Fig 1: Human OSTF1 controls Myo1e localization at sites of endocytosis.A) Cartoon representation of the 3’ end of the MYO1E gene. The orange arrow points to the position where the gRNA was designed to target Cas9 for gene editing. Numbers correspond to exons. Green arrows indicate positions of primers used to verify insertion of the tag by PCR analysis. B) PCR verification of the cell line with endogenously tagged MYO1E. C) Immunoblot analyses of whole cell lysates from wildtype and Myo1e-mNG cells with anti-a-tubulin (loading control) and anti-Myo1e. D) Fixed-cell TIRFM imaging of Myo1e-mNG cells stained with Alexa Flour 594-transferrin (Tfn-T94). Scale bar on left images, 5 µm and scale bar on zoomed in section on right, 1 µm. E) Live-cell TIRFM of Myo1e-mNG cells expressing mCherry or OSTF1-mCherry. Scale bar, 10 µm. F) In wildtype cells (left) myosin-1 exists in a cytoplasmic pool bound by Ank1/OSTF1, and a membrane bound pool at sites of endocytosis that lack Ank1/OSTF1. When yeast cells lack ank1+ (middle), myosin-1 constitutively binds along the entire PM and these cells have defects in endocytic internalization. Overproduction of Ank1 or OSTF1 (right) causes all the myosin-1 to be cytoplasmic and not localize to sites of endocytosis. In yeast cells this leads to severe endocytic internalization defects. Created with BioRender.com.
Fig 2: Human OSTF1 is an likely Ank1 ortholog.A) A schematic, drawn to scale, of OSTF1 and Myo1e. Myo1e contains the following C-terminal domains: isoleucine-glutamine (IQ), tail homology 1 (TH1), tail homology 2 (TH2) and src homology 3 (SH3). OSTF1 contains an N-terminal proline rich region (PRR), an SH3 domain, three ankyrin repeats and a C-terminal acidic tail. B) AF predicted structure of the OSTF1-Myo1e complex. Full-length proteins were used to model the interaction. Myo1e is in gray except the TH1 domain is blue. The numbers on the model indicate the three predicted interaction interfaces between the proteins. OSTF1 PRR-Myo1e SH3 (1) OSTF1 ankyrin repeats-Myo1e lever arm (2) and OSTF1 acidic tail-Myo1e TH1 (3). OSTF1 is magenta. C-D) Sequence alignments of the indicated amino acids for the indicated proteins. S. pombe (S.p.), S. cerevisiae (S.c.), H. sapiens (H.s.), M. musculus (M.m.).
Supplier Page from Sino Biological, Inc. for Human OSTF1 Gene ORF cDNA clone in cloning vector