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doi: 10.1242/10.1242/jcs.00112

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Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development

¹ Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
² Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
³ Gene Function Research Center, Tsukuba Central #4, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan

View larger version (89K): [in a new window]   Fig. 1. Expression of Dd-Wt, Ch-Ac and Ch-Sm in HS1 null cells. (A) Schematic representation of Dd-Wt, Ch-Sm and Ch-Ac heavy chains (modified figure reproduced with permission from Liu et al., 2000). (B) Filaments of purified Dd-Wt, Ch-Sm and Ch-Ac myosins (reproduced with permission from Liu et al., 2000). (C) Coomassie-blue SDS-PAGE of total cell extracts of AX3 cells and HS1 cells expressing Dd-Wt, Ch-Ac or Ch-Sm. (D) Westerns of SDS-PAGE of total cell extracts. AX3, HS1 and Dd-Wt cells were blotted with anti-DdMII, Ch-Ac cells were blotted with anti-Acanthamoeba myosin II and Ch-Sm cells were blotted with anti-smooth muscle myosin II. Anti-Acanthamoeba myosin and anti-smooth muscle myosin did not react with HS1 cells (not shown).

View larger version (66K): [in a new window]   Fig. 2. Ch-Ac and Ch-Sm support growth and cytokinesis in suspension culture of transfected HS1 cells. (A) Growth rates of cells determined by cell number. (B) Representative cells stained with 4,6-diamidino-2-phenylindole after 3 days in suspension culture showing that transfected cells have many fewer nuclei than HS1 cells. (C) Quantification of number of nuclei per cell; the data are the mean of three experiments in each of which at least 100 cells were scored for each cell type.

View larger version (61K): [in a new window]   Fig. 3. Localization of Ch-Sm differs from localization of Dd-Wt and Ch-Ac in interphase and dividing cells. (A) Interphase AX3 cells and HS1 cells expressing Dd-Wt, Ch-Ac and Ch-Sm. (B) Dividing cells. (C) HS1 cells expressing Ch-Sm at interphase, early telophase and late telophase. (D) HS1 cells expressing the tail of Acanthamoeba myosin II at interphase, anaphase and telophase. Myosins were localized by indirect immunofluorescence.

View larger version (43K): [in a new window]   Fig. 4. Dynamics of localization of Ch-Ac and Ch-Sm to the cleavage furrow during cytokinesis. (A) Time course of localization of GFP-Ch-Ac to the cleavage furrow of a typical, live cell undergoing cytokinesis. Time is in seconds. (B) Gallery of images showing the localization by indirect immunofluorescence of Ch-Sm in cells at different stages of cytokinesis. The cells in B were aligned by matching them to similar images in the Ch-Ac sequence (A). Whereas Ch-Ac relocated symmetrically from the cortex to the cleavage furrow, the cleavage furrow in HS1 cells expressing Ch-Sm seemed to form at the site of the cortical patch of Ch-Sm, which remained on one side of the cleavage furrow until the furrow was highly constricted.

View larger version (62K): [in a new window]   Fig. 5. Ch-Ac and Ch-Sm rescue ConA-capping but Ch-Sm does not redisperse after capping. Distribution of ConA and myosins 5 minutes (A) and 20 minutes (B) after initiating capping by addition of ConA. (C) Percentage of cells with myosin caps as a function of time after initiating capping by addition of ConA. The ConA caps did not disperse in any of the cell lines. ConA, red; myosins, green.

View larger version (26K): [in a new window]   Fig. 6. Plaque expansion and phagocytosis assays. (A) The deficiency of HS1 cells in the plaque expansion assay was largely reversed by expression of Dd-Wt, less well by expression of Ch-Ac and only slightly by expression of Ch-Sm. Seven to ten drops of Dictyostelium amoebae in a suspension of heat-killed bacteria were spotted onto black filters and micrographs were taken 5 days later when the expanding amoeba plaques had ingested the bacteria and exposed the black filter. (B) Phagocytosis of latex beads was only 25% inhibited in HS1 cells and was completely rescued by expression of Dd-Wt and Ch-Ac. HS1 cells expressing Ch-Sm were no different than HS1 cells. Each point is the mean of five independent experiments for AX3 and three independent experiments for the other cell lines. Standard deviations were about 10% for the later time points in experiments with AX3, Dd-Wt and Ch-Ac, and 20-30% for HS1 and Ch-Sm. Uptake was based on protein concentration rather than cell number to compensate for any differences in cell size. Cells expressing Ch-Sm were larger than the other cell lines, which were all about the same size.

View larger version (117K): [in a new window]   Fig. 7. Assays of cell streaming and chemotaxis. (A) In the cell streaming assay, micrographs were taken 7-8 hours after plating the amoebae on non-nutrient medium. (B) In the chemotaxis assay, cells were pulsed with 75 nM cAMP (final concentration after each pulse) for the time required for maximum expression of cAMP receptors (see text), washed and allowed to adhere to a coverslip, and photographed at 15-second intervals. The micropipette contained 10 µM cAMP.

View larger version (61K): [in a new window]   Fig. 8. Ch-Ac and Ch-Sm concentrate in the posterior region of migrating cells. Cells were pulsed with 75 nM cAMP (final concentration) as in Fig. 9. At the times of maximum cAR1 concentration (see text), cells were washed and placed on a coverslip and allowed to migrate towards a pipette containing 10 µM cAMP, as in Fig. 7. Micrographs of cells expressing GFP-myosins were taken while the cells were under observation so the direction of movement was known. The localization of Ch-Sm was determined by indirect immunofluorescence of fixed cells and the direction of movement inferred from the orientation of the pseudopods. The cells shown are typical. All were moving from right to left. GFP-Dd-Wt-AX3, AX3 cell expressing GFP-Dd-Wt; GFP-Dd-Wt, HS1 cell expressing GFP-Dd-Wt; GFP-Ch-Ac, HS1 cell expressing GFP-Ch-Ac; Ch-Sm, HS1 cell expressing Ch-Sm.

View larger version (130K): [in a new window]   Fig. 9. Ch-Ac and Ch-Sm do not support full development to fruiting bodies. AX3 cells developed to fruiting bodies; HS1 never progressed beyond the mound stage; Dd-Wt rescued full development in HS1 cells; HS1 cells expressing Ch-Ac did not develop beyond the mound stage; Ch-Sm-transfected HS1 cells developed a few aberrant fingers but never developed to fruiting bodies. All micrographs were taken at 48 hours after plating the amoebae.

View larger version (83K): [in a new window]   Fig. 10. Alignment of tail sequences of Acanthamoeba, Dictyostelium and chicken smooth muscle myosins reveals limited similarities. Sequences beginning with the invariant Pro that defines the head/tail junction (Korn, 2000) were aligned by the default mode of the PC version of Clustal X (Thompson et al., 1997). Amino acids that occur at the same positions in all three myosins are in red, those that occur only in Acanthamoeba and Dictyostelium myosins are in blue, and those that occur in smooth muscle and Dictyostelium myosins are in green. The region of highest similarity in the three myosins and the corresponding region of chicken skeletal muscle myosin are enlarged at the bottom of the figure. Amino acids in skeletal muscle myosin that are the same as those in the corresponding positions of the three other myosins are in red, those that are the same as in Acanthamoeba and Dictyostelium myosins are in blue, those that are the same as in smooth muscle and Dictyostelium myosins are in green and those that are the same only in skeletal and Dictyostelium myosins are in purple. The GenBank accession numbers are: Acanthamoeba myosin, P05659; Dictyostelium myosin, A26655; smooth muscle myosin, P01587; skeletal muscle myosin, P13538.