(a–b) Expressing GFP-JMY in U2OS cells significantly increases their motility in wound healing assays. Stable lines of GFP-JMY and GFP-JMYΔCA were wounded by scraping with micropipette tips. Images were acquired at 0, 2, 4, 6, and 12 hours after wounding. (b) Migration rate from 0–6 hours was averaged from a minimum of 4 replicates on each of 3 days. GFP-JMY expression induces cells to migrate 16.6% faster than wild-type cells (n=3, p<0.003). Cells expressing a truncation of JMY lacking the Arp2/3-interacting CA domain migrate at the same rate as wild-type cells. Scale bars, 100 µm.
(c–e) Wound healing assays in U2OS cells indicate that knocking-down JMY by RNAi impairs cell migration. Cells were transfected with JMY or control non-targeting 2 (Dharmacon) siRNA (C) and wounded (red dashed line) as in a. Images were taken at the same position 0, 3, 6, and 24 h after wounding. (d) Wound size at each time point for all conditions. (n=4). (e) Western blots show RNAi efficiency in U2OS cells.
(f) Knocking down JMY expression decreases cellular levels of F-actin. HEK 293 cells were transfected with a vector encoding both GFP and a JMY-specific shRNA and fixed and stained with Alexa Fluor 568-phalloidin. Average phalloidin intensity in GFP-negative (non-RNAied) and GFP-positive (RNAied) cells is plotted (n=396, p<0.03, see Methods). Error bars, s.e.m.
(g) JMY localizes to the leading edge of U2OS cells. Cells were grown as above, and fixed and stained for JMY 15 minutes after wounding. JMY is primarily nuclear, but it is also enriched at the leading edge (indirect immunofluorescence, green) where it colocalizes with a subset of actin filaments (Alexa Fluor 568-phalloidin, red). Inset: leading edge, contrast enhanced to show actin filaments and JMY. Scale bar, 10 µm.
(h) Models of in vivo role of JMY. Top, Model 1: JMY nucleates filaments that then serve as substrates for dendritic nucleation by Arp2/3. Bottom, Model 2: JMY evolved to nucleate actin in different cellular contexts. In the nucleus it nucleates unbranched filaments, and in the cytoplasm it both nucleates filaments and activates Arp2/3.