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: The organization, dynamics and phosphorylation of CAV-1 are regulated by contractile actin assemblies. (A) Immunofluorescence staining of endogenous F-actin and CAV-1 vesicles in wild type (WT), myosin II inhibition (NMII inh.) and Arp2/3 complex inhibition (Arp2/3 inh.) cells, respectively. The representative analysis of CAV-1 positive dots detected by Imaris of magnified yellow boxes 1, 2, and 3 on the top panels are marked as balls which are randomly colored on the lower panels. The size of the color balls indicates the calculated sizes of CAV-1 vesicles. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). (B) The length distribution of CAV-1 vesicles. The number of vesicles in each group of size is divided by the total CAV-1 number of the same cell. n = 25,602 vesicles from 32 WT cells, 13,456 vesicles from 31 myosin II inhibition cells, and 26,103 vesicles from 29 Arp2/3 complex inhibition cells. (C) Quantification of the movement rate of CAV-1 vesicles in wild-type ( n = 26), myosin II inhibition ( n = 22), and Arp2/3 complex inhibition ( n = 23) cells. (D) FRAP analysis of CAV-1-mGFP dynamics in WT, NM II inh. and Arp2/3 inh. cells. Magnified regions represent time-lapse images of the bleached regions. Bars, 10 μm (in cell images) and 5 μm (in the magnified time-lapse image). (E) Normalized average FRAP recovery curves of CAV-1-mEGFP in WT ( n = 23), NM II inh. ( n = 22), and Arp2/3 inh. ( n = 22) cells. (F) The representative 200 s duration dot tracking analysis of mEGFP tagged CAV-1, CAV-1(Y14F) and CAV-1(Y14D) vesicles in CAV-1 KO cells by Imaris. Color-coded bar from blue to red indicates the tracked mean speeds ranging from 0 to 0.4 μm/s. Bars, 10 μm. (G) Quantification of the movement rate of CAV-1 positive vesicles. n = 419/496/432 vesicles from 10 CAV-1/CAV-1(Y14F)/CAV-1(Y14D)-mEGFP expressing cells. (H,I) Western blot analysis (H) and quantifications (I) of phosphorylated CAV-1 (Tyr14) (compared to total CAV-1) in WT, NM II inh. and Arp2/3 inh. cell lysates. The obtained intensity value from wild-type cells was set to 1. n = 3. Data in panel (C,G,I) are presented as mean ± SD. *** P ≤ 0.001; N.A., not significant (one-way ANOVA). 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: Phospho-proteomics, Immunofluorescence, Staining, Inhibition, Expressing, Western Blot

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

Journal: Science advances

Article Title: Chaperone-mediated autophagy regulates adipocyte differentiation.

doi: 10.1126/sciadv.abq2733

Figure Lengend Snippet: Fig. 2. Blocking CMA impairs adipocyte differentiation. (A) Immunoblot of 3T3L1 cells control (CTR) or stably knocked down for L2A [L2A(−)] and densitometric quantifi- cation. n = 5 i.e. (B) Phase contrast images at the indicated differentiation days. Green fluorescent protein (GFP) shows transduction efficiency. (C) Percentage of differentiated cells at day 8 from (B). n = 10 i.e. (D) Immunoblot of the indicated proteins and densitometric quantification during adipocyte differentiation. n = 3 − 6 i.e. (E) DPH, PLIN1, and PLIN2 staining at day 9 of adipocyte differentiation. (F and G) Quantification of area occupied by LD (F) and PLIN1 intensity (G) from (E). n = 4 (F) and 3 (G) i.e. (H) % eWAT/body weight for WT and L2AKO mice. n = 10 mice. (I and J) Hematoxylin and eosin (H&E) staining (I) and size distribution of cells (J) of eWAT. Zoom: Higher magnification. n = 3 mice. (K) eWAT immunostaining for Mac2 and caveolin1 and quantification of number of macrophages. Arrowheads: Macrophages (Mac2+ cells). Nuclei are highlighted with Hoechst. n = 4 mice. (L and M) mRNA expression of indicated genes (L) and H&E staining (left) and quantification of crown-like structures (CLS; arrowheads) (right) (M) in eWAT from WT and L2AKO mice on HFD. n = 4 mice. (N) Staining for BODIPY 493/503 (to stain LD) and quantification of LD area in in vitro differentiated primary preadipocytes from WT and L2AKO mice eWAT at day 9. Nuclei are highlighted with DAPI. Full-field images are in fig. S1K. n = 4 mice with 15 fields each. All insets are boxed areas at higher mag- nification. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using two-way ANOVA (D and J) or unpaired t test (the rest of the panels).

Article Snippet: Antibodies used for tissue imaging were caveolin1 (1:1000; Cell Signaling Tech nology, 3267), CD31 (1:200; Cell Signaling Technology, 77699), Ki67 (1:500; Abcam, ab15580), Mac2 (1:400; Thermo Fisher Scientific, 13530182), and PreF1 (1:500; SigmaAldrich, MABN671).

Techniques: Blocking Assay, Western Blot, Control, Stable Transfection, Transduction, Staining, Immunostaining, Expressing, In Vitro

Journal: Science advances

Article Title: Chaperone-mediated autophagy regulates adipocyte differentiation.

doi: 10.1126/sciadv.abq2733

Figure Lengend Snippet: Fig. 3. CMA is required for preadipocyte commitment to differentiation. (A) mRNA expression of indicated genes of 3T3L1 cells control (CTR) or stably knocked down for lamp2a [L2A(−)] at indicated times of adipocyte differentiation. n = 3 to 10 i.e. (B and C) mRNA expression of indicated preadipocyte (B) and adipocyte (C) genes of eWAT from WT and L2AKO mice. n = 5 mice. (D) Flow cytometry for WT and L2AKO eWAT adipose progenitor cells showing percentage of Lin− PDGFR+ adipose progenitors. n = 3 mice. (E) Left: Immunostaining for PreF1 and caveolin1 of eWAT from WT and L2AKO mice. Arrowheads: PreF1+ cells. Right: Quantification of percentage of PreF1+ cells. Nuclei are highlighted with Hoechst. n = 4 mice. Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ns P > 0.05 using two-way ANOVA (A) and unpaired t test (B to E).

Article Snippet: Antibodies used for tissue imaging were caveolin1 (1:1000; Cell Signaling Tech nology, 3267), CD31 (1:200; Cell Signaling Technology, 77699), Ki67 (1:500; Abcam, ab15580), Mac2 (1:400; Thermo Fisher Scientific, 13530182), and PreF1 (1:500; SigmaAldrich, MABN671).

Techniques: Expressing, Control, Stable Transfection, Flow Cytometry, Immunostaining