Figure 5.
Figure 5. Motor-independent roles of Myo1 in promoting actin ring assembly and furrow ingression. (A) Cytokinesis defects in bni5Δ and different myo1 mutants. Cells of strains listed below were grown in YPD at 23°C and scored for cytokinesis defects as follows: mild cytokinesis defect, cells with 3–4 cell bodies linked together; strong cytokinesis defect, cells with more than 4 cell bodies linked together. The number of cells scored for each strain was 500. The strains were XDY41 (MYO1-GFP), XDY254 (MYO1-GFP bni5Δ), YJL335A (myo1-Tail-GFP), YJL222A (myo1-TD2-GFP), YJL488A (myo1-TD2Δ-GFP), YEF6322 (myo1-TD2Δ-GFP bni5Δ), and YEF1804 (myo1Δ). (B) Myo1 tail is sufficient for promoting actin ring assembly. Cells of the strains YEF6307 (MYO1-GFP NUP57-GFP; top) and YEF6309 (myo1-Tail-GFP NUP57-GFP; bottom) were grown in SC-Ura media at 23°C, fixed, and stained for actin, and then imaged by spinning-disk confocal microscopy along the Z-axis with a 0.1-µm increment (71 and 46 Z-sections for wild-type and myo1-Tail cells, respectively). For clear visualization of the actin ring, images of a single focal plane near the middle of the cell were presented (see Video 7 for 3D construction of the actin ring in a myo1-Tail cell). Myo1*-GFP indicates GFP-tagged Myo1 or its tail. (C) AMR constriction (marked by Myo1-RFP) is spatiotemporally coupled with membrane trafficking (marked by Myo2-GFP) and PS formation (marked by Chs2-GFP). Cells of strains YKT662 (MYO2-GFP) and YEF5762 (CHS2-GFP) carrying pRS316-MYO1-mCherry were grown in YPD at 23°C and then imaged by time-lapse microscopy at 23°C with a 1-min interval. Representative montage images of the time-lapse data are shown here. Arrowheads indicate the start of Myo1 ring constriction. (D) Septum morphology in wild-type and different myo1 mutants. The indicated strains listed in A were processed to visualize septum formation by transmission EM (left) and the orientation of the PS in different strains was measured (diagram; see footnote of Table I for details) and plotted. CW, cell wall; PM, plasma membrane; SS, secondary septum.

Motor-independent roles of Myo1 in promoting actin ring assembly and furrow ingression. (A) Cytokinesis defects in bni5Δ and different myo1 mutants. Cells of strains listed below were grown in YPD at 23°C and scored for cytokinesis defects as follows: mild cytokinesis defect, cells with 3–4 cell bodies linked together; strong cytokinesis defect, cells with more than 4 cell bodies linked together. The number of cells scored for each strain was 500. The strains were XDY41 (MYO1-GFP), XDY254 (MYO1-GFP bni5Δ), YJL335A (myo1-Tail-GFP), YJL222A (myo1-TD2-GFP), YJL488A (myo1-TD2Δ-GFP), YEF6322 (myo1-TD2Δ-GFP bni5Δ), and YEF1804 (myo1Δ). (B) Myo1 tail is sufficient for promoting actin ring assembly. Cells of the strains YEF6307 (MYO1-GFP NUP57-GFP; top) and YEF6309 (myo1-Tail-GFP NUP57-GFP; bottom) were grown in SC-Ura media at 23°C, fixed, and stained for actin, and then imaged by spinning-disk confocal microscopy along the Z-axis with a 0.1-µm increment (71 and 46 Z-sections for wild-type and myo1-Tail cells, respectively). For clear visualization of the actin ring, images of a single focal plane near the middle of the cell were presented (see Video 7 for 3D construction of the actin ring in a myo1-Tail cell). Myo1*-GFP indicates GFP-tagged Myo1 or its tail. (C) AMR constriction (marked by Myo1-RFP) is spatiotemporally coupled with membrane trafficking (marked by Myo2-GFP) and PS formation (marked by Chs2-GFP). Cells of strains YKT662 (MYO2-GFP) and YEF5762 (CHS2-GFP) carrying pRS316-MYO1-mCherry were grown in YPD at 23°C and then imaged by time-lapse microscopy at 23°C with a 1-min interval. Representative montage images of the time-lapse data are shown here. Arrowheads indicate the start of Myo1 ring constriction. (D) Septum morphology in wild-type and different myo1 mutants. The indicated strains listed in A were processed to visualize septum formation by transmission EM (left) and the orientation of the PS in different strains was measured (diagram; see footnote of Table I for details) and plotted. CW, cell wall; PM, plasma membrane; SS, secondary septum.

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