pearson’s correlation coefficient for GFP versus mCherry/mApple >0.7. (D) MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Rab11a shRNA and GFP-Munc13-4 were left untreated or were stimulated with 1.25 µM ionomycin for 5 min, fixed, and imaged by confocal microscopy. (E) SDS-PAGE Western blot of indicated proteins in cells stably expressing control shRNA (Ctrl) or Rab11a shRNA. (F) Culture media supernatants (as in ) from control (Ctrl) or MDA-MB-231 cells stimulated with 1.25 µM ionomycin for 30 min were filtered onto membrane and immunoblotted for CD63 and GM130. (G) Quantification of CD63 + exosome release as percentage of cellular total indicated as mean values ± SE for n = 5. **, P < 0.01 for comparison of ionomycin-treated samples. Bars, 5 μm.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Immunolabeling, Confocal Microscopy, Expressing, Stable Transfection, shRNA, SDS Page, Western Blot, Membrane, Comparison

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Munc13-4 regulates MVB size. (A and B) MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA were fixed for immunofluorescence detection of endogenous CD63 (red) and LAMP1 (green) by confocal microscopy (A) or SIM (B). Bars, 5 μm. (C) Mean diameter of CD63 + structures in each indicated cell type imaged by confocal or SIM shown as mean values ± SE for three cells/group from three separate preparations (*, P < 0.05). (D) SIM showing orthogonal (top) and 3D reconstruction view of CD63 (red) and LAMP1 (green) in MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4. Bars, 5 μm. (E) 3D reconstruction of SIM images from Ctrl MDA-MB-231 cell in D of a single MVB labeling CD63 (red) and LAMP1 (green). Linescan of indicated channels is shown on the bottom. Bars, 1 µm.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Stable Transfection, Expressing, shRNA, Immunofluorescence, Confocal Microscopy, Labeling

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Munc13-4 function in exosome release is dependent on Rab11a. (A) MDA-MB-231 cells expressing GFP-Rab11, GFP-Rab11 Q70L, or GFP-Rab11 S25N were immunolabeled for myc-Munc13-4 (red) and endogenous CD63 (cyan). Bar, 5 µm. (B) Pearson’s correlation coefficient for GFP-Rab11 versus CD63 immunolocalization. Mean values ± SE (15 cells/group from three separate preparations) are shown. *, P < 0.05; **, P < 0.01. (C) MDA-MB-231 cells expressing GFP, GFP-Rab11, or GFP-Rab11 S25N were immunoblotted for the indicated proteins. Arrow indicates GFP-Rab11; lower band in upper panel corresponds to Rab11. (D) Cell medium supernatants (as in ) from untreated MDA-MB-231 cells or cells treated with 1.25 µM ionomycin were filtered onto membrane and analyzed for CD63 and GM130. (E) Quantification of CD63 exosome release as percentage of total cellular shown as mean values ± SE ( n = 3) with *, P < 0.05 for comparison to GFP-alone samples. (F) Model for the generation of secretion-competent MVBs as arising from the transient fusion of Rab11 + endosomes with MVB precursors, which requires Munc13-4, Ca 2+ , and Rab11a. MT1-MMP (green bar) is found in the recycling endosome (RE) and delivered to the MVB dependent on Munc13-4 for incorporation to ILVs and exosome release. The secretion-competent MVBs generated acquire components for Ca 2+ -dependent fusion with the plasma membrane. Bars, 5 μm.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Expressing, Immunolabeling, Membrane, Comparison, Generated

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: Direct TIRF imaging of exosome secretion dependent on Munc13-4. (A) pH-sensitive pHluorin (pHl) was inserted into loop 1 of CD63, which localizes to both the limiting membrane and ILVs of MVBs. Initially quenched at the low pH in the MVB, CD63-pHluorin increases fluorescence upon fusion of MVBs with the plasma membrane. (B) MDA-MB-231 cells stably expressing control shRNA (Ctrl, upper row) or Munc13-4 shRNA (Munc13-4 KD, lower row) were transfected with CD63-pHluorin plasmid, stimulated with 1.25 µM ionomycin (at zero time), and imaged for 15 min by TIRF microscopy. The surface of single cells is shown. In the upper row, ionomycin treatment elicited the release of brightened CD63-pHluorin–containing ILVs released into TIRF field. Bar, 5 µm. Inset shows one of the MVBs releasing ILVs as exosomes. Bar, 1 µm. See Videos 2 and 3. (C) Intensity of CD63-pHluorin signal is quantified as fold increase over initial fluorescent value reported as mean values ± SE (≥9 cells/group; *, P < 0.05).

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Imaging, Membrane, Fluorescence, Stable Transfection, Expressing, shRNA, Transfection, Plasmid Preparation, Microscopy

Journal: The Journal of Cell Biology

Article Title: A Ca 2+ -stimulated exosome release pathway in cancer cells is regulated by Munc13-4

doi: 10.1083/jcb.201710132

Figure Lengend Snippet: ECM degradation by exosomal MT1-MMP is dependent on Munc13-4. (A) Exosomes (100,000- g pellet) and 400 µg cell lysate were prepared from MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA (Munc13-4 KD) and analyzed for indicated proteins by SDS-PAGE Western blotting. Exosomes secreted basally or from cells stimulated with 1.25 µM ionomycin for 1 h were compared. (B) MDA-MB-231 cells expressing CD63-mKATE2 and MT1-MMP-pHluorin were imaged by TIRF microscopy before (0 min) and after treatment with 1.25 µM ionomycin for 5 min. (C) Control or Munc13-4 KD cells expressing MT1-MMP-pHluorin and CD63-mKATE2 or mApple-Rab11a were fixed, permeabilized to brighten pHluorin, and imaged by confocal microscopy. (D) Pearson’s correlation coefficient for pHluorin versus mKATE2 or mApple is indicated as mean values ± SE (15 cells/group from three separate preparations are shown). (E) MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA were grown on cover slides coated with Oregon-Green-Gelatin for 16 h and labeled with phalloidin and DAPI. Zoomed images from representative cells are shown. (F) Gelatin degradation was calculated as a percentage of clearing of gelatin from the larger field per cell, shown as mean values ± SE (nine fields of view [5–25 cells]/group from three separate preparations). *, P < 0.05. Bars, 5 μm.

Article Snippet: Antibodies used were rabbit monoclonal Munc13-4 (residue 20–80; EPR4914) from Abcam and goat polyclonal Munc13-4 antibody (residue 967–980; NB100-41385) from Novus Biologicals.

Techniques: Stable Transfection, Expressing, shRNA, SDS Page, Western Blot, Microscopy, Confocal Microscopy, Labeling

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: UNC13D regulates the migration of pancreatic cancer cells via focal adhesion (FA) turnover. A , B Boyden chamber and wound healing assay of cell migration capacity upon UNC13D knockdown in ASPC-1 and PANC-1. Data are presented as the mean ± SD. Two-tailed unpaired Student T test: ** p < 0.01 and *** p < 0.001. The quantification data shown are representative of three independent experiments. C Analysis of cell invasion capacity upon UNC13D knockdown in MIA PaCa-2 and PANC-1 cells. The quantification data are presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student T test: ** p < 0.01. D Gene set enrichment assay (GSEA) of differentially expressed genes (DEGs) between UNC13D-high vs -low groups in the TCGA cohort indicates the enrichment of the integrin pathway and FA pathway. E FA disassembly assay using nocodazole (NDZ) shows UNC13D regulates FA turnover of pancreatic cancer cells. UNC13D knockdown slows FA disassembly. CAPAN-1 shSCR or shUNC13D were starved, left untreated or incubated with 10 μM nocodazole. After 4 h, nocodazole was washed out at different times to allow microtubule regrowth to trigger FA disassembly. The cells were then fixed and immunostained with anti-PXN antibody. Quantification of FAs number was obtained from an average of ten cells of each condition from three independent experiments. Data were shown as mean ± SD of three independent experiments. Two-tailed unpaired Student T test: * p < 0.05. Scale bar, 10 μm. F FAK autophosphorylation and its total form were evaluated by western blot at different times of nocodazole washed-out as in E . FAK autophosphorylation was quantified and normalized to total FAK expression

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Migration, Wound Healing Assay, Knockdown, Two Tailed Test, Incubation, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: Expression level and prognostic significance of UNC13D and FAK (PTK2) in pancreatic cancer. A Comparison between cancer and matched normal pancreas tissue from GSE28735 cohort (n = 45, Wilcoxon signed rank test). B Comparison between cancer and normal pancreas tissues from TCGA and GTEx cohort (n = 350, Mann–Whitney U test). C Comparison between cancer and normal pancreas tissues from GEO cohorts in GENT database (n = 429, Mann–Whitney U test). D Expression level of UNC13D in patients of each subgroup of pancreatic cancer (TCGA, n = 178). E Kaplan–Meier survival analysis of HCC patients according to the UNC13D and FAK (PTK2) gene expression. Overall survival in TCGA (n = 178), ICGC (n = 91), GSE21501(n = 102), and GSE28735 (n = 45) were examined according to the UNC13D, FAK (PTK2), or both expression (Log-rank test). The p value is provided inside each graph

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Comparison, MANN-WHITNEY, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: The Cox regression analysis of four independent cohorts depending on the expression levels of UNC13D and FAK (PTK2)

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: RAB11-UNC13D-FAK axis in recycling endosomes regulates the phosphorylation of FAK. A UNC13D knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by p-FAK, p-PXN, FAK, and PXN. Data are presented as the mean ± SD. The experiments were independently performed at least three times. Statistical analysis was performed using Student’s unpaired T test. ** p < 0.01, *** p < 0.001. B Co-immunoprecipitation analysis of the binding between exogenous Flag-UNC13D and HA-FAK in co-transfected HEK293 cells indicates the direct interaction of them. IgG was incubated with cell lysates as negative controls. C Immunoprecipitation of endogenous FAK from CAPAN-1 and PANC-1 cells confirms the binding between endogenous UNC13D and FAK. D Immunoprecipitation of endogenous RAB11 from PANC-1 overexpressing Flag-UNC13D cells indicates the binding between UNC13D and endogenous RAB11. E Co-immunoprecipitation analysis of the binding among RAB11, UNC13D, and FAK indicates the formation of RAB11-UNC13D-FAK complex. F UNC13D knockdown decreased the binding of RAB11 with FAK. RAB11 was immunoprecipitated from PANC-1 expressing shUNC13D or shSCR to examine the dependency of the interaction between RAB11 and FAK on UNC13D. G Co-localization of FAK, UNC13D, ITGB1, and RAB11 in PANC-1 cells. GFP-FAK, mOrange-UNC13D, and mCerulean-Rab11A cells were transduced. For the active integrin staining, 12G10 anti-ITGB1 was used. The data shown are representative of three independent experiments. Scale bar: 10 μm. H RAB11A knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by pFAK, pPaxillin (p-PXN), FAK, and Paxillin (PXN). The quantification of western blotting in ( G ) is presented as the mean ± SD of three independent experiments. Student’s unpaired T test, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Phospho-proteomics, Knockdown, Immunoprecipitation, Binding Assay, Transfection, Incubation, Expressing, Staining, Western Blot

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: Analysis of the interaction domains of FAK and UNC13D. A Co-immunoprecipitation assay after the transduction of full-length FAK or its truncation mutants in HEK293 cells. Each group was purified by immunoprecipitation with HA beads, followed by a western blot to detect FLAG-UNC13D with the anti-FLAG antibody. B Co-immunoprecipitation analysis after transduction of full-length UNC13D or its truncation mutants in HEK293 cells. Each group was purified by immunoprecipitation with HA beads, followed by a western blot to detect FLAG-UNC13D with the anti-FLAG antibody

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Co-Immunoprecipitation Assay, Transduction, Purification, Immunoprecipitation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: The interaction of UNC13D with FAK is calcium-dependent. A The modulation of calcium-regulated FAK/PXN signaling was measured by the expression level of p-FAK, p-SRC, p-PXN, and p-ERK. The level of calcium was modulated by the treatment with ionomycin 1.25 μM or EGTA 1 mM for 5 min. B The interaction of UNC13D with FAK is modulated by the calcium. The binding between UNC13D and FAK in HEK293 cells was examined by immunoprecipitation after the modulation of calcium concentration by ionomycin 1.25 μM or EGTA 1 mM for 5 min. The quantification data were shown as mean ± SD of three independent experiments. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. * p value < 0.001 ( C ) Mutations of calcium-binding domains (C2A or C2B) in UNC13D regulated its interaction with FAK. UNC13D wild type or C2 mutants (C2A or C2B) in HEK293 cells were immunoprecipitated with HA beads and immunoblotted by anti-FLAG antibody for the detection of Flag-UNC13D. The quantification data were shown as mean ± SD of three independent experiments. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. ** p value < 0.001 ( D ) The abstract figure indicating recycling machinery of integrin coupled with FA turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells. Continuous recycling of integrin via exocytosis and endocytosis of vesicles is required for migration of cancer cells. Through exocytosis of endosomal vesicles, endocytosed integrin reaches at the plasma membrane of leading edge. During the assembly of FA, integrins interact with the extracellular matrix and recruit many proteins including talin, vinculin, paxillin and FAK. During the disassembly of FA, FA complex proteins are disintegrated and integrin is endocytosed into endosomal vesicles in a FAK-dependent manner. During the recycling process of integrin, UNC13D plays roles in tethering and priming of endosomal vesicles to the plasma membrane. Moreover, it can regulate disassembly of FA via RAB11-UNC13D-FAK axis. UNC13D knockdown inhibits exocytosis of endosomal vesicles and disassembly of FA, and finally cellular migration

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Concentration Assay, Comparison, Migration, Clinical Proteomics, Membrane, Knockdown

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: Clinicopathological characteristics of patients and their association with UNC13D expression (TCGA, n = 178)

Article Snippet: The antibodies and dilutions used in this study included an anti-UNC13D antibody (16905–1-AP, 1:1000) purchased from Proteintech (Chicago, USA).

Techniques: Expressing