Figure 2. Myo1 targeting before... | Rockefeller University Press

Figure 2.

$Figure 2. Myo1 targeting before cytokinesis depends on its interaction through mTD1 with Bni5. (A) Localization of Myo1 and myo1-mTD1 in wild-type and bni5Δ cells. Cells carrying MYO1-GFP or myo1-mTD1-GFP in wild-type (BNI5) (YXD41 and YJL489A) or bni5Δ (XDY254 and XDY258) backgrounds were grown in YPD at 23°C and observed by DIC and fluorescence microscopy. (B) Strains YEF6321 (MYO1-GFP BNI5-RFP; left) and YEF6326 (myo1-mTD1-GFP BNI5-RFP; right) were grown in YPD at 23°C and then imaged by 3D dual-color time-lapse microscopy at 23°C with a 2-min interval. Montage images of the GFP, RFP, and merged channels from the representative time-lapse data are shown here. Arrowhead indicates the start of Myo1 ring constriction. (C) myo1-mTD1 interacts with Bni5 in yeast. GFP-tagged Myo1 fragments were immunoprecipitated using anti-GFP antibody from cell lysates of yeast strains (XDY189, XDY190, XDY191, XDY192, and XDY194; see Table II). Myo1 fragments (IP) and Bni5-3HA (Bound) in the precipitates were detected by Western blot analyses using antibodies against GFP and HA, respectively. (D) myo1-mTD1 interacts with Bni5 in vitro. Equal amounts of MBP, MBP-myo1-TD1, MBP-myo1-mTD1, and MBP-myo1-TD2 bound to amylose beads were mixed individually with the same amount of GST-Bni5. The pulled down MBP and MBP-myo1 fragments as well as GST-Bni5 in the bound and input fractions were detected by Western blot analyses using antibodies against MBP and GST, respectively.$

Myo1 targeting before cytokinesis depends on its interaction through mTD1 with Bni5. (A) Localization of Myo1 and myo1-mTD1 in wild-type and bni5Δ cells. Cells carrying MYO1-GFP or myo1-mTD1-GFP in wild-type (BNI5) (YXD41 and YJL489A) or bni5Δ (XDY254 and XDY258) backgrounds were grown in YPD at 23°C and observed by DIC and fluorescence microscopy. (B) Strains YEF6321 (MYO1-GFP BNI5-RFP; left) and YEF6326 (myo1-mTD1-GFP BNI5-RFP; right) were grown in YPD at 23°C and then imaged by 3D dual-color time-lapse microscopy at 23°C with a 2-min interval. Montage images of the GFP, RFP, and merged channels from the representative time-lapse data are shown here. Arrowhead indicates the start of Myo1 ring constriction. (C) myo1-mTD1 interacts with Bni5 in yeast. GFP-tagged Myo1 fragments were immunoprecipitated using anti-GFP antibody from cell lysates of yeast strains (XDY189, XDY190, XDY191, XDY192, and XDY194; see Table II). Myo1 fragments (IP) and Bni5-3HA (Bound) in the precipitates were detected by Western blot analyses using antibodies against GFP and HA, respectively. (D) myo1-mTD1 interacts with Bni5 in vitro. Equal amounts of MBP, MBP-myo1-TD1, MBP-myo1-mTD1, and MBP-myo1-TD2 bound to amylose beads were mixed individually with the same amount of GST-Bni5. The pulled down MBP and MBP-myo1 fragments as well as GST-Bni5 in the bound and input fractions were detected by Western blot analyses using antibodies against MBP and GST, respectively.