Figure 2: NM1 and Myo1c are enriched at the plasma membrane.

(A) Wild-type NM1-EYFP or Myo1c-EYFP and their respective mutants (actin-binding or PI-binding) were stably expressed in U2OS cells, and their localization was observed by wide-field fluorescence microscopy. Plasma membrane association is disrupted upon mutation of the PI-binding site of myosins. (B) NM1 wild type and KO skin fibroblasts were fixed and immunostained with NM1 and actin antibodies. NM1 is co-localizing with actin in focal adhesions and membrane ruffles. Deletion of NM1 does not cause any alteration in actin cytoskeleton structure. (C) NM1-Flag and Myo1c-V5 were co-expressed in the U2OS cells and detected by confocal fluorescence microscopy. Both proteins co-localize at the plasma membrane and in membrane ruffles (arrows). (D) Localization of NM1-Flag and Myo1c-V5 was inspected by SIM. Both proteins are localized at the plasma membrane in a mosaic pattern and do not overlap entirely. (E) Visualisation of co-localization measurement of Myo1C and NM1 from inset pictures in 3C and 3D. PM – plasma membrane, MR – membrane ruffles. Scale bars, 5 μm (C) and 2 μm (D).