EVL tip localization is necessary and sufficient for DF motility. (A and B) Glial cells expressing iRFP670-LifeAct, EVL-iLID, and either CAAX-iLID (A) or MITO-iLID (B). Cells were photostimulated with 488 nm light for 5 min, and imaged for 5 min before, during, and after photostimulation. Row 1: Localization of LifeAct and EVL-iLID during indicated phases of photostimulation. Inset: mVenus-CAAX-iLID or mVenus-MITO-iLID localization. Scale bars = 10 µm. Rows 2–3: Localization of EVL-iLID and LifeAct in magnified region of lamellipodia indicated in Row 1. Individual channels displayed with black subtraction for ease of morphological comparison. Arrowheads indicate regions which exhibited altered dynamics following photostimulation (lamellipodia by empty arrowheads, filopodia by filled arrowheads). Row 4: Maximum intensity projection of temporally color-coded binary mask outline. Row 5: Kymograph of cell’s edge. 15 min duration, 5 s interval, vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. See also Fig. 6 and Video 6.