Journal: bioRxiv

Article Title: Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

doi: 10.1101/2022.03.04.483020

Figure Lengend Snippet: (A) Widefield fluorescence image of a representative Rat2 cell transfected with pEGFP-N1-IRSp53L (IRSp53-eGFP) and pmTagRFP-VASP (VASP-RFP). Brackets indicate some filopodia where IRSp53 is present along them. White arrows indicate the same filopodia to demonstrate that VASP is enriched in their tips. Scale bar, 5 μm. (B) Time-lapse images of a filopodium formation. Magnification of the indicated area shown in (A), cyan boxes. The white arrow indicates the appearance of a IRSp53 cluster followed by a VASP cluster indicated by a cyan arrow in the onset of filopodia formation. White boxes indicate the selected area used to generate outlines of plasma membrane positions over time shown in (C). Scale bar, 2 μm. (C) Colored outlines of membrane positions in the region indicated by the white boxes shown in (B). Total 27 frames, frame interval 2 sec. (D) Adaptive kymograph maps replot the detected membrane profiles in (C) in the y-axis and the corresponding time points in the x-axis to show the dynamics of IRSp53 ( Left ) and VASP ( Right ) on the plasma membrane over time. Y-axis shows the membrane positions of the proteins, and the x-axis shows the time (in second, total 27 frames). Color maps: low fluorescence intensity in blue, and high fluorescence intensity in red. Circled numbers correspond to the frames indicated in (B).

Article Snippet: Time-lapse image series for Rat2 cells transfected with either pEGFP-N1-IRSp53L/pmTagRFP-VASP or pEGFP-N1-IRSp53L/fascin-pmCherry, or pEGFP-N1-IRSp53L/empty-pmCherry were obtained using Widefield microscope GE DeltaVision Ultra with extra sensitive cameras and Solid State Illuminators (SSI) for fluorescence excitation, equipped with incubation system set at +37 0 C and 5% CO2 and 63x or 100x oil objectives.

Techniques: Fluorescence, Transfection

Journal: bioRxiv

Article Title: Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

doi: 10.1101/2022.03.04.483020

Figure Lengend Snippet: (A) Left: Representative confocal images of GUVs incubated with all the protein ingredients (IRSp53, VASP, actin, capping protein and profilin) and in the absence of Fascin. Right: Percentages of GUVs having actin-filled membrane tubes in the absence (“No Fascin”) and the presence (“With Fascin”) of Fascin. GUV numbers: N = 53, 32, 54 “No Fascin” and 56, 45, 39 “With Fascin”. n = 3 sample preparations. Statistical tests: chi-squared test on data pooled from the 3 sample preparations, p = 0.8652, paired t test considering the 3 sample preparations individually, p = 0.6906 (shown in the figure). (B) Widefield fluorescence image of a representative Rat2 cell transfected with pEGFP-N1-IRSp53L (IRSp53-GFP) and fascin-pmCherry (fascin-Cherry). Scale bar, 2 µm. (C) Time-lapse images of a single filopodia formation. Magnification of the indicated area shown in (B), white boxes. The white arrow indicates the appearance of IRSp53 clusters followed by the recruitment of fascin in filopodia formation. Scale bars, 2 µm. Time in sec. (D) Left panel: Representative kymograph of IRSp53 and fascin fluorescence signals on growing filopodia from Rat2 cell transfected with pEGFP-N1-IRSp53L (IRSp53-GFP) and fascin-pmCherry (fascin-Cherry). Number 1 and 2 in the kymograph indicate filopodia growth before (Number 1) and after (Number 2) the presence of fascin inside filopodia. Right panel: Quantification of filopodial growth rate before (“Before Fascin arrived”) and after (“After Fascin arrived”) the emergence of fascin inside the filopodia. N = 26 filopodia. Statistical test: Mann-Whitney nonparametric test, p = 0.00001633. (E) Frequency of filopodia retractions in Rat2 cells transfected with pEGFP-N1-IRSp53L (IRSp53-GFP) and with either empty-pmCherry (“Control”) or fascin-pmCherry (“Fascin”). The frequency of each event was calculated for the period of filopodia growth as the number of retractions per sec. The numbers of filopodia analysed were n=13 (“Control”) and n=30 (“Fascin”). Statistical test: Mann-Whitney nonparametric test, p = 0.000083802.

Article Snippet: Time-lapse image series for Rat2 cells transfected with either pEGFP-N1-IRSp53L/pmTagRFP-VASP or pEGFP-N1-IRSp53L/fascin-pmCherry, or pEGFP-N1-IRSp53L/empty-pmCherry were obtained using Widefield microscope GE DeltaVision Ultra with extra sensitive cameras and Solid State Illuminators (SSI) for fluorescence excitation, equipped with incubation system set at +37 0 C and 5% CO2 and 63x or 100x oil objectives.

Techniques: Incubation, Fluorescence, Transfection, MANN-WHITNEY