Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. ( B ) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. ( C ) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). ( D ) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. ( E ) 2D 1 H- 15 N HSQC spectra of 13 C/ 15 N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). ( F ) Peak intensity alteration of 13 C/ 15 N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after ( V ) and before ( V 0 ) addition of unlabeled MUN. ( G ) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( H ) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. ( I ) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Variant Assay, Binding Assay, Labeling, Mutagenesis, Residue

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. ( B to D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E ) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca 2+ , and Munc13-1 (C 1 C 2 BMUN). Donor (NBD) fluorescence was monitored at 538 nm. ( F to H ) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C 1 C 2 BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Labeling, Fluorescence, Liposomes

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

Journal: Molecules

Article Title: Cocaine Self-Administration and Abstinence Modulate NMDA Receptor Subunits and Active Zone Proteins in the Rat Nucleus Accumbens

doi: 10.3390/molecules25153480

Figure Lengend Snippet: Changes in the concentration of Rab3A, RIM1 and Munc13 in the nucleus accumbens in rats following cocaine self-administration. Ras-related protein A (Rab3A), Rab3 interacting molecules 1 (RIM1) and mammalian uncoordinated protein 13 (Munc13). All data are expressed as mean ± SEM. N = 6 rats/group. * p < 0.05 vs. yoked saline. ( a ), Rab3A [ng/mg protein]; ( b ), RIM1 [ng/mg protein]; ( c ), Munc13 [ng/mg protein].

Article Snippet: Quantitative measurement of RIM1, Munc13 and Rab3A in tissue homogenates was performed using Rat Regulating synaptic membrane exocytosis protein 1 (RIM1) ELISA Kit (CK-bio-20387; Shanghai Coon Koon Biotech Co., Shanghai, China), Rat Protein unc-13 (Munc13) ELISA Kit (CK-bio-20385; Shanghai Coon Koon Biotech Co., Shanghai, China) and Rat Ras-related protein Rab3A (Rab3A) ELISA Kit (CK-bio-20386; Shanghai Coon Koon Biotech Co., Shanghai, China) following the manufacturer’s protocol.

Techniques: Concentration Assay, Saline

Journal: Molecules

Article Title: Cocaine Self-Administration and Abstinence Modulate NMDA Receptor Subunits and Active Zone Proteins in the Rat Nucleus Accumbens

doi: 10.3390/molecules25153480

Figure Lengend Snippet: Changes in the concentration of Rab3A. RIM1 and Munc13 in the nucleus accumbens in rats following different conditions of cocaine forced abstinence: (a) cocaine abstinence in an enriched environment; (b) cocaine abstinence in an isolated condition; (c) cocaine abstinence with extinction training; or (d) cocaine abstinence without instrumental task.

Article Snippet: Quantitative measurement of RIM1, Munc13 and Rab3A in tissue homogenates was performed using Rat Regulating synaptic membrane exocytosis protein 1 (RIM1) ELISA Kit (CK-bio-20387; Shanghai Coon Koon Biotech Co., Shanghai, China), Rat Protein unc-13 (Munc13) ELISA Kit (CK-bio-20385; Shanghai Coon Koon Biotech Co., Shanghai, China) and Rat Ras-related protein Rab3A (Rab3A) ELISA Kit (CK-bio-20386; Shanghai Coon Koon Biotech Co., Shanghai, China) following the manufacturer’s protocol.

Techniques: Concentration Assay, Isolation, Saline