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Genetic and morphological evidence for two parallel pathways of cell-cycle-coupled cytokinesis in Dictyostelium

Akira Nagasaki1, Eugenio L. de Hostos2 and Taro Q. P. Uyeda1,*

1 Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan
2 Department of Cell Genetics, Exelixis Inc., South San Francisco, CA 94083-0511, USA



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  		Fig. 2. Disruption constructs targeting mhcA and amiA, and confirmation of disruption using genomic PCR. (A) The targeting vector used to knockout amiA was constructed by replacing part of its coding region and promoter with the blasticidin S [pKO amiA(Bsr)] or G418 [pKO amiA(Neo)] resistance gene. In the targeting vector for mhcA, a portion of the motor domain was replaced with the G418 resistance cassette. Expression of all drug resistance genes was driven by the actin 15 promoter. Thick lines indicate coding sequences of amiA and mhcA. (B) Mutant cells were identified by a shift in size of the PCR products. (Left) Knockout of amiA in mhcA- cells (HS1), yielding HTU1; (middle) knockout of amiA in corA- cells, yielding HTU8; (right) knockout of corA in mhcA- cells (HS1), yielding HTU7. Arrows in A show the positions of the primers used for genomic PCR.

 


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  		Fig. 1. Three modes of cytokinesis in Dictyostelium. A series of time-lapse phase contrast images obtained at 30 second intervals are shown. (A) Wild-type AX2 cells were embedded in low melting temperature agarose and cultured without solid surfaces. In this condition, wild-type cells divide using cytokinesis A. (B) mhcA- cells were cultured on a plastic dish to allow adhesion to a solid surface. Division of mhcA- cells is driven by cytokinesis B. (C) Multinucleate mhcA- cells grown in suspension for 3 days were then placed on a plastic dish. These giant cells divide by cytokinesis C. Bars, 10 µm.

 


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  		Fig. 3. Phenotypic analysis of wild-type and mutant cells in which cytokinesis A and/or B has been disrupted. (A,B) Histograms showing the distributions of nuclei/cell among cells grown in suspension (B) or on glass surfaces (A). Cells of all three strains shown were cultured in suspension before being transferred to plastic Petri dishes with thin glass bottoms; fixation, DAPI staining and counting of nuclei were performed immediately (B) or after 3 days of continued growth on the glass surface (A). (C-I) Photomicrographs of cells grown for 3 days on a glass surface. Unlike cells shown in A and B, these cells were precultured on plastic Petri dishes, suspended by streams of medium using micropipettes, diluted, and replated to plastic Petri dishes with thin glass bottoms because cells carrying the mhcA- mutation were unable to grow in suspension. Wild-type (C) and mhcA- cells (D) were mostly mononucleate. AmiA- (E), corA- (G) and amiA-/corA- (I) cells were somewhat larger and flatter than wild-type cells, indicating moderate disruption of cytokinesis. The greater enlargement of amiA-/mhcA- (F) and corA-/mhcA- (H) double knockout cells suggests cytokinesis is more severely affected in these strains. (J) A histogram showing the distributions of nuclei/cell among the cells shown in C-I. More than half of the amiA-/mhcA- and corA-/mhcA- cells are multinucleate, and more than 30% of them are highly multinucleate (more than five nuclei per cell), confirming severe disruption of cytokinesis in these cells. Magnifications in panels C-I are the same. Bar, 10 µm.

 


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  		Fig. 4. Sequences of morphological changes during division on a solid substrate. Cells were attached to plastic dishes for 2 days before observation. Each panel shows a series of phase contrast images recorded with intervals of 60 seconds between frames. (A-E) Mitotic wild-type (A), mitotic mhcA- (B), mitotic amiA- (C), mitotic corA- (D) and amiA-/mhcA- (E) cells. The cell cycle stage of the cell in E was not determined, but is most probably interphase. Bars, 10 µm. Magnifications of A-D are the same.

 


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  		Fig. 5. Visualization of the cell cycle as a function of GFP-histone expression. (A) Structure of the GFP-tagged histone H1 chimeric gene. (B,C) Confocal images of the nucleus were identified in interphase (B) and mitotic (C) cells by GFP-H1 fluorescence. (D-F) A series of time-lapse micrographs of GFP fluorescence images superimposed on phase contrast images. They show differing cytokinesis processes in three strains of GFP-H1-expressing cells grown on solid substrates: mhcA- cells undergoing cytokinesis B (D); amiA- cells failing to divide (E); and amiA-/mhcA- cells undergoing cytokinesis C (F). Bars, 10 µm.

 


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  		Fig. 6. A schematic diagram depicting two pathways leading to cell-cycle-coupled cell division in Dictyostelium cells. (A) Cytokinesis A. Mitotic AmiA- or coronin- cell carries out cytokinesis by active contraction of the cleavage furrow which depends on actin and myosin II. (B) Cytokinesis B. A mitotic myosin II-null cell divides by passive contraction of the cleavage furrow. In this case, cytoplasm in equatorial region is withdrawn indirectly (white arrows inside the cell) by traction forces generated along polar peripheries (black arrows). (C) Summary of three methods of cytokinesis in Dictyostelium. Cytokinesis A requires myosin II expression, but adhesion to a substrate is not necessary. Cytokinesis B is not dependent on myosin II but adhesion is indispensable. These two mechanisms of cell division occur immediately following nuclear division and are somehow coordinated in wild-type cells. The third pathway, cytokinesis C, is cell cycle independent and occurs during interphase.

 

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