The Myo1p tail alone provides the full function of Myo1p during cytokinesis in the BF264-Du strain background. (A) Diagram of the myosin-II domain structure shown for a hypothetical myosin-II sequence. The N-terminal catalytic domain or head binds ATP and actin filaments. The light chain domain binds the ELC (IQ1) and RLC (IQ2). The C-terminal tail forms a dimer that assembles into bipolar filaments. The arrow indicates the site at the beginning of the tail where an NcoI site was engineered into the coding sequence of MYO1, myo2, and myp2 to facilitate construction of plasmids containing chimeras and the headless Myo1Tp (lacking both the catalytic and light chain domains). (B) Differential interference contrast (DIC) (left) and fluorescence (right) micrographs of budding yeast myo1Δ cells expressing GFP-Myo1p (pGFP-MYO1), GFP (pPGT), GFP-Myo2H-Myo1Tp (pGFP-myo2H-MYO1T), or GFP-Myp2H-Myo1Tp (pGFP-myp2H-MYO1T). Cells were grown and mounted in CSM-Trp^- medium. (C) In a separate experiment budding yeast cells (as in B and F) were grown in CSM-Trp^- medium and scored for cytokinesis defects measured as the fraction of cells forming connected cell chains, as described by Tolliday et al. (2003). (D) DIC (left) and fluorescence (right) micrographs of budding yeast myo1Δ she4Δ cells expressing GFP-Myo1p (pGFP-MYO1), GFP (pPGT), or GFP-Myo2H-Myo1Tp (pGFP-myo2H-MYO1T). (E) In a separate experiment, budding yeast cells (as in D) were grown in CSM-Trp^- medium and scored for cytokinesis defects. (F) DIC (left) and fluorescence (right) micrographs of budding yeast myo1Δ cells expressing GFP-Myo1Tp (Myo1p tail). Bars, 5 μm.

The Myo1p tail alone provides the full function of Myo1p during cytokinesis and cell growth in the W303a background. DIC (top left and bottom) and fluorescence (top right) micrographs of W303a budding yeast myo1Δ haploids expressing GFP-Myo1p (pGFP-MYO1) (A) and GFP-Myo1Tp (pGFP-MYO1T) (B). (C) In a separate experiment budding yeast cells (as in A and B) were grown in CSM-Trp^- medium and scored for cytokinesis defects by quantitating the percent of cells forming connected cell chains. Bar, 5 μm.

Coiled-coil predictions for different myosin-II tails. Predictions were calculated using the COILS program (Lupas et al., 1991). The tails of Homo sapiens nonmuscle myosin-II-B, S. pombe Myp2p, S. cerevisiae Myo1p, S. pombe Myo2p, and four uncharacterized fungal type-II myosins (from A. fumigatus, N. crassa, C. albicans, and C. neoformans) were compared. x-axis, amino acid position. y-axis, probability of forming a coiled-coil.

Dynamics of the Myo1p tail contractile rings are similar to those of full-length Myo1p contractile rings. (A) Time-lapse spinning disk confocal fluorescence micrographs of myo1Δ strain (BF264-Du) cells expressing GFP-Myo1p (top) or GFP-Myo1Tp (bottom) during contractile ring constriction. Stacks of six confocal z sections at intervals of 0.5 μm were collected (projected at maximum intensity) at each time point. Dividing cells are outlined in white in the first panels. (B) Plot of average contractile ring circumference versus time for 20 GFP-Myo1p cells (closed circles) and 20 GFP-Myo1Tp cells (open circles). Time zero represents the point at which rings initiate contraction. The slopes of these plots are the rates of ring constriction. (C) Confocal fluorescence micrograph of two cells carrying the GFP-Myo1Tp construct (as described in A). The lower cell is orientated such that a complete, uninterrupted contractile ring is apparent. Bars, 5 μm.

The head and tail of fission yeast Myp2p are required for its function. (A) DIC micrographs show the cell morphology for a fission yeast strain carrying a chromosomal GFP-myp2 fusion (JW 994), a myp2Δ strain (TP5), and strains with chromosomal GFP-myp2 head (EL 102) or GFP-myp2 tail (EL 101) fusions replacing the myp2 gene. Strains were grown under restrictive conditions for testing Myp2p function (EMM medium, 20°C). (B) In a separate experiment, cells were stained with Hoescht dye and scored for the number of nuclei per cell. (C) Fluorescence micrographs of GFP-Myp2p (JW 994, left), GFP-Myp2Hp (EL 102, center), and GFP-Myp2Tp (EL 101, right). Cells are shown after growth under permissive conditions (YE5S, 30°C). (D) DIC (left) and fluorescence (right) micrographs for a strain (FY 527) bearing p81-myo2H encoding the Myo2Hp-GFP fusion. Cells are shown after growth in EMM Ura^- medium. Bars, 5 μm.