Figure 1 | Scientific Reports

Figure 1

From: ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain

Figure 1

Glucose-starvation induction of filopodia formation is mediated by ROS. (a) Left panel: Filopodia induced by glucose-starvation are inhibited by supplementation of antioxidants to the starvation media. emLifeact (fluorescently tagged actin binding protein) was transiently expressed in U2OS cells to visualize actin and filopodia. The cells were then starved in starvation medium without supplements or supplemented with either catalase (60 µg/ml), Propyl Gallate (PG, 20 µM) or a combination of both (EtOH was used as vehicle). Right panel: ROS induces filopodia formation. emLifeact expressing U2OS cells were exposed to either PG or 0.2 mM H2O2 for two hrs, inducing filopodia formation in complete growth media. Note the lack of effect of PG in complete medium compared to starvation medium. Bar is 20 µm. (b) Quantification of the number and length of the filopodia in (a). Over 300 filopodia were manually counted for each condition from three independent experiments. The significance of the change in both number and length of filopodia was assessed via two tailed Student’s T-test. pVal for filopodia number per cell = 1.4 × 10−6, 0.083, 0.0001, 3.91 × 10−11, 3.57 × 10−11, 0.4.pVal for filopodia length = 1.27 × 10−14, 0.01, 0.093, 0.0027, 0.0035, 0.161 (same order as the chart). (c) Immunofluorescence for endogenous Myo19 in H2O2 stimulated U2OS cells reveals that it localizes with mitochondria at filopodia tips. Blue – nuclei, green – αMyo19, red – phalloidin stained actin, Magenta – The mitochondrial marker ATP5A (ATP-synthase subunit alpha). Bar is 10 µm. Zoom – magnification of the area indicated by the yellow rectangle. We note that the staining of ATP5A is also nuclear, however this is due to the secondary antibody used for detection of ATP5A stains which stains nuclei even in the absence of the ATP5A antibody.