primary antibody anti laminin α1  (R&D Systems)

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) UMAP visualization of all the cells colored by sample. ( B ) UMAP visualization of all the cells from dy H /dy H and WT brains colored by cluster identity. ( C ) Cell composition proportion of every annotated cell cluster in dy H /dy H and WT separately. ( D ) Violin plots of the expression of Lama2 gene in WT and dy H /dy H in each annotated cell cluster. ( E ) Immunofluorescence of laminin α2, GFAP (astrocytes), and PLP1 on P14 mouse brain slices. DAPI was for nuclei staining. White arrows indicated the cortical surface. ( F ) Gene Ontology (GO) enrichment of differentially expressed genes in vascular and leptomeningeal fibroblasts, vascular smooth muscle cells, and astrocytes. ( G ) Cell-cell communications among cell clusters. Total number of inferred cell-cell interaction counts (left) and strength (right) in dy H /dy H and WT, respectively. Heatmap of ligand-receptor interaction counts in all pairwise cell clusters in WT and dy H /dy H brains.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Immunofluorescence, Staining

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) The ligand-receptor pairs in laminin pathway. ( B ) Violin plots showed the expression of Mbp , Mobp , Plp1 , Slc1a2 , Mt3 , and Slc6a13 in every annotated cell cluster in dy H /dy H and wild-type (WT) separately. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05. ( C ) Hypothesis that cortical dysplasia (cobblestone malformation) and neuronal over-migration in the cortex due to a defective gliovascular basal lamina of the blood-brain barrier caused by the lack of laminin α2 in LAMA2 -MD.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Migration

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) Quantitative analysis of immunofluorescence staining for myosin heavy chain (MYHC), MYOG, desmin, MYOD1, β-tubulin, and MYF-5 in wild-type (WT) and dy H /dy H muscles at P14. ( B ) Quantitative analysis of Western blot for laminin α2, F-actin, α-actin, MYH2, MYHC, desmin, β-tubulin, MYOG, and MYOD1 in P14 WT and dy H /dy H muscles. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Immunofluorescence, Staining, Muscles, Western Blot

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: Colocalization of α-dystroglycan (red fluorescence, blue arrow) and laminin α2 chain (green fluorescence, yellow arrow) showed that α-dystroglycan protein was normally located and expressed in the cell membrane both in the wild-type (WT) and dy H /dy H muscles. Scale bars: 40 µm.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Fluorescence, Membrane, Muscles

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: Laminin α2 deficiency in the basement membrane due to pathogenic variants in the LAMA2 gene leads to loss of dystroglycan/integrin-matrix scaffolds and extracellular matrix networks. Then, a series of molecular changes associated with the cytoskeleton, extracellular matrix, fibrosis, inflammation, apoptosis, pyroptosis, and mitochondrial energy metabolism occur.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Membrane

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Western blotting of integrin subunits in PC3 cells and sEVs and spreading assay of the cells that secreted sEVs in this study on all three ECMs (fibronectin, laminin, and collagen type I) coated on glass. (A) sEVs were isolated from the cell culture supernatant of intact PC3 cells or from cells in which an integrin subunit (β1, β4, α2, or α6) was knocked out via the CRISPR-Cas9 method. (B) The correlation map of integrin heterodimers and the ECM. (C) dSTORM images of the ECM (left-top: fibronectin, left-bottom: collagen type Ⅰ, and right: laminin) coated on glass. (D) Images of HeLa cells attached to glass coated with ECM components (fibronectin, collagen type Ⅰ, and laminin) or casein after 0, 30, or 60 min of incubation. (E) The time course of the area of five tumor cell lines on glass coated with ECM components (fibronectin, collagen type Ⅰ, and laminin) or casein ( n = 8 cells). Data are presented as the mean ± SE. Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Western Blot, Isolation, Cell Culture, CRISPR, Incubation

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Integrin α2β1 in sEVs derived from PC3 cells is responsible for the binding of CD63-containing sEVs to collagen type I, and integrins α6β1 and α6β4 are responsible for the binding to laminin. (A, C, E, and G) Single-particle fluorescence images of sEV–CD63Halo7-SF650T on glass coated with fibronectin, collagen type I, and laminin before and after the KO of integrin β1 (A), integrin α2 (C), integrin α6 (E), and integrin β4 (G). (B, D, F, and H) The numbers of sEVs attached to glass coated with these ECM molecules before and after the KO of integrin β1 (B), integrin α2 (D), integrin α6 (F), and integrin β4 (H) ( n = 16 images). Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Derivative Assay, Binding Assay, Single Particle, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: The integrin β1, integrin α6, and integrin β4 subunits in sEVs containing CD81 or CD9 derived from PC3 cells are responsible for the binding of the sEVs to laminin, and the integrin α2 subunit is responsible for the binding of the sEVs to collagen type I. (A–H) The numbers of intact PC3 cell–derived sEVs attached to glass coated with fibronectin, collagen I, and laminin were compared with those of sEVs derived from integrin β1 (A and B), integrin α2 (C and D), integrin α6 (E and F), and integrin β4 (G and H) KO cells. CD81-Halo7 (A, C, E, and G) and CD9-Halo7 (B, D, F, and H) in sEVs were labeled with SF650T. Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Derivative Assay, Binding Assay, Labeling

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Neither laminin nor fibronectin is present on PC3-derived sEV surfaces. (A) Schematic diagram of the isolation of specific sEVs. sEVs were isolated by ultracentrifugation at 200,000 × g for 4 h (UC sample). Then, special sEVs were isolated from sEVs by a bead-conjugating antibody (IP: immunoprecipitation sample). (B) Western blot analysis of tetraspanin (CD63, CD81, and CD9), fibronectin (FN), and laminin (LN) in UC and IP samples. sEVs were isolated by IP using an anti-CD63 antibody. (C) Western blot analysis of sEVs isolated by IP using anti-fibronectin antibody. Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Derivative Assay, Isolation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Tumor-derived sEVs bind much more strongly to laminin than to fibronectin. (A) The diameters of sEVs, mEVs, and MVs derived from the 4175-LuT, PC3, BxPC3, HeLa, and SKBR3 cell lines were measured by qNano. (B) Single-particle fluorescence images of sEV–CD63Halo7-SF650T particles derived from the 4175-LuT, PC3, BxPC3, HeLa, and SKBR3 cell lines on glass coated with fibronectin or laminin. These sEVs were purified by ultracentrifugation (200,000 × g for 4 h). (C–E) The numbers of EV–CD63Halo7-SF650T particles (sEVs [C], mEVs [D], and MVs [E]) attached to glass coated with ECM components ( n = 16 images). mEVs and MVs were isolated by low-speed centrifugation (50,000 × g for 30 min) and (10,000 × g for 30 min), respectively. Data are presented as the mean ± SE. The numbers of sEVs, mEVs, and MVs derived from all cell lines applied to laminin- or fibronectin-coated glass were adjusted to the same. (F) Western blotting of integrin subunits in sEVs, mEVs, and MVs derived from 4175-LuT, PC3, BxPC3, HeLa, and SKBR3 cells. Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Derivative Assay, Single Particle, Fluorescence, Purification, Isolation, Centrifugation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: GM1 is responsible for the binding of sEVs to laminin. (A) (top) Schematic diagram of gangliosides and (bottom) chemical structure of GM1. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG). (B) Dot blotting of GM1 and GM3 in PC3 cells and PC3-derived sEVs. (C) Western blot analysis of integrin subunits in B78 cell lines with abundant expression of one type of ganglioside—GM1, GM2, GM3, GD2, or GD2/GD3—and sEVs derived from these cells. (D and E) The numbers of sEVs attached to glass coated with laminin (D) and the numbers normalized to the ratio of integrin α6/CD81 in the sEVs (E). (F and G) The numbers of sEVs attached to glass coated with fibronectin (F) and the numbers normalized to the ratio of integrin α5/CD81 in the sEVs (G). (H) Single-particle fluorescence images of DMPC liposomes containing GM1, GM2, GM3, GD1a, GD2, or GD3 on glass coated with fibronectin or laminin. (I and J) The numbers of liposomes attached to glass coated with laminin (I) or fibronectin (J). (K) Single-particle fluorescence images of sEVs-PC3-CD63Halo7-TMR and sEVs-PC3-ITGB1KO-CD63Halo7-TMR on laminin before or after treatment of the GM1’s glycan. (L) The numbers of sEVs bound to laminin before or after treatment with a high concentration of the GM1 glycan moiety (0.5 mM final). Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided). In I and L, due to the necessity of multiple statistical tests, the significance level was corrected by the Bonferroni method and determined to be 0.025 (=0.05/2). Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Derivative Assay, Western Blot, Expressing, Single Particle, Fluorescence, Liposomes, Glycoproteomics, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Integrins β1 and α6 in sEVs and the ECM are responsible for the binding of sEVs to cell PMs. (A) Immunofluorescence images of fibronectin, collagen type I, laminin, laminin α5, and laminin α1 in human normal embryonic lung fibroblast (MRC-5) cells and human bone marrow stromal (HS-5) cells. (B) The fluorescence intensities of laminin on cells in 1,000 μm 2 ( n = 4 images). (C) TIRF images of MRC-5 cells expressing mCherry and sEV–CD63Halo7-SF650T particles (arrowhead) attached to cell membranes after 10, 20, and 30 min of incubation. sEVs were isolated from the cell culture supernatant of intact PC3 cells (top panels) and from the cell culture supernatant of integrin β1 KO PC3 cells (bottom panels) bound to the basal surface of MRC-5 cells. (D) TIRF images of HS-5 cells and sEV–CD63Halo7-SF650T particles (arrowhead) attached to the basal surface of cell membranes after 30 min of incubation. sEVs were isolated from intact PC3 cells (top panels) and integrin β1 KO PC3 cells (bottom panels). (E) TIRF images of MRC-5 cells and the attached sEV–CD63Halo7-SF650T particles derived from intact PC3 cells (top panels) and from integrin α6 KO PC3 cells (bottom panels) after 30 min of incubation. (F–H) Time course of the numbers of intact sEVs and integrin β1 KO sEVs attached to the basal surface of the MRC-5 cell membrane ( n = 8 images) (F) and to the basal surface of the HS-5 cell membrane ( n = 15 images) (G) per 1,000 μm 2 . (H) Time course of the numbers of intact sEVs and integrin α6 KO sEVs attached to the basal surface of the MRC-5 cell membrane per 1,000 mm 2 ( n = 12 images). Data are presented as the mean ± SE. (I) Fluorescence images of MRC-5 cells expressing GFP and sEV–CD63Halo7-TMR by confocal microscopy. sEVs were isolated from the cell culture supernatant of WT PC3 cells (top panels) and from that of integrin β1 KO PC3 cells (bottom panels). The cells were fixed after treatment of the sEVs for 1 h. (J) The numbers of sEVs attached to MRC-5 and HS-5 cell PMs by confocal fluorescence microscopy ( n = 10 images). In , since not all cells necessarily express mCherry or GFP, many fluorescent spots of sEVs were observed either in regions containing non-expression cells or potentially on the glass surface. Nevertheless, we can quantify the number of sEVs bound to both the apical and basal surfaces of the cell PM by counting the number of fluorescent EV spots on cells expressing mCherry or GFP. Data are presented as the mean ± SE. n.s., nonsignificant difference; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Immunofluorescence, Fluorescence, Expressing, Incubation, Isolation, Cell Culture, Derivative Assay, Membrane, Confocal Microscopy, Microscopy

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Integrins in sEV bind to laminin through conventional integrin–laminin interactions. (A and B) TIRF images of sEV-PC3-CD63Halo7-TMR stained with anti-activated integrin β1 HUTS-4 (A) and anti-integrin β1 P5D2 (B) on uncoated glass (top) or laminin-coated glass (bottom). (C) Colocalization ratio of fluorescent spots of sEVs stained with the antibody to those of sEV-PC3-CD63Halo7-TMR ( n = 10 images). (D) Western blot analysis of sEVs derived from WT, integrin α6 KO, integrin α6-rescued, and integrin α6 R155A-expressing PC3 cells. (E) Fluorescence images of the sEVs derived from WT, integrin α6 KO, integrin α6-rescued, and integrin α6 R155A-expressing PC3 cells bound to laminin on glass. (F) Quantification of sEVs bound to laminin on glass under the conditions described in E ( n = 8 images). Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided). In F, due to the necessity of multiple statistical tests, the significance level was corrected by the Bonferroni method and determined to be 0.0125 (=0.05/4). Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Staining, Western Blot, Derivative Assay, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: sEVs bind to laminin on the PMs of living MRC-5 cells, as revealed by pseudo real-time super-resolution movie observation. (A) Schematic diagram for generating merged movies of dSTORM images of the ECM structures and single-particle images of sEVs. Data acquisition was performed by observing single-fluorescent molecules of ECM components immunostained with SF650B-conjugated antibodies at 200 frames/s (3,504 frames), and dSTORM images were reconstructed using the data acquired every 5.0 s (=1,002 frames). The first dSTORM image was reconstructed using frames 1–1,002, and the process was then repeated by shifting the initial frames backward by 6 frames; thus, 417 dSTORM images were obtained. These dSTORM still images were connected to construct the dSTORM movie. The single-particle movies of sEVs were subjected to a rolling average for 6 frames and synchronously merged with the dSTORM movie. (B–D) Conventional immunofluorescence images (top-left) and dSTORM images (bottom-left) of the ECM structures (B: fibronectin, C: collagen type I, and D: laminin) on the cells. dSTORM images of the ECM structures (magenta) and single-particle images of sEV–CD63Halo7-TMR particles (green) were merged (right). sEVs localized near (<100 nm) the boundary of ECM structures and sEVs localized alone are indicated by yellow and white arrowheads, respectively. (E) The image sequence (every 0.3 s) of laminin obtained by dSTORM (magenta) and a single sEV–CD63Halo7-TMR particle (green) on the living MRC-5 cell membrane. sEVs colocalized with laminin, as indicated by the yellow arrowhead. (F) Colocalization analysis method. We measured the nearest distance from an edge of the ECM structure to a centroid of the sEV spot and performed this measurement for all pairs of ECM structures and sEV particles. The normalized relative frequency was defined as the ratio of the average value of the spatial pair correlation function of the actual image to that of randomly distributed spots generated by a computer. We obtained histograms showing the distribution of the normalized relative frequency of sEVs at each distance from the edge of the ECM structures. Zero on the x axis indicates the contour of the ECM structures in the dSTORM images determined by the KDE method. When sEVs are enriched near the ECM structures, the normalized relative frequency is >1. (G) Probability density analysis of the sEVs and ECM structures. The colored areas indicate regions within the ECM structures. The sEV–CD63Halo7-TMR particles localized near the contour of laminin ( n = 20 cells).

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Single Particle, Construct, Immunofluorescence, Sequencing, Membrane, Generated

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Video showing the simultaneous observation of sEV-PC3-CD63Halo7-TMR particles (green) and immunostained laminin (magenta) on a living MRC-5 cell. By connecting the dSTORM image sequences of laminin structure, we generated a pseudo real-time dSTORM movie, which was superimposed with a movie of sEV particles (33 frames/s). sEVs localized near (<100 nm) the boundary of laminin structure and sEVs localized alone are indicated by yellow and white arrowheads, respectively. Real-time replay; frame rate, 33 frames/s. Related to , right.

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Generated

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Enlarged video of the simultaneous observation of laminin structure by dSTORM (magenta) and a single sEV-PC3-CD63Halo7-TMR particle (green) on a living MRC-5 cell membrane. The field of view in this video is different from that of . Real-time replay; frame rate, 33 frames/s. Related to .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Membrane

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Talin-1 in sEVs does not regulate the binding affinity of integrins for laminin. (A) Western blot analysis of PC3 and BxPC3 cells after talin-1 KD by siRNA. (B and C) Cell spreading assay of WT and talin-1 (Tln1) KD PC3 cells (B) and BxPC3 cells (C) on glass coated with ECM components: fibronectin (FN), laminin (LN), or collagen typeⅠ (COL1). Cells were observed after 2 h of incubation, and cell areas were quantified ( n = 35 cells). (D) Western blot analysis of PC3 cell–derived sEVs after talin-1 KD by siRNA or overexpression of talin-1. (E–G) The fluorescence images (E) and the numbers of PC3-sEVs attached to glass coated with laminin before and after (F) talin-1 KD or (G) overexpression of talin-1 ( n = 14 images). Halo7-integrin β1 in sEVs was labeled with SF650T. (H–J) The fluorescence images (H) and the numbers of CD63-labeled sEVs attached to glass coated with laminin before and after (I) talin-1 KD and (J) overexpression of talin-1 ( n = 16 images). (K and L) The numbers of EV–CD63Halo7-SF650T particles attached to glass coated with collagen type I before and after (K) talin-1 KD or (L) overexpression of talin-1 ( n = 21 images). (M) Western blot analysis of BxPC3 cell–derived sEVs after talin-1 KD by siRNA. (N–P) Fluorescence images (N) and the numbers of sEVs-BxPC3 bound to laminin (O) or collagen type I (P) on glass before and after talin-1 KD ( n = 7 images). The membranes of sEVs were stained with Exosparkler DeepRed. (Q) Western blot analysis of the phosphorylation of Ser425 on talin-1 in PC3 cells and sEVs. Roscovitine: an inhibitor of CDK5 that phosphorylates Ser425 of talin-1. (R) Western blot analysis of kindlin-2 in PC3 cells and PC3-derived sEVs before and after kindlin-2 KD and talin-1 KD. Data are presented as the mean ± SE. n.s., nonsignificant difference; *P < 0.05; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided). Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Western Blot, Incubation, Derivative Assay, Over Expression, Fluorescence, Labeling, Staining, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Cholesterol impairs the binding of sEVs to laminin and the MRC-5 cell PM. (A) Fluorescence images of sEV–CD63Halo7-SF650T particles bound to laminin (LN) on glass before and after cholesterol depletion by MβCD and the numbers of attached sEVs per image (82 × 82 μm). The cholesterol content in PC3 cell–derived sEVs was reduced to 16% after treatment with MβCD. (B and C) The numbers of sEV–CD63-Halo7-SF650T particles attached to glass coated with laminin before and after treatment with saponin (B) and the addition of cholesterol by MβCD–cholesterol complex (C).The cholesterol content was increased to 186% after treatment with the MβCD–cholesterol complex. (D) Fluorescence images of an MRC-5–GFP cell and sEV–CD63Halo7-SF650T particles on the MRC-5 cell after 30 min of incubation. (E and F) Time course of the number of sEV–CD63-Halo7-SF650T particles per 1,000 μm 2 attached to the MRC-5 cell membrane before and after treatment with MβCD ( n = 16 cells) (E) or the MβCD–cholesterol complex ( n = 8 cells) (F). Data are presented as the mean ± SE. n.s., nonsignificant difference; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Fluorescence, Derivative Assay, Incubation, Membrane

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: CD151 and cholesterol regulate the binding affinity of sEVs for laminin. (A) Western blot analysis of CD151 and integrin subunits in PC3 cells and sEVs after CD151 KD. The amount of cell proteins loaded in one lane was 2.5 times greater than that of the sEVs in the other lane. (B) Images of WT PC3 cells and CD151 KD cells on glass coated with ECM components (fibronectin [FN], laminin [LN], or collagen type Ⅰ [COL1]) after 2 h of incubation. The areas of the cells were quantified ( n = 20 cells). (C) Single-particle fluorescence images of sEV–CD63Halo7-SF650T particles bound to laminin (LN) on glass before and after CD151 was knocked down and cholesterol was depleted by MβCD (left). The number of attached sEVs increased (right) ( n = 21 images). (D) The binding affinity ratio of cholesterol-depleted sEVs to intact sEVs was compared with that of CD151 KD sEVs. (E) Single-particle fluorescence images and the number of sEVs attached to collagen type I on glass before and after CD151 KD ( n = 19 images). (F) Western blot analysis of integrin–CD151 complex in PC3-derived sEVs before and after treatment with MβCD. The complex was immunoprecipitated with anti-integrin β1 and blotted with anti-integrin α6, α3, or CD151 antibodies. Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided). In C, due to the necessity of multiple statistical tests, the significance level was corrected by the Bonferroni method and determined to be 0.025 (=0.05/2). Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Western Blot, Incubation, Single Particle, Fluorescence, Derivative Assay, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Laminin-mediated binding of PC3-derived sEVs induces endothelial morphogenesis in HUVEC. (A) Western blot analysis of laminin γ1 and total laminin levels in laminin γ1 KD HUVEC. (B) Fluorescence images of WT or laminin γ1 KD HUVECs expressing GFP and sEVs-PC3-CD63Halo7-TMR bound to the cells, observed by confocal microscopy. Cells were fixed after 1-h incubation with sEVs. Since not all cells necessarily express GFP, many fluorescent spots of sEVs were observed either in regions containing non-expressing cells or potentially on the glass surface. Nevertheless, the number of sEVs bound to both the apical and basal surfaces of the cell PM can be quantified, as shown in . (C) Quantification of sEV particles bound to both apical and basal PM of WT or laminin γ1 KD HUVEC by confocal microscopy ( n = 15 cells). (D) Time course analysis of the number of sEV particles bound to the basal PM of live WT or laminin γ1 KD HUVEC, monitored using TIRFM ( n = 12 cells). (E) Fluorescence images of HUVEC-expressing GFP after 12 h of treatment with PC3-derived sEVs or 10 ng/ml VEGF. Yellow arrowheads show branched protrusions of HUVEC. (F) WT (+)sEV image from E showing protrusion lengths measured along yellow lines as indicated. (G) Average protrusion lengths across all examined cells before and after treatment with sEVs or VEGF. (H) Changes in average protrusion length after treatment with sEVs or VEGF, relative to untreated conditions ( n = 16 cells). (I) Average total protrusion length per cell before and after treatment with sEVs or VEGF. (J) Variations in average total protrusion length per cell after treatment with sEVs or VEGF, relative to untreated conditions ( n = 16 cells). Data are presented as mean ± SE. n.s., nonsignificant difference; *P < 0.05; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided). Source data are available for this figure: .

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Derivative Assay, Western Blot, Fluorescence, Expressing, Confocal Microscopy, Incubation

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Schematic model showing the binding of EVs to the recipient cell PM. (A) Integrin heterodimers in EVs, α6β1/α6β4 and α2β1, can bind to laminin and collagen type I, respectively, but integrin heterodimers in EVs, such as α5β1, bind only weakly to fibronectin. CD151 enhances the binding of integrins α6β1 and α6β4 in sEVs to laminin. Cholesterol in sEVs suppresses laminin binding through a CD151-dependent mechanism. (B) Because Ser425 of talin-1 is hardly phosphorylated in EVs, talin-1 is inactive and is not involved in promoting the ECM-binding activity of integrin heterodimers. Kindlin-2 is also not involved in the activation of integrin heterodimers in EVs.

Article Snippet: The cells were then incubated with 10 μg/ml primary antibodies against fibronectin (F3648; Sigma-Aldrich), laminin (L9393; Sigma-Aldrich), collagen type I (ab34710; Abcam), laminin α1 (sc-74418; Santa-cruz biotech), or laminin α5 (ab17107; Abcam) in MEM at 37°C for 30 min. Next, the cells were washed twice with HBSS and incubated with 12 μg/ml rhodamine-conjugated anti-rabbit IgG antibody (55666; Cappel) or 10 μg/ml FITC-conjugated anti-mouse IgG antibody (55514; Cappell) in MEM at 37°C for 30 min. After washing twice with HBSS, fresh HBSS was added to the glass-based dishes.

Techniques: Binding Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Tumour-derived LAMA5 is critical for tumour initiation and controls progression and phenotype in luminal breast cancer

doi: 10.1101/2024.12.26.630367

Figure Lengend Snippet: a) Differential pathway cluster enrichment of Lama5 fl/fl and Lama5 fl/+ tumors compared to Lama5 +/+ control tumors. b) Heatmap showing differential expression of ECM signature genes in Lama5 fl/fl tumors compared to Lm5 tumors. c) Heatmap showing differential expression of FGF family genes in indicated MEC populations in Lama5 fl/+ and Lama5 fl/fl compared to Lm5 tumors.

Article Snippet: Probes for either laminin α1, α3, α4 or α5 (Advanced Cell Diagnostics) were hybridized for 2 h at 40°C.

Techniques: Control, Quantitative Proteomics