Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Schematic diagram of podosome structure. b, Dyn2 forms belt-like structures around the actin cores of synaptic podosomes. Myoblasts were seeded on laminin-coated coverslips and subjected to differentiation. Differentiated myotubes were fixed and stained to visualize endogenous Factin, Dyn2, and Tks5. Images were acquired from z-stack confocal microscopy. Boxed areas were magnified and shown in lower panels to display the Z-projection and orthogonal view of a single podosome. Arrowhead, podosome with a Dyn2 belt. Arrow, podosome without a Dyn2 belt. Scale bar, 2 μm. c, Dyn2 localizes differently from other podosome components. Differentiated myotubes were fixed and stained to visualize endogenous F-actin, Dyn2, Arp2, cortactin, vinculin, and myosin IIA. Scale bar, 2 μm. d-f, Level of Dyn2-belt formation is correlated with podosome height and width. Podosomes were grouped into three categories according to amounts of Dyn2 surrounding the actin core. Data are presented as mean ± s.d. of podosome height and width. Each dot represents one podosome. At least 40 podosomes in 10 different cells were analyzed. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test. ** P < 0.01; *** P < 0.001. The representative images are shown in the lower panels, with Dyn2 in green and F-actin in red. g-h, Snapshots of time-lapse images of a single podosome showing the temporal distribution of Cortactin and Dyn2 in myotubes. Myoblasts were transfected with Lifeact-GFP and Cortactin-mCherry (g) or Dyn2 WT -mCherry (h), respectively, and were seeded on laminin-coated glassbottom dishes, subjected to differentiation for 5-7 days, and imaged by inverted fluorescence microscopy at 37 °C with 1 min frame intervals. Scale bar, 2 μm. i, Frequency distribution of podosome lifespan in myotubes (n = 37 podosomes). j, Percentage of Dyn2 appearance during podosome lifespans in myotubes. Each dot represents one podosome.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Staining, Confocal Microscopy, Transfection, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Dyn2 bundles actin filaments. Reconstituted F-actin (5 μM) was incubated with Dyn2 at indicated concentrations for 30 min and subjected to 20 min centrifugation at 14,000 x g . S, supernatant; P, pellet. b, Dyn2 (1 μM) shows significant actin bundling activity. Mean ± s.d. of percentage of sedimented actin was quantified in ImageJ as the ratio of actin in the pellet versus total actin. c-d, F-actin sedimentation assay and quantification result of Dyn2 bundling activity. Reconstituted F-actin (5 μM) was incubated with 1 μM Dyn2 for 30 min followed by 15 min incubation with 1 mM GTP or GMPPCP. e, Real-time visualization of the actin bundling activity of Dyn2 under confocal microscopy. Scale bar, 10 μm. f, TEM images of negative-stained actin-Dyn2 bundles. Scale bar, 100 nm. g, TEM images of negative-stained branched actin-Dyn2 bundles. Scale bar, 100 nm. h-k, Branched F-actin sedimentation assay and quantification result of Dyn2 bundling activity. Concentrations of individual components were 5 μM actin, 1 μM Dyn2, 30 nM Arp2/3 complex, 80 nM VCA and 1 mM nucleotides. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test. ns, not significant; * P < 0.05; *** P < 0.001. l, Morphology of Dyn2-bundled linear and branched actin under confocal microscopy. Scale bar, 1 μm.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Incubation, Centrifugation, Activity Assay, Sedimentation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Distribution of phospho-deficient Dyn2 Y597F -mCherry in myotubes. b, Percentage of synaptic podosomes with Dyn2-mCherry enrichment in myotubes (n = 19 podosomes per condition). c, Actin bundling ability of Dyn2 mutants with or without GTP. The percentage of sedimented actin was quantified and is shown in d. e, TEM images of negative-stained actin-Dyn2 bundles with or without 15 min GTP incubation. Scale bar, 100 nm. f, Dyn2 functions as a molecular girdle and checkpoint for podosome maturation and turnover, respectively. (i) Interactions between ECM and integrins initiate podosome formation. Actin polymerization proteins such as Arp2/3 complex and cortactin drive podosome initiation. (ii) Tks5 is recruited to the nascent podosome and promotes podosome maturation. (iii) Phosphorylated Dyn2 is recruited to the podosome to bundle the actin core and facilitate its growth. Dyn2 forms a belt-like structure around the actin core to enhance the function of podosome. (iv, v) After dephosphorylation, GTP hydrolysis triggers Dyn2 dissociation from the podosome and induces its turnover. g, Functional role of Dyn2 in postsynaptic NMJ morphogenesis. (i) During NMJ prepatterning, AChR clusters are induced by extracellular signals, such as laminin or Wnt. (ii) During NMJ development, perforation and remodeling of AchR clusters is facilitated by synaptic podosomes whose maturation and turnover is governed by Dyn2.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Staining, Incubation, De-Phosphorylation Assay, Functional Assay

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) ECAD internalization was monitored by IF in MCF10A-EPN3 cells treated with AP2µ KD or mock. Top, representative images; internalized ECAD (green), DAPI (blue). Bar, 20 µm. Bottom, quantification of relative internalized ECAD fluorescence intensity/cell in individual field of views, normalized to mock control. N (fields of view): Mock=4, AP2µ=35; n=3. B) ECAD internalization in MCF10A-EPN3 cells subjected to single or double Eps15/Eps15L1 KDs. Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): Mock=42, Eps15 KD=39, Eps15L1 KD=40, Double KD=35, n=3. C) Left panels: PM, representative immuno-EM images showing PM-ECAD-positive (gold-labeled) tubular invaginations (indicated by arrows) in MCF10A-EV and MCF10A-EPN3 cells; scale bar, 200 nm; Int, representative immuno-EM images showing internalized ECAD-positive (gold-labeled) structures (indicated by arrows and enlarged in the insets) in MCF10A-EV and MCF10A-EPN3 cells; scale bar, 250 nm. Right upper panel: quantification of internalized ECAD expressed as a percentage of PM-ECAD. N (cells): EV=24, EPN3=27. Right-lower panel: gold-labelled ECAD-positive clathrin-coated pits (CCPs) and tubular invaginations (TIs) expressed as percentage of total number of structures in 100 µm PM length/cell. N (cells): EV=27, EPN3=25. D) Effects of inhibitors on ECAD internalization in MCF10A-EPN3 cells. Cells were pre-treated with the indicated compounds or vehicle (DMSO) before measuring ECAD internalization as in (A): CK666 (50 µM, 1 h), Genz-123346 (4 µM, 6 days), Lactose (100 mM, 1h), I3 (20 µM, 10 min). Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): CK666=42 (DMSO control=44) (n=5); Genz=20 (DMSO control=20) (n=3); Lactose=33 (mock=40) (n=3); I3=40 (DMSO control=53) (n=6). E) ECAD internalization in MCF10A-EPN3 cells subjected to I3 treatment as in (D), Gal3 KD or Gal3 KD/I3 treatment. Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): Mock=34, I3=23, Gal3 KD=26, Gal3 KD/I3=28, n=2. F) Co-internalization of Gal3–ECAD was monitored for 10 min in MCF10A-EV and -EPN3 cells. Left: representative confocal images, Gal3-Alexa488 (green), anti-ECAD (red), DAPI (blue). Bar: 20 µm. Right: Manders overlap coefficient of internalized Gal3-ECAD. N (cells): EV/EPN3=69; n=2. In all panels, results are shown as mean±SD, except panel for E in which median ± max/min values are shown. p-values (unpaired Student’s t-test, two-tailed): **** <0.0001; *** <0.001; ** <0.01; * <0.05, ns, not significant.

Article Snippet: The following inhibitors of endocytic players were used in this study: CK666 (Arp2/3 complex inhibitor, 50 μM, SML0006 Merck), EIPA (NHE inhibitor, 50 μM; used as macropinocytosis inhibitor, A3085 Merck), I3 (non-permeable Galectin-3 inhibitor, 20 μM, GalectoBiotech), Genz-123346 (ceramide synthase inhibitor, 4 μM, 5.38285 Merck), and (+)-Lactose (pan-galectin inhibitor, 100 mM, L3750 Merck).

Techniques: Fluorescence, Control, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Internalization of Tf-488 was monitored in MCF10A-EV and -EPN3 cells with or without AP2µ KD. Results are shown normalized to mock control. N (fields of view): EV, Mock=56, AP2µ KD=28 (n=3); EPN3, Mock=44, AP2µ KD=28 (n=3). B) Quantification of Tf-488 internalization in MCF10A-EV and - EPN3 cells subjected to single or double Eps15/Eps15L1 KDs. N (fields of view): EV, Mock=35, Eps15 KD=35, Eps15L1 KD=35, Double KD=35 (n=3); EPN3, Mock=36, Eps15 KD=35, Eps15L1 KD=35, Double KD=35 (n=3). C) Negative controls for inhibitor screening (supporting ). Quantification of Tf-488 internalization in MCF10A-EV and -EPN3 cells pre-treated with the following compounds or vehicle control: CK666 (50 µM, 1 h), Genz (4 µM, 6 days), I3 (20 µM, 10 min). N (fields of view): EV, DMSO=15, CK666=15, Genz=15, I3=15 (n=3); EPN3, DMSO=15, CK666=15, Genz=15, I3=14 (n=3). D-E) Positive controls for inhibitor screening (supporting ). (D) CD44 internalization was monitored in vivo by IF using an anti-CD44 antibody in MCF10A-EV and -EPN3 cells pre-treated with I3 (20 µM, 10 min) or vehicle control. Quantification of relative CD44 fluorescence intensity is shown normalized to control. N (fields of view): EV, DMSO=18, I3=16; EPN3, DMSO=18, I3=16 (n=3). (E) Shiga-toxin (STXB) endocytosis and binding was monitored by continuous incubation of STXB-488 conjugated ligand in MCF10A-EV and- EPN3 cells pre-treated with Genz (4 µM, 6 days). Quantification of relative STXB fluorescence intensity is shown normalized to control. N (fields of view): EV, DMSO=12, Genz=12; EPN3, DMSO=12, Genz=12 (n=3). F) Quantification of Transferrin internalization in MCF10A-EV and -EPN3 cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=20, I3=20, Gal3 KD=20, Gal3 KD/I3=20 (n=2); EPN3, mock=21, I3=20, Gal3 KD=21, Gal3 KD/I3=21 (n=2). G) Quantification of CD44 internalization in MCF10A-EV and -EPN3 cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=20, I3=20, Gal3 KD=21, Gal3 KD/I3=20 (n=2); EPN3, mock=20, I3=19, Gal3 KD=20, Gal3 KD/I3=20 (n=2). H) ECAD internalization was monitored in MCF10A-EV cells subjected to Eps15/Eps15L1 single or double KD. Quantification of relative ECAD fluorescence intensity/cell normalized to mock control. N (fields of view): Mock=42, Eps15 KD=39, Eps15L1 KD=37 and Double KD=40 (n=3). I-M) ECAD internalization was monitored in MCF10A-EV cells pre-treated as in panel C. Top, representative images showing internalized ECAD (green) and DAPI staining (blue). Bar, 20 µm. Bottom, quantification of relative ECAD fluorescence intensity is shown normalized to control. N (fields of view): (I) DMSO=18, CK666=18; (L) DMSO=15, Genz=15; (M) DMSO=19, I3=17 (n=3). N) ECAD internalization was monitored in MCF10A-EV cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=26, I3=27, Gal3 KD=26, Gal3 KD/I3=28 (n=2). p-values in the relevant panels (Unpaired Student’s t-test, two-tailed): ****, <0.0001; ** <0.001, * <0.05

Article Snippet: The following inhibitors of endocytic players were used in this study: CK666 (Arp2/3 complex inhibitor, 50 μM, SML0006 Merck), EIPA (NHE inhibitor, 50 μM; used as macropinocytosis inhibitor, A3085 Merck), I3 (non-permeable Galectin-3 inhibitor, 20 μM, GalectoBiotech), Genz-123346 (ceramide synthase inhibitor, 4 μM, 5.38285 Merck), and (+)-Lactose (pan-galectin inhibitor, 100 mM, L3750 Merck).

Techniques: Control, In Vivo, Fluorescence, Binding Assay, Incubation, Staining, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration

doi: 10.3389/fcell.2021.665919

Figure Lengend Snippet: Actin protrusive network is upregulated by AMPK-Rac1-PAK1-Cofilin signaling cascade in cells lacking CAV-1. (A) Immunofluorescence microscopy analysis demonstrating that more pronounced endogenous ARPC2 colocalizes with F-actin (visualized by Alexa 568 phalloidin) on the lamellipodia protrusions in CAV-1 deficient cells. Magnified regions of cell edges on the right show the distribution of ARPC2 in WT and CAV-1 KO cells. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). Quantification of width of lamellipodial protrusions are shown on the right. n = 16 regions from 16 cells for each group. (B) Representative images of membrane ruffling by time-lapse microscope. 1-pixel-wide areas were cut out to generate a 300 frame 2 s interval kymograph. Yellow dashed lines indicate the track of cell movement. An enlarged region is displayed on the right, vertical dashed lines show the membrane protrusion distance, while horizontal dashed lines mark the duration of protrusion. Bars, 10 μm (in cell images) and 2 μm (in the magnified box). Quantification of protrusion rate are shown on the right. n = 16 regions from 16 cells for each group. (C) P-AMPK (Thr172) and total AMPK were detected from the lysates of each group by western blotting. Please note that CAV-1(Y14F)-mEGFP can’t be detected by using phospho-CAV-1(Tyr14) antibody. Asterisk denotes the non-specific band. Quantification of P-AMPK (Thr172) levels (compared to total AMPK) from each group was shown on the right panel. n = 3. (D) Pull-down assays were performed for WT, CAV-1 KO, and CAV-1 KO; CAV-1-mEGFP re-expressed cells. Proteins bound to GST-PAK binding domain were analyzed by western blots and further quantified (compared to total Rac1) based on the band’s intensity. n = 3. (E) Western blot analysis and quantification (compared to total PAK1 and Cofilin) of the levels of phosphorylated PAK1 (Thr423) and Cofilin (Ser3) in WT and CAV-1 KO cell lysates. n = 3. (F) Immunostaining and quantification of endogenous P-Cofilin (Ser3) and F-actin distribution in WT and CAV-1 KO cells. A 16 μm length line was used to generate a line profile to illustrate the co-localization of P-Cofilin (Ser3) and F-actin. The lamellipodia region was enlarged on the right, and 2 μm width region was chosen to analyze the mean intensity of P-Cofilin (Ser3) on the leading edge. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (G) Immunostaining and quantification of endogenous P-cofilin (Ser3) distribution upon compound C treatment in WT and CAV-1 KO cells. Magnified regions represent the lamellipodia region. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (H) Western blot analysis and quantification of the phosphorylated levels of AMPK (Thr172), PAK1 (Thr423), Cofilin (Ser3) and activity of Rac1 and RhoA upon compound C treatment. n = 3. In panel (C–E,H) , the obtained intensity value from wild-type cells was set to 1. All the data are presented as mean ± SD. In (C,G,H) , *** P < 0.001; * P < 0.05; N.A., not significant (one-way ANOVA). In (D–F) , *** P < 0.001; ** P < 0.01 (unpaired t -test). All the data are from three independent experiments.

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit antibody (1:1,000 dilution for WB, 1:200 for IF; #3267, Cell Signaling, Beverly, MO, United States); Phospho-CAV-1 (Tyr14) rabbit antibody (dilution 1:1,000 for WB; #3251, Cell signaling); AMPK rabbit antibody (dilution 1:500 for WB; #SAB4502329, Sigma, St. Louis, MO, United States); P-AMPK (Thr172) rabbit antibody (dilution 1:500 for WB, 1:100 for IF; #2531S, Cell Signaling); Cofilin (E-8) mouse antibody (dilution 1:1,000 for WB; #sc-376476, Santa Cruz, Dallas, TX, United States); Phospho-Cofilin (Ser3) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #3313, Cell signaling); p190RhoGAP rabbit antibody (dilution 1:2,000 for WB; #26789, Proteintech, Rosemont, IL, United States); FAK rabbit antibody (dilution 1:1,000 for WB; #3285, Cell Signaling); Phospho-FAK (Tyr397) rabbit antibody (dilution 1:1,000 for WB; #3283, Cell Signaling); PAK1 rabbit antibody (dilution 1:1,000; #2602, Cell Signaling); Phospho-PAK1 (Thr423)/PAK2 (Thr402) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #2601, Cell Signaling); Tpm4.2 (LC24) mouse antibody (dilution 1:500 for WB and IF; a kind gift from Peter W. Gunning, UNSW Australia); Phospho-myosin light chain 2 (Thr18/Ser19) rabbit antibody (dilution 1:500 for WB, 1:200 for IF; #3674, Cell Signaling); Myosin light chain mouse antibody (dilution 1:1,000 for WB; #M4401, Sigma); Vinculin mouse antibody (dilution 1:100 for IF; #V9131, Sigma); ARPC2 rabbit antibody (dilution 1:1,000 for WB and IF; #15058, Proteintech, Rosemont, IL, United States); Myosin-18B rabbit antibody (dilution 1:500 for WB; #HPA000953, Sigma); Rab8 rabbit antibody (dilution 1:100 for IF; #R5530, Sigma); and GAPDH mouse polyclonal antibody (dilution 1:1,000 for WB; #G8795, Sigma).

Techniques: Immunofluorescence, Microscopy, Membrane, Western Blot, Binding Assay, Immunostaining, Activity Assay