W140 is a conserved residue in Myo19 motor domain which is essential for filopodia tip localization. (a) Sequence alignment of human myosin sequences deposited in Swiss-Prot shows the presence of a unique Tryptophan instead of the consensus Valine or Glutamic acid, the unique Tryptophan is preserved in almost all Myo19 homologs (38/39). (b) Homology based structure prediction of Myo19 (cyan) and Myo19W140V(tan) via the Phyre2 server, aligned with two solved human Myo5 structures (not shown, RCSB: 4ZG4, 1W7J) with bound ADP (shown) in the P-Loop. (c) Co-localization of Halo tagged Myo19 or the Myo19W140V mutant with the mitochondrial stain, MitoTracker Green-FM. The intensity plots generated from the yellow line reveal that the mutant localizes to mitochondria similarly to the wild-type. Bar is 20 µm. Blue – nuclei, green – MitoTracker Green-FM stained mitochondria, red – Halo tagged Myo19 or Myo19W140V. (d) H2O2 stimulation of U2OS cells over-expressing Halo tagged Myo19 or Myo19W140V results in the localization of Myo19 to filopodia tips, but not of Myo19W140V. Bar is 20 µm. Zoom - magnification of the area indicated by the yellow rectangle. The absence of mitochondria is due to them being in a higher focal plane, whereas these filopodia form at the base of the cell. The weak diffuse signal is most likely resulting from Myo19 over-expression or saturation of mitochondria. Blue – nuclei, green – emLifeAct, red – Halo tagged Myo19 or Myo19W140V. (e) Rescue of Myo19W140V mutant phenotype by WT Myo19. U2OS cells were co-transfected with Halo tagged Myo19W140V and either WT emMyo19 or an eGFP control and induced with H2O2. As previously shown, Myo19W140V was unable to reach filopodia tips. To our surprise, Myo19 was able to rescue the localization defect and promoted the localization of the mutant to the filopodia tips. Blue – nuclei, green – GFP or emMyo19, red – Halo tagged Myo19W140V, Magenta – phalloidin stained actin. Bar is 10 µm.