Journal: bioRxiv
Article Title: Arp2/3 inhibition switches Eps8’s network associations to favour longer actin filament formation necessary for tunneling nanotubes
doi: 10.1101/2022.08.24.504515
Figure Lengend Snippet: ( a ) Experimental setup for pulling membrane nanotubes using a concanavalin A (Con-A)-coated bead (~3 m in diameter) trapped in an optical tweezer (OT). CAD cells stably expressing the small F-actin-binding peptide F-tractin fused to EGFP (green) were exogenously labelled with the lipophilic Cell Mask™ Deep Red plasma membrane stain (magenta). Intensity profiles of the F-actin fluorescence were measured along the nanotube axis and plotted against the nanotube length. ( b, d ) Representative time-lapse images of a nanotube pulled from a DMSO-treated (mock control) (Supplementary Video 5) and a CK-666-treated cell (Supplementary Video 6). White arrowheads annotate the progression of actin development within the nanotube in d . ( c, e ) Plot of actin profiles within pulled nanotubes for mock- and CK-666-treated cells. Insets show a magnified view at the tube extremity to better highlight the greater presence of F-actin in the CK-666 condition as compared to the mock condition. Mock condition, 11 tubes; CK-666 condition, 12 tubes. ( f ) Left: Exponential fits to the actin intensity profiles were performed to determine a characteristic decay length (2 ℓ ) at which the initial intensity at X = 0 decays to a value of 1/ e . For visualization purposes of the analysis, exponential fits are shown for the mean actin profiles computed from the individual plots presented in e and f . Upper and lower limits of the intensity range are shaded. Right: Dot plot of the characteristic decay lengths (2 ℓ ) for mock- and CK-666-treated cells. Data is represented as the mean ± SEM. Mock (11 tubes), 2.78 ± 0.50; CK-666 (12 tubes), 5.80 ± 0.73. Statistical analysis was performed using an unpaired Mann-Whitney test. ( g ) Top: Force plot of a pulled nanotube from a mock-treated cell showing no F-actin development. Solid teal line, 10-point moving average curve. Bottom: Associated images of the indicated time points (g1, g2). ( h ) Top: Force plot of a pulled nanotube from a CK-666-treated cell showing F-actin development spanning the entire nanotube length. Peaks in the force plot (black arrowheads), with magnitudes of Δ F , arise when retrograde flows outcompete actin polymerization (at the nanotube tip) causing bead displacement towards the cell body (recorded as a positive rise in the force in the lab frame). Solid teal line, 10-point moving average curve. Shaded grey region corresponds to a magnified view on the right. Bottom: Associated images of the indicated time points (h1, h2). ( i ) Histogram of the force peak magnitudes (Δ F ). Sample size, 33 peaks. The trapped bead is annotated by a dotted white circle when not clearly visible. Scale bars, 5 μm.
Article Snippet: Beads were then resuspended in PBS to a concentration of 0.05% w/v, and an appropriate amount of a 1 mg mL −1 biotin-conjugated concanavalin A (ConA) solution (C2272, Sigma-Aldrich) was added to the bead suspension assuming a binding capacity of 10 μg ConA per mg of beads.
Techniques: Stable Transfection, Expressing, Binding Assay, Staining, Fluorescence, MANN-WHITNEY
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