The myosins and their mutants used in this study are shown in Table 1. Base clones for each myosin 1 gene (myoC, myoE, myoD, and myoF ) were generated by TA cloning (Strataclone system; Agilent) of a full-length genomic fragment generated by PCR using AX2 genomic DNA as a template. Q5 mutagenesis (New England Biolabs) was then used to introduce mutations into the region of each gene encoding one or both of the BH regions (see Supplemental Table S1). The sequence of all PCR-generated clones was verified (University of Minnesota BioMedical Genomics Center). The wild-type or mutant full-length genes were then restriction enzyme cloned into a low copy number origin of replication extrachromosomal expression plasmid carrying a G418 resistance cassette, pTX-GFP (Levi et al., 2000 (link)). The resulting plasmid encoded each of the myosin 1s with GFP fused at its N-terminus. The plasmid carrying RFP-LifeAct and hygromycin resistance was described earlier (Riedl et al., 2008 (link); Veltman et al., 2009 (link); Brzeska et al., 2014 (link)).