Membrane binding of endocytic myosin-1s is inhibited by a class of ankyrin repeat proteins

Figures

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  FIGURE 1:. Ank1 associates with and controls the localization of Myo1. (A) A summary of data from mass spectrometry analysis of proteins associated with Myo1-GFP. (B) Anti-GFP immunoblot (IB; top) and anti-myc IB (middle) of anti-GFP immunoprecipitations (IPs) from the indicated strains. α-tubulin was used as a lysate control (bottom). Part of this blot is also shown in Supplemental Figure S1F. (C) Representative live-cell images of cells expressing myo1-GFP, mNG-cam1, cam2-mNG, LifeAct-mCherry, or fim1-mNG in wild-type and ank1Δ cells grown at 25°C before imaging. (D) Quantification of the percent of Fim1-mNG that internalized into the cell in wild-type and ank1Δ cells from live-cell time-lapse imaging of the strains imaged with 1-s intervals for a total of 1 min. Error bars represent SEM, ** p < 0.01; t test. n of patches ≥ 74 from n of cells ≥ 8. (E) Representative tetrads and schematic from the indicated genetic cross. F) Live-cell imaging of the indicated strains grown at 25°C before imaging. The blue arrows indicate Ank1-GFP and Myo1-mCherry colocalized at the SPB when Ppc89-GBP was present. Scale bars, 5 µm.

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  FIGURE 2:. Myo1 and Ank1 physically associate to inhibit Myo1 membrane binding. (A) A schematic, drawn to scale, of the domain layouts of Ank1 and Myo1 proteins. Myo1 contains the following C-terminal domains: IQ, TH1, TH2, SH3 and CA. The numbers on Ank1 indicate the three ankyrin repeats. (B) Anti-GFP IB (top) and anti-myc IB (middle) of anti-GFP IPs from the indicated strains. α-tubulin was used as a lysate control (bottom). (C) Live-cell imaging of Myo1Δ(1-964)-mNG in wild-type or ank1Δ cells grown up at 25°C before imaging. Scale bar, 5 µm. (D) In vitro binding assay with bead bound recombinant Myo1(1-964)-FLAG-Cam1-Cam2 with recombinant Ank1. Samples were washed, resolved by SDS–PAGE, and stained with Coomassie blue. The second lane is a control with empty beads incubated with Ank1. (E) Left, AF predicted structure of the Myo1–Ank1 complex. The Myo1 motor domain, lever arm and TH1 domain (residues 1-964) were modeled with full-length Ank1. The motor domain and lever arm of Myo1 are gray and the Myo1 TH1 domain is blue. Ank1 is magenta. Right, a zoomed in view of the interaction interface of Ank1 and Myo1 predicted by AF where the ankyrin repeats bind the Myo1 lever arm. The AF model…

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  FIGURE 3:. Structure–function analysis of Ank1. (A) Live-cell imaging of myo1-mNG fim1-mCherry with either an empty vector (EV) or a vector inducing nmt1-ank1+ for 24 h at 32°C. (B) Quantification of the percent of Fim1-mCherry that internalized into the cell in myo1-mNG fim1-mCherry cells expressing EV or nmt1-ank1+ from live-cell time-lapse imaging of the strains from A imaged with 1-s intervals for a total of 1 min. Error bars represent SEM, *** p < 0.001; t test. n of patches ≥ 85 from n of cells ≥ 9. (C) Anti-GFP IB (top) and anti-myc IB (second from top) of anti-GFP IPs from the indicated strains. anti-myc IB from lysates is shown in the blot second from the bottom. α-tubulin was used as a lysate control (bottom). (D) Live-cell imaging of cells expressing Myo1-mCherry with Ank1-GFP, Ank1Δ(1-155)-GFP, or Ank1-R17E-GFP. Cells were grown up at 25°C before imaging. (E) Quantification of the percent of Fim1-mNG that internalized into the cell in wildtype, ank1-R17E and ank1Δ(1-155) cells from live-cell time-lapse imaging of the strains imaged with 1-s intervals for a total of 40 s. Error bars represent SEM, ** p < 0.01; *** p < 0.001; t test. n of patches ≥ 109 from n of cells ≥ 14. (F)…

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  FIGURE 4:. Human OSTF1 is a likely Ank1 orthologue. (A) A schematic, drawn to scale, of OSTF1 and Myo1e. Myo1e contains the following C-terminal domains: IQ, TH1, TH2, and SH3. OSTF1 contains an N-terminal 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 and D) Sequence alignments of the indicated amino acids for the indicated proteins: S. pombe (S.p.), S. cerevisiae (S.c.), Homo sapiens (H.s.), and Mus musculus (M.m.).

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  FIGURE 5:. 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) IB analyses of whole cell lysates from wildtype and Myo1e-mNG cells with anti-α-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 wild-type 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…