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Video 1. Mitosis and cytokinesis in a wild-type cell of Dictyostelium discoideum. The cell expressed GFP-α-tubulin (green) together with mRFP-LimE∆ as an actin label (red). The left panel shows confocal dual-color fluorescence images, the right panel DIC bright-field images. The cell has been slightly flattened by agarose overlay. Focus has been changed at the 417-s to 420-s frames. The 5-s frame in the video corresponds to the 0-s frame in Figure 1B. Frame-to-frame interval is 1 s. Bar, 10 µm. Video 2. Animation of three stages of cell division of a 3D rendered wild-type cell of D. discoideum. The cell expressed GFP-α-tubulin (green) together with mRFP-LimE∆ as an actin label (red). Time is indicated in seconds. Bar, 10 µm. Video 3. A multinucleate wild-type cell produced by electric-pulse induced fusion. The cells fused expressed mRFP-α-tubulin (red) and GFP-myosin-II heavy chains (green). The left panel shows confocal dual-color fluorescence images, the right panel DIC bright-field images. The 19-s frame in the video corresponds to the 0-s frame in Figure 2. At the beginning of the sequence, an interphase cell is seen to be surrounded by the syncytium. In the 49-s to 180-s frames, spindles are bent when the aster microtubules interact with the cell cortex. Frame-to-frame interval is 1 s. Bar, 10 µm. Video 4. Successful (top) and failing (bottom) cytokinesis in myosin-II-null cells. Mitoses of mononucleate cells on a glass surface are shown. The cells expressed GFP-cortexillin I (green), together with mRFP-histone 2B as a label of the chromosomes. The left panels show confocal dual-color fluorescence images, the right panels DIC bright-field images. In the upper panels the 1-s frame in the video corresponds to the 0-s frame in Figure 4A. In the lower panels the 57-s frame in the video corresponds to the 0-s frame in Figure 4B. The cell on bottom has been slightly flattened by agarose overlay. Frame-to-frame interval is 1 s. Bar, 10 µm. Video 5. Synchronous mitosis and division of a multinucleate myosin-II-null cell. This cell expressed GFP-cortexillin I (green) and mRFP-histone 2B (red). The left panel shows confocal dual-color fluorescence images, the right panel DIC bright-field images. At the 446-s and 597-s frames the focus has been on the bottom surface of the cell. The 60-s frame in the video corresponds to the 0-s frame in Figure 5. The cell has been flattened by agarose overlay. Frame-to-frame interval is 1 s. Bar, 10 µm. Video 6. A multinucleate myosin-II-null cell. The cell expressed GFP-cortexillin I (green), together with mRFP-histone 2B as a label of the chromosomes. The left panel shows confocal dual-color fluorescence images, the right panel DIC bright-field images. Division on the left side fails, and three nuclei slip through the furrow toward the right. The 9-s frame in the video corresponds to the 129-s frame in Figure 6. Frame-to-frame interval is 1 s. Bar, 10 µm.

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.

Background. Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans. Results In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC. Conclusions Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.

Background The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape. Results Using particles that consist of two lobes separated by a neck, we found that Dictyostelium cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR) domain in combination with an Src homology (SH3) domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis. Conclusions Phagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle there, or extending the cup along the particle surface to identify the very end of the object to be ingested. Our data illustrate the flexibility of regulatory mechanisms that are at the phagocyte's disposal in exploring an environment of irregular geometry.

The Dictyostelium kinase DPAKa is a member of the p21-activated kinase (PAK) family, consisting of an N-terminal domain characterized by a coiled-coil region and proline-rich motifs, a Rac-binding CRIB-domain, and a highly conserved C-terminal kinase domain. In this study we show that cells overexpressing a C-terminal DPAKa fragment comprising the kinase domain are significantly impaired in motility and phagocytosis, whereas DPAKa-null cells display no obvious phenotypic change. We analyzed the in vivo localization of full-length and truncated DPAKa tagged with green fluorescent protein (GFP). The N-terminal fragments show a highly dynamic cortical localization without a permanent polarized enrichment, whereas the C-terminal fragment is homogenously distributed throughout the cell. The localization of full-length DPAKa is similar to that of myosin II at the rear end of locomoting cells and at the base of phagocytic cups. During mitosis DPAKa is gradually recruited to the cell cortex starting at metaphase, which also parallels the dynamics of myosin II cortical recruitment. However, in contrast to myosin II, DPAKa does not accumulate in the cleavage furrow but stays uniformly distributed throughout the cell cortex. This finding contrasts with previous work claiming accumulation of DPAKa in the cleavage furrow of dividing cells. Our results suggest that the N-terminus directs DPAKa to the cortex, and the C-terminus is necessary for restricting its localization to the rear of moving cells during chemotaxis. Therefore, DPAKa may play distinct roles in myosin II regulation during cell movement and cell division.