Fig. 4

Cellular morphology and motility are regulated by the subcellular localization of wildtype MLC1. a Representative image of COS-7 cells expressing MLC1-GFP either in the intracellular organelles (*) or on the plasma membrane (***). COS-7 cell showing intermediate distribution pattern of MLC1 is located on the top of the image (**). Plasma membrane-targeted MLC1 was stained using anti-Myc antibody (red, surface MLC1, before permeabilization). Total MLC1 expression was visualized using GFP (green, total MLC1, after permeabilization), while nuclear expression was examined using DAPI (blue). Scale bar: 20 μm (upper), 5 μm (bottom). b The polarized distribution of surface MLC1-GFP and filopodia. The histogram for each signal was analyzed using ImageJ. Scale bar: 20 μm. c Kymographs of COS-7 cells expressing PM-MLC1 and ER-MLC1. Scale bar: 20 μm. Motility of cells expressing PM-MLC1 (n = 6) or ER-MLC1 (n = 6), as determined via live cell imaging. Mean velocity (d) and directionality (e) were analyzed. f Time-lapse imaging of the subcellular localization of MLC1-GFP in moving COS-7 cells (yellow, merged with mCherry). mCherry (red) was used as a marker of transfection, allowing us to determine cellular morphology using a fluorescence microscope. Scale bar: 25 μm