ITSN1 recruitment stimulates cellular FN deposition to stabilize lamellipodia. ITSN1 recruitment stimulates cellular FN deposition to stabilize lamellipodia.

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ITSN1 recruitment stimulates cellular FN deposition to stabilize lamellipodia. ITSN1 recruitment stimulates cellular FN deposition to stabilize lamellipodia. (A) Representative fluorescence micrographs depicting optogenetic recruitment of ITSN1 DH/PH in a fibroblast co-expressing Venus–paxillin and Halo–iLID-CAAX. The dotted white box in top panel represents the enlarged area displayed in bottom panels. Blue arrows denote the area of activation. (B) Representative western blot analysis of cells treated with control siRNA (siCTRL) or FN siRNA (siFN) #1 or #2. (C) Mean distance for the ITSN1 DH/PH-induced protrusions on FN or poly-L-lysine (PolyLL) in fibroblasts treated with the denoted siRNA as determined from kymographs. (D) Representative fluorescence micrographs depicting optogenetic recruitment of ITSN1 DH/PH in a fibroblast co-expressing YPET–FN and Halo–iLID-CAAX. The dotted white box in top panel represents the enlarged area displayed in the bottom panels. Blue arrows denote the area of activation. The purple line represents the cell edge. Purple arrows demark deposited YPET–FN. (E) Plot of the number of YPet–FN puncta counted under each induced protrusion. (F) Rose plots, FMI graphs and velocity plots representing migration vectors for ITSN1-Micro- and iLID-CAAX-expressing fibroblasts plated on poly-L-lysine and treated with the denoted siRNA. (G) Representative western blot of cells treated with the denoted shRNA (shNS is a non-specific shRNA). (H) Mean distance for the ITSN1 DH/PH-induced protrusions on FN or poly-L-lysine in fibroblasts treated with the denoted shRNA. Error bars are the 95% c.i. *P<0.05. Scale bars: 50 µm. Refer to Tables S1 and S2 for experimental details. Seth P. Zimmerman et al. J Cell Sci 2017;130:2971-2983 © 2017. Published by The Company of Biologists Ltd