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In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other

¹ National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Biochemistry and Genetics, National Institutes of Health, Building 8, Room 403, Bethesda, Maryland 20892, USA
² National Heart, Lung, and Blood Institute, Laboratory of Cell Biology, National Institutes of Health, Building 50, MSC 8017, Bethesda, Maryland 20892, USA

View larger version (21K): [in a new window]   Fig. 1. Schematic view of septation in wild-type (A-D) and in chs2 or myo1 mutants (A,E-G). The neck region between the mother and daughter cell is represented, with the cell walls as the grey area and the plasma membrane as the brown line. The red spots indicate the location of Chs2p. Chitin is shown in green. In (B), the membrane invaginates and chitin is laid down in the invagination. Continuation of this process leads to generation of the primary septum disk and to pinching off of the membrane (C). Next, secondary septa (yellow) are built up from both the mother and daughter cell sides. A trilaminar septum results (D). In chs2 and myo1 mutants, invagination of the plasma membrane in a small area and growth of a primary septum do not take place. Instead, inward growth of cell wall material over a large portion of the plasma membrane pushes the membrane toward the center of the channel (E,F), finally closing the latter and generating a thick and uniform septum (G). In our hypothesis, this represents growth of secondary septa, which would occur at 90° to the normal direction. Thus, in the cell cycle, stage E would start between the normal times for (C) and (D). The green spots in (E-G) designate chitin formed by the action of chitin synthase III that is required for the remedial septa but not for the normal secondary septa. In (G), a lacuna resulting from the uneven fusion of the advancing secondary septum is also shown.

View larger version (169K): [in a new window]   Fig. 2. Electron microscopy of septa in wild-type (YPH499), chs2 (YMS11) and myo1 (YMS57) strains. (A) early invagination in cytokinesis of wild-type; (B) completed primary septum in wild-type; (C) completed trilaminar septum in wild-type; (D) an advancing abnormal septum (right) and a completed septum (left) in the chs2 mutant; (E) a completed septum in the chs2 mutant, showing a lacuna; (F), an advancing abnormal septum in the myo1 strain; (G), an advancing septum (left) and two completed septa in the myo1 mutant. White arrows indicate an advancing primary septum; black arrows point to lacunae. Bars represent 1 µm.

View larger version (71K): [in a new window]   Fig. 3. Morphology of single and double mutants in MYO1 and CHS2 as seen by differential interference contrast. The strains used are YMS11 (chs2) (A), YMS57 (myo1) (B) and YMS135 (chs2 myo1) (C). The generation time in YEPD at 30°C was 145 minutes for YMS11, 226 minutes for YMS57 and 230 minutes for YMS135.

View larger version (105K): [in a new window]   Fig. 4. Localization of Chs2p or Myo1p in strains lacking the other protein. (A) Myo1p-GFP rings in wild-type (YPH499[pMS55]). (C) Myo1p-GFP rings in a chs2 strain (YMS11[pMS55]). (E,G) Chs2p-GFP rings in wild-type (YMS167) and in a myo1 strain (YMS171), respectively. The pictures on the right (B,D,F,H) are the corresponding frames, as seen under phase contrast. Bars represent 5 µm.

View larger version (65K): [in a new window]   Fig. 5. Morphology by differential interference contrast (A) and temperature sensitivity (B) of single and double mutants in MYO1 and CHS3. In (A), arrows point to cells that are enlarged and lysed. In (B), successive tenfold dilutions are shown. The strains used are ECY46-4-1B (chs3), YMS57 (myo1) and YMS75 (myo1 chs3). The generation times of the strains in YEPD at 30°C were 160 minutes for ECY46-4-1B, 226 minutes for YMS57 and 800 minutes for YMS75.

View larger version (27K): [in a new window]   Fig. 6. There are low levels of some proteins (Bud3p, Bud4p and Spa2p) in chs2 and myo1 mutants (A). In (B), the decrease in level of Bud4p, when fused to GFP, is much less than with the native protein. The strains used are YPH499 (wild-type), YMS11 (chs2), YMS57 (myo1), YMS160 (Bud4p-GFP), YMS164 (chs2, Bud4p-GFP) and YMS166 (myo1, Bud4p-GFP). For the detection of Spa2p-HA and Myo1p-c-myc, YPH499 containing pMS53 or pMS54, respectively, was used. Hsp104p was detected as a loading control. In this figure, relevant genotypes are listed above the blot and detected proteins on the right side of it. For the western blotting technique used see Materials and Methods.

View larger version (121K): [in a new window]   Fig. 7. Fluorescent rings of Bud3p-GFP (A,C,E) and Bud4p-GFP (G,I,K). (B,D,F,H,J,L) are phase-contrast pictures of the same cells. For Bud3p,(A) and (B) show wild-type (YMS159), C and D chs2 (YMS161), E and F myo1 (YMS163). For Bud4p, G and H represent wild-type (YMS160), I and J chs2 (YMS164), K and L myo1 (YMS166). Double rings in myo1 and chs2 mutants are more separated from each other (about 1.5 times) than those in wild-type.