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.

Distinct targeting mechanisms impart distinct Myo1 localization dynamics during the cell cycle. (A) Effects of bni5Δ and myo1 truncations on the dynamics of Myo1 localization during the cell cycle. Cells of strains XDY286 (MYO1-GFP; n = 33), XDY287 (MYO1-GFP bni5Δ; n = 38), XDY288 (myo1-Tail-GFP; n = 88), XDY289 (myo1-TD2Δ-GFP; n = 42), and XDY290 (myo1-TD2-GFP; n = 31) were grown in YPD at 23°C and then imaged by 3D dual-color time-lapse microscopy at 23°C with a 1-min interval. Montage images from the representative time-lapse data are shown here. Top row in each image panel: Cdc3-RFP (red); middle row: Myo1*-GFP (asterisk indicates Myo1 or its fragment; green); bottom row: merged images of septin-RFP and Myo1*-GFP. The merged images were used to determine the symmetry of Myo1*-GFP ring during its localization and/or constriction. Arrowheads indicate septin-hourglass splitting. Images within each image panel were processed with the same magnification. (B) Constriction rates of Myo1 and its fragments in wild-type and/or bni5Δ cells. “a” indicates that only cells showing symmetric Myo1 constriction were used for the calculation.

Actin ring plays a role in guiding PS formation. (A) Actin ring assembly in iqg1-CHDΔ mutant. Cells of strains RNY2594 (iqg1Δ MYO1-GFP, pRS315-IQG1-GST) and RNY2595 (iqg1Δ MYO1-GFP, pRS315-IQG1[Δ2-411]-GST) were grown in SC-Leu media at 23°C and then fixed and stained for actin and DNA. (B) Abnormal constriction of the Myo1-GFP ring in iqg1-CHDΔ mutant. Cells of the strains RNY2596 (iqg1Δ MYO1-GFP CDC3-mCherry, pRS315-IQG1-GST) and RNY2597 (iqg1Δ MYO1-GFP CDC3-mCherry, pRS315-IQG1[Δ2-411]-GST) were grown in SC-Leu media at 23°C and then imaged by time-lapse microscopy at 23°C with a 1-min interval. Arrowheads indicate septin-hourglass splitting. (C) PS formation in iqg1-CHDΔ mutant. PS of the strains listed in A were visualized by transmission EM and the orientation of the PS was quantified as in Fig. 5 D.

Myo1 is a two-headed myosin and its tail contains two distinct targeting domains. (A) Myo1 heavy chain forms a dimer in yeast. Myo1-C-TAP and myo1-TD2Δ-C-TAP were purified from strains XDY7 and XDY99, respectively (left, silver staining), and observed by rotary-shadowing EM (right). (B) Recombinant myo1-Tail forms a dimer. MBP-myo1-Tail-TD2Δ was purified from E. coli (left, Coomassie blue staining) and observed by rotary-shadowing EM (right). (C) Summary of localization data on Myo1 fragments. mTD1, minimal targeting domain 1; TD2, targeting domain 2; AD, assembly domain. (D) Localization of different Myo1 fragments during the cell cycle. Yeast strains (XDY41, YJL335A, YJL221A, YJL489A, YJL222A; see Table II) were used for observation. Bar, 1 µm.

Myo1 targeting during cytokinesis depends on Mlc1 and Iqg1. (A) myo1-TD2 localization depends on Mlc1 and Iqg1. Wild-type (WT) (XDY154), mlc1-11 (XDY173), and iqg1Δ (XDY218) cells carrying plasmid pRS316-MYO1-TD2-GFP were grown in SC-Ura medium at 23°C and then examined for myo1-TD2 localization. (B) Full-length Myo1 localization depends on Mlc1 and Iqg1. Strains YEF6179 (mlc1-11 MYO1-GFP CDC3-RFP; left) and YEF6325 (iqg1Δ CDC3-RFP, pRS316-MYO1-C-GFP; right) were grown in liquid SC-Leu media at 23°C and then imaged by 3D dual-color time-lapse microscopy at 23°C with a 1-min interval. Montage images of the GFP, RFP, and merged channels from the representative time-lapse data are shown here. Arrowheads indicate the splitting of septin hourglass into two cortical rings, which coincides with mitotic exit and the onset of cytokinesis. (C) myo1-TD2 localization in cells containing different iqg1 alleles. Plasmid pRS315 derivatives carrying the indicated iqg1 alleles (see Table III) were transformed into strain XDY218. Transformants were grown in SC-Ura-Leu medium at 23°C and then quantified for myo1-TD2 localization. APC, APC/C recognition site; CHD, calponin-homology domain; IQ, IQ motifs; Ras-GAP, RasGAP-related domain; RasGAP-C, RasGAP C terminus–related domain.

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.

Biphasic targeting of Myo1 and the guiding role of the actomyosin ring in membrane deposition and ECM remodeling during cytokinesis. (A) A model for the biphasic targeting of Myo1 during the cell cycle. Myo1 targeting before cytokinesis is mediated solely by the septin-binding protein Bni5. Myo1 targeting during cytokinesis depends on Mlc1 and Iqg1. Iqg1, Myo1, and actin filaments at the bud neck likely define the minimal components of the AMR. (B) A model for the guiding role of the AMR in PS formation. In addition to force generation, the AMR may function as a “compass” to guide membrane deposition and ECM remodeling (PS formation in yeast) during cytokinesis by capturing and distributing vesicles and their cargoes along the division site. This motor-independent guidance role of the AMR is sufficient to ensure membrane closure during cytokinesis for small cells such as budding yeast, but not for the larger ones such as fission yeast and animal cells.