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: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: SPIN90 selectively activates ArpC5L containing Arp2/3 complexes (A) SPIN90 efficiently activates ArpC5L but not ArpC5 containing Arp2/3 complexes. Representative plots of pyrene-actin polymerization assays with 10 nM Arp2/3 complex, 100 nM SPIN90 and 2.5 µM actin (5 % pyrene labelled). It demonstrates that ArpC5L- but not ArpC5-containing Arp2/3 complexes activated by SPIN90 induce rapid actin polymerization, regardless of the ArpC1 isoform. The experiments were repeated independently three times and yielded similar results. (B) Representative TIRF images demonstrate that ArpC5L- but not ArpC5-containing complex activated by SPIN90 efficiently generate actin filaments. Scale bar = 20 µm A mixture of 20 nM Arp2/3 complex, 200 nM SPIN90 and 0.5 µM actin (15 % Alexa 488 labelled) was observed overtime. The graph shows quantitative analysis of filament density over time from three independent technical replicates. The points indicate the mean density, and the error bars represent the standard deviation.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: SPIN90 has a higher affinity for Arp2/3 complexes with ArpC5L than ArpC5 (A) Surface representation of the Arp2/3 complex from the SPIN90-Arp2/3 complex structure (PDB: 6DEC). Residues in Arp2/3 complex subunits contacting the two SPIN90 molecules are shown in darker colours. (B) Immunoblots using an ArpC2 antibody show the amount of Arp2/3 complex containing ArpC1B pulled down with GST–SPIN90-Cter. Both blots were run on the same gel and developed on the same membrane to allow a direct comparison of signal intensity. (C) The graph shows the quantification of average normalized ArpC2 intensity from three independent replicates, with error bars resenting standard deviation. The data was fit with Hill equation.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Western Blot, Membrane, Comparison, Standard Deviation

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: SPIN90 localizes at the leading edge and integrates into lamellipodial actin networks. (A) Quantitative immunoblot analysis of endogenous levels of ArpC5 isoforms in B16 cells using recombinant His-tagged ArpC5 and His-tagged ArpC5L as standards. The average concentrations and standard deviations from three independent replicates are shown in the table on the right. (B) Schematic shows the two Ruby-tagged SPIN90 constructs expressed in cells. Representative live images show B16 cells overexpressing GFP (volume marker) together with Ruby–SPIN90 (upper) or with Ruby-SPIN90 Nter (lower). Scale bars = 5 µm. Signal intensity (right) is quantified by normalizing the intensity at the leading edge (solid line) compared to that 2□µm from the leading edge (dashed line). Each pair of points represents measurements from the same cell with the different colours representing data from three independent replicates. A two-tailed paired t-test is used to estimate the p-value.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Western Blot, Recombinant, Construct, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: SPIN90 enhances the recruitment of ArpC5L but not ArpC5 to the leading edge Representative images of live cells expressing endogenous NeonGreen–ArpC5L (A) or ArpC5-NeonGreen (B) after treatment with the indicated siRNAs. Allstar represents siRNA control. The graphs show quantification of the ArpC5L or ArpC5 signal intensity at the leading edge in the different conditions. Each point represents an individual cell, with different colours indicating 3 independent experimental replicates. For each repeat, 8-15 cells were imaged and quantified. The mean of each experiment is shown as a circle, and error bars are standard deviation. A two-tailed paired t-test was used to calculate the p-value. Scale bars = 5 µm

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Expressing, Control, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: The organisation of actin filaments at the leading edge of B16 cells. (A) Schematic representation of ρ (rho) and ψ (psi) in the organization of the actin network. (B) Representative images of an Alexa-488 phalloidin stained B16 cell for each cell line as indicated. The coloured region in the lamellipodium is the result of polarimetry analysis within a 0.65 µm (10-pixel) region from the leading edge to determine the mean actin filament orientation in each pixel. The mean filament orientation per pixel is represented with a stick whose orientation corresponds to filament orientation (angle rho, ρ) and whose color depicts filament orientation with respect to the leading edge (angle rho c , ρ c ) within the 0–180° range according to the colour bar. The stick maps of a representative ROI (labelled with white rectangles) are zoomed in and shown. (C) The polar histogram shows the statistical distribution of actin filament angles (ρ c ) per pixel within the 0–90° range relative to the leading edge of all the cells, with 38 to 55 cells analysed per condition across three independent repeats. The average < ρ c > and standard deviation for each cell line are indicated. The colours correspond to the colour bar shown in panel A. (D) Violin plot showing the statistical distribution of actin filament angles (ρ c ) per pixel within the 0–90° range, as shown in panel B. A two-tailed Mann–Whitney test was used to calculate the p-value between individual cells under two conditions. The medians and interquartile ranges are shown as black lines, while the means and standard deviations are shown as blue dots with error bars. (E) The statistical results of the average lamellipodial actin filament angle < ρ c > relative to the leading edge within the 0–90° range in individual wildtype, ArpC5, ArpC5Lor SPIN90 knockout cells. Each point represents the mean angle of actin filaments relative to the leading edge in a single cell from three independent experiments, the latter shown in different colours. Mean ± SD for each experimental condition is indicated. A two-tailed Welch’s t-test is used to estimate the p-value between the means under two conditions. (F) The statistical results of the standard deviation (SD) of actin filament angles relative to the leading edge within the 0–90° range in individual wildtype, ArpC5, ArpC5L, or SPIN90 knockout cells. Each point represents the SD of actin filaments relative to the leading edge in a single cell from three independent experiments, the latter shown in different colours. Mean ± SD for each experimental condition is indicated. A two-tailed Mann Whitney test is used to estimate the p-value between individual cells under two conditions. (G) Representative images of Alexa-488 phalloidin stained B16 cell (right) overexpressing Ruby-SPIN90 (left). The zoomed-in image shows the mean actin filament orientation per pixel within the ROI (white rectangle) as in panel A. (H) The polar histogram shows the statistical distribution of actin filament angles (ρ c ) per pixel within the 0–90° range relative to the leading edge in all cells overexpressing either Ruby-SPIN90 (61 cells) or Ruby-SPIN90 Nter (59 cells), across three independent experimental repeats. The average < ρ c > and standard deviation for each experimental condition are indicated. The colours correspond to the colour bar shown in panel A.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Staining, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Knock-Out

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: SPIN90 impacts the lamellipodia protrusion and cell motility. (A) Representative image stills of WT, ArpC5, ArpC5Land SPIN90 knockout B16 cells expressing endogenously tagged GFP β-actin. (B) The quantification of lamellipodia width, lamellipodial actin density. Each point represents an individual cell, with different colours indicating independent experimental replicates. The mean of each experiment is shown as a circle, and error bars are standard deviation. A two-tailed paired t-test was used to estimate the p-value between the means under two conditions. (C) Representative image still (upper) from a FRAP assay on a B16 cell with endogenously GFP tagged β–actin. The kymograph (lower) shows the cell protrusion (indicated by straight line) and rearward flow (indicated by dashed line), made visible by the FRAP region. (D) The quantification of lamellipodial protrusion rate, rearward flow and protrusion efficiency. Each point represents an individual cell, with different colours indicating independent experimental replicates. The mean of each experiment is shown as a circle, and error bars are standard deviation. A two-tailed paired t-test was used to estimate the p-value between the means under two conditions. (E) Quantification of B16 cell random migration speed. Each point represents an individual cell, with different colours indicating 3 independent replicates. The mean of each experiment is shown as a circle, and error bars represent standard deviation. A two-tailed paired t-test was used to estimate the p-value between the means under two conditions. (F) Representative images (left panels) of the leading edge of B16 cells overexpressing either Ruby, or Ruby-SPIN90. The right panel shows the protrusion rate of these cells. Each point represents an individual cell, with different colours indicating independent experimental replicates. The mean of each experiment is shown as a circle, and error bars are standard deviation. A two-tailed paired t-test was used to estimate the p-value between the means under two conditions.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Knock-Out, Expressing, Standard Deviation, Two Tailed Test, FRAP Assay, Migration

Journal: bioRxiv

Article Title: SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion

doi: 10.64898/2025.12.01.691495

Figure Lengend Snippet: Schematic summary (A) In migrating cells, SPIN90 recruits the ArpC5L-, but not the ArpC5-containing complex, to the lamellipodial leading edge . Meanwhile, both ArpC5L- and ArpC5-containing complexes generate branches at the lamellipodia . Thus, SPIN90 and Arp2/3 iso-complexes integrate into the lamellipodial actin network, contributing to the formation of a complex actin architecture that ensures efficient protrusion . (B) ArpC5 KO cells (left panel), the ArpC5L-containing complex participates in the formation of both linear and branched actin filaments. Consequently, the distribution of filament orientations in their lamellipodia is much fig than that of the wild type. In contrast, in ArpC5L KO cells (right panel), the ArpC5-containing complex generates only branched actin filaments, resulting in a narrower distribution of filament orientations.

Article Snippet: Other antibodies are including: ARPC2/p34-Arc polyclonal (Millipore, 07-227), ARPC5 monoclonal (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β-actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: