Research Article 4345 The Ste20-like kinase SvkA of discoideum is essential for late stages of cytokinesis

Meino Rohlfs, Rajesh Arasada*, Petros Batsios, Julia Janzen and Michael Schleicher‡ Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, Germany *Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA ‡Author for correspondence (e-mail: [email protected])

Accepted 2 October 2007 Journal of Cell Science 120, 4345-4354 Published by The Company of Biologists 2007 doi:10.1242/jcs.012179

Summary The of the social Dictyostelium discoideum demonstrate that GFP-SvkA is enriched at the centrosome encodes ~285 kinases, which represents ~2.6% of the total and localizes to the midzone during the final stage of cell genome and suggests a signaling complexity similar to that division. This distribution is mediated by the C-terminal of yeasts and humans. The behavior of D. discoideum as an half of the kinase, whereas a rescue of the phenotypic amoeba and during development relies heavily on fast changes requires the active N-terminal kinase domain as rearrangements of the actin cytoskeleton. Here, we well. The data suggest that SvkA is part of a regulatory describe the knockout phenotype of the svkA gene encoding pathway from the centrosome to the midzone, thus severin kinase, a homolog of the human MST3, MST4 and regulating the completion of cell division. YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to Supplementary material available online at multinucleated cells sometimes with >30 nuclei. The defect http://jcs.biologists.org/cgi/content/full/120/24/4345/DC1 arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage Key words: Severin kinase, Ste20 kinases, Cytokinesis, Actin furrow and to sever the last cytosolic connection. We cytoskeleton, Severin, Dictyostelium, Midbody

Introduction is the D. melanogaster kinase Misshapen, and its vertebrate Ste20-like kinases are ubiquitous Ser/Thr kinases subdivided homolog Nck-interacting kinase. These kinases regulate the into two families, the p21-activated kinases (PAKs) numbering formation of filopodia (Ruan et al., 2002) and the localization

Journal of Cell Science six and the germinal centre kinases (GCKs) numbering 27 of dynein in flies (Houalla et al., 2005), as well as recruitment representatives in humans (Manning et al., 2002). GCKs of actin and myosin II during fish epiboly (Köppen et al., 2006) harbor the catalytic domain close to the N-terminus, whereas or in mammalian embryos (Xue et al., 2001). the PAK family is characterized by a C-terminal kinase domain They are also involved in the formation of lamellipodia upon and an N-terminal region that facilitates interaction with Rho- stimulation by growth factors (Baumgartner et al., 2006). related GTPases by means of PDB or CRIB domains The GCK-III (or YSK) subfamily is one of eight GCK (Hofmann et al., 2004). By contrast, the role of the highly subfamilies and has three representatives in mammals: diverse C-terminal region of GCKs is not well understood. YSK1/SOK1 (Osada et al., 1997; Pombo et al., 1996), MST3 There are, however, indications that the C-terminal domain (Schinkmann and Blenis, 1997) and MST4 (Lin et al., 2001; regulates activity, localization or intermolecular interactions Qian et al., 2001). The role of these kinases in MAPK signaling (Arasada et al., 2006; Dan et al., 2001). cascades is a matter of debate (Lin et al., 2001; Pombo et al., Kinases from both families are believed to be upstream 1997; Qian et al., 2001; Schinkmann and Blenis, 1997), but regulators of mitogen-activated protein kinase (MAPK) recent data suggest very clearly a regulatory function for MST4 cascades (Raman and Cobb, 2003). In addition, GCKs play in the ERK pathway (Ma et al., 2007). YSK1/SOK1 was important roles as activators and repressors of demonstrated to influence cell migration by phosphorylating (Hipfner and Cohen, 2004; Huang et al., 2005; McCarty et al., the dimeric adaptor protein 14-3-3␨ (Preisinger et al., 2004). 2005). Well characterized is the role of Hippo, the Drosophila In addition, YSK1/SOK1 is involved in organization of the melanogaster homolog of human MST2, that plays a Golgi and shuttles between cytosol and Golgi, modulated by fundamental role during the development of the eye (reviewed an interaction with the Golgi matrix protein GM130 (Preisinger in Edgar, 2006). Further functions for GCKs are the regulation et al., 2004). An RNAi screen in D. melanogaster showed that of ion transport and cell volume (reviewed in Gagnon et al., the knockdown of the only fly GCK-III representative 2006; Strange et al., 2006) and the response to various cellular (CG5169) causes changes in the actin cytoskeleton (Kiger et stresses (Lehtinen et al., 2006; Pombo et al., 1996; Taylor et al., 2003). This correlates well with data from Dictyostelium al., 1996; Tsutsumi et al., 2000). Several GCKs interact with discoideum, where one member of the GCK-III subfamily was cytoskeletal proteins (Eichinger et al., 1998; Mitsopoulos et al., purified to near homogeneity and shown to phosphorylate the 2003; Nakano et al., 2003; Storbeck et al., 2004). A thoroughly gelsolin-like F-actin-fragmenting protein severin (Eichinger et studied example for the regulation of cytoskeletal components al., 1998). This severin kinase (SvkA) has an overall identity 4346 Journal of Cell Science 120 (24)

of 50% to the stress-dependent human GCK-III kinase Phenotype of the svkA mutant YSK1/SOK1. Furthermore, the kinase domains are 74% The svkA-knockout strain had a slight growth defect in shaking identical. culture and on lawn of Klebsiella aerogenes. , The social amoeba D. discoideum is a valuable model adhesion and random motility were normal. The most dramatic organism for investigating the influence of Ste20-like kinases phenotypes were a developmental and a cytokinesis defect, as in cytoskeletal reactions because it is highly mobile as single discussed below. cell and during multicellular development, it is amenable to In wild-type cells, SvkA is expressed at about equal levels molecular genetics, and data on the complete genome are throughout the developmental cycle (Fig. 2A). Interestingly, available (Eichinger et al., 2005). The amoeba contains four multinucleated svkA-knockout cells (Fig. 2B) separated into PAK proteins, 13 GCK proteins that represent the subfamilies smaller cells within 30-60 minutes after transfer to low-salt GCK-II, -III and -VI (Arasada et al., 2006) and several D. phosphate buffer and remained connected by cytoplasmic discoideum-specific GCK subfamilies (Goldberg et al., 2006). bridges, sometimes for several hours (Fig. 2C,D, An investigation of the Ste20-like kinase Krs1, homologous to supplementary material Movie 1). A direct result of this Hippo and MST2, revealed an inhibitory function of the C- behaviour was an uncoordinated movement towards a cAMP terminal domain. Furthermore, Krs1 is necessary for direct cell source and the appearance of multiple leading fronts migration during (Arasada et al., 2006; Muramoto (supplementary material Movie 2). Consequently, the et al., 2007). formation of normal streams and early aggregates was delayed Here, we characterize the role of the Ste20-like SvkA protein by several hours (Fig. 3A). The fruiting bodies were smaller during cytokinesis in D. discoideum. The disruption of the gene and, in particular, the stalks were broader and distorted. impairs cell division and leads to multinucleated cells. In Expression of a full-length kinase restored the normal addition, knockout of svkA affects directed slug movement and phenotype (Fig. 3B), whereas expression of a kinase-dead the formation of fruiting bodies, suggesting an important role mutant could not rescue the phenotype (data not shown). for this kinase not only in single cells but also during Mixtures of svkA-knockout and wild-type cells improved the development. morphology of the fruiting body gradually, but it required a composition of >50% wild-type cells for development into Results essentially normal fruiting bodies (data not shown). SvkA knockout and rescue A transient step in the developmental cycle is the slug stage To examine the cellular activities affected by SvkA, we in which the strongly polarized multicellular aggregate moves disrupted the svkA gene by homologous recombination (Fig. in a positively thermotactic and phototactic fashion. The 1A). The svkA gene disruption of several independent clones knockout of svkA produced drastic changes in phototactic was confirmed by PCR and immunoblots (Fig. 1B,C). motility, and the defect could be rescued again by expression Expression of a full-length (FL) kinase fused to GFP under the of full-length SvkA (Fig. 3C). The overall motility of svkA- control of the actin 15 promoter was used to rescue the svkA- knockout slugs was reduced, but aggregates moved on a null phenotype (Fig. 1C,D). Despite the good reaction of the straight path towards the light source. This indicates that

Journal of Cell Science polyclonal antiserum in immunoblots, its specificity in reception of the light signal in mutant slugs is still intact. immunofluorescence was poor. Therefore, we used mainly GFP-fusion constructs to study the cellular localization of Defects in cytokinesis are most pronounced in the SvkA. Partially purified GFP-SvkA-FL phosphorylated the phenotype of the mutant already described in vitro substrates domains 2 and 3 of severin The loss of SvkA resulted in cells that were up to ten times (DS211C) or myelin basic protein (MyBP) (Eichinger et al., larger than average wild-type cells (Fig. 2B), with sometimes 1998), which showed that the fusion to GFP did not abolish >30 nuclei and a corresponding number of centrosomes (data SvkA function (Fig. 1D). not shown). A comparison between submerged and shaking

Fig. 1. Disruption of the svkA gene encoding severin kinase. (A) Two fragments of the kinase were amplified by PCR from genomic DNA, ligated to the blasticidin-resistance cassette and electroporated into AX2 wild-type cells. (B) Blasticidin- resistant clones were tested for knockout events by PCR1 (primers 1 and 2, 1385 bp for knockout) and PCR2 (primers 1 and 3, 2038 bp as a wild-type control). Under the conditions used, PCR2 did not ever amplify ~3400 bp, including the blasticidin-resistance cassette, in the knockout, but the lack of a PCR product indicated a disruption of the svkA gene as well. (C) Western blot of 2ϫ105 cells per lane, visualized with kinase-specific antibodies confirms the knockout at the level of SvkA protein and also shows the expression of the GFP fusion protein in the wild-type (first lane) and the mutant background (fourth lane). (D) GFP-SvkA-FL, immunoprecipitated from rescue cells, phosphorylated domains 2 and 3 of severin (DS211C, left), myelin basic protein (MyBP, right) and itself (both lanes). SvkA in Dictyostelium 4347

Fig. 2. Expression of SvkA during development and induction of cytofission. (A) Western blot with antibodies against SvkA showing the presence of the kinase throughout the 24 hours of wild-type development (2ϫ105 cells per lane). (B) The knockout cells grown on a plastic surface were considerably larger than the wild-type cells and the rescue cells. (C,D) SvkA-knockout cells were shifted from HL-5 medium to low-salt phosphate buffer (PB) and observed for several hours. The large and multinucleated cells separated only partially. Arrows indicate persistent connections between cells. Bars, 50 ␮m (B,C); 10 ␮m (D); see also supplementary material Movie 1.

cultures (150 rpm) showed that cells in shaking cultures formation of structures reminiscent of a four-part clover leaf

Journal of Cell Science remained multinucleated (Fig. 4A,B). At higher velocity (220 (Fig. 5B) that either divided into mono- and multinuclear cells rpm), the number of large cells (more than four nuclei) was or frequently failed to separate altogether. reduced in favor of mono- and binucleated cells (data not To mark key events of cell division, we transformed svkA- shown). Apparently, an increased shear force helps to rupture knockout cells with vectors encoding different GFP constructs the thin cytoplasmic bridges between incompletely separated (alpha-tubulin, coronin, cortexillin, histone 2B and myosin II). cells. In cultures grown on solid surfaces until confluence or None of these constructs altered the aberrant cytokinesis, and on a lawn of K. aerogenes, it was noted that the cells tended the cells remained large and multinucleated. Furthermore, to become smaller again, possibly because so-called ‘midwife overexpression of the homologous Ste20-like kinase Krs1 as a cells’ assisted in cell division (supplementary material Movie GFP fusion protein (Arasada et al., 2006) or the in vitro 3). substrate GFP-severin did not alter the phenotype. The knockout cells progressed normally through the first steps of Single-cell analysis of cell division cell division as judged by: (1) the separation of chromosomes To analyze cell division at higher resolution, we recorded (GFP-histone 2B), (2) the assembly and disassembly of a phase-contrast time-lapse movies of individual svkA-knockout spindle (GFP-alpha-tubulin), and (3) the first indications of a cells in HL-5 medium. Frequently, dividing cells followed at cleavage furrow (GFP-cortexillin I). The only mislocalization first the usual sequence of events: rounding, spreading, in comparison with normal cell division was observed polarization and formation of a cleavage furrow (Fig. 5A). In occasionally in knockout cells expressing GFP-cortexillin I 10–20% of the observed cell divisions, formation of the (Fig. 5C; supplementary material Movie 4). cleavage furrow was asymmetric. Initially, the cleavage furrow Immunostaining of fixed svkA-knockout cells showed that appeared normal, but then further constriction progressed dividing cells became polarized, as judged by the localization asymmetrically, leading to a crescent-shaped intermediate. The of F-actin (Fig. 6A,B) and coronin (Fig. 6G). Cortexillin I was final cytoplasmic bridge did not sever and was sometimes still enriched in the midzone of the cleavage furrow (Fig. 6C,E), visible after more than 1 hour. In many cases, the thin myosin II was observed at the polar ends and the midzone of connection allowed a subsequent fusion of the two pseudo- the cell as expected (Fig. 6D), DGAP1 was found in the late daughter cells, thus accounting for the appearance of cells with cleavage furrow (Fig. 6F), and alpha-tubulin was visualized at increasing numbers of nuclei. The corrupted formation of the the centrosome, the mitotic spindle and in the midzone, also as cleavage furrows in binucleated mutant cells led to the expected (Fig. 6A-G). 4348 Journal of Cell Science 120 (24) Journal of Cell Science

Fig. 3. Developmental defects in the svkA-null mutant. (A) The development of starved svkA-null cells on phosphate agar showed a delay, whereas the wild-type and rescue strains developed as expected into loose and tight aggregates (6 hours/9 hours), slugs (12 hours) and mature fruiting bodies (24 hours). (B) Typical fruiting bodies obtained after three days on phosphate agar (wild-type and rescue strains: one each at the right; knockout cells: nine examples on the left). (C) Phototaxis of D. discoideum slugs on phosphate agar towards a light source at the right (*).

The localization of SvkA supports its role during the late The localization of SvkA is a function of the C-terminal half stages of cytokinesis of the kinase because a GFP-SvkA-CT construct in the svkA- Fixed cells expressing GFP-SvkA-FL in the knockout null background, which could not rescue the aberrant background showed an overall localization of the kinase in the cytokinesis phenotype, still localized at the centrosome (data cytosol, to a lesser extent also in the nuclei and with a not shown) and, during later phases of cytokinesis, localized somewhat diffuse, but clear, enrichment around the centrosome to the cleavage furrow (Fig. 8B). The expression of a kinase- (Fig. 7A). This centrosomal localization was also observed in dead GFP-SvkA-K134A construct could not rescue the fixed wild-type cells expressing the C-terminal half of the cytokinesis defect, although it localized to the midzone (Fig. kinase (GFP-SvkA-CT; Fig. 7B). In dividing cells, GFP-SvkA- 8C). This highlights the combined importance of proper FL formed a cloud around the spindle and nuclei, without clear localization and SvkA kinase activity for cytokinesis. colocalization with the microtubules (Fig. 7C). In an analysis of live cells undergoing cell division, GFP- Discussion SvkA-FL was enriched in the cleavage furrow during the late Disruption of the svkA gene in D. discoideum led to defects in stage of cytokinesis (Fig. 8A), which is the most affected stage development, phototaxis and, most prominently, in cytokinesis. in the svkA knockout (Fig. 5C). The remnants of a midbody- SvkA-null cells became multinucleated on solid surfaces as like structure with its enriched GFP-SvkA-FL were observed well as in shaking cultures. The appearance of multinucleated for several minutes at the surface of the two daughter cells (Fig. cells seemed to be the consequence of incomplete cytokinesis 8A). because svkA-null cells started to divide normally but SvkA in Dictyostelium 4349

Fig. 4. Defective cytokinesis in svkA-null cells. (A,B) Cells grown on a solid surface or in a shaking culture (150 rpm) were fixed and stained with DAPI for quantification of nuclei per cell. More than 1000 cells were counted for each cell line. In the mutant, >50% of the total number of nuclei were found in multinucleated cells under both conditions.

frequently failed to sever the final connecting bridge between The observed severe phenotype of the svkA mutant cannot the two daughter cells. This led to a frequent re-fusion of easily be explained by absent phosphorylation of the F-actin- daughter cells, resulting in multinucleated cells. The fragmenting protein severin. First, the phosphorylation of enrichment of GFP-tagged SvkA at centrosomes and in the late severin that coined the name ‘severin kinase’ is based on in cleavage furrow suggests that the kinase regulates the final vitro data only (Eichinger et al., 1998). Second, disruption of stages of cytokinesis. All defects could be rescued by the gene encoding severin resulted in mutants with only subtle expression of a full-length GFP-SvkA kinase fusion protein. defects (André et al., 1989). Thus it is reasonable to conclude Journal of Cell Science

Fig. 5. Asymmetric cytokinesis. (A,B) Time-lapse images of svkA-knockout cells dividing on a plastic surface. The increasingly asymmetric cleavage furrow inhibits complete separation of the daughter cells (bars, 20 ␮m). (C) Time-lapse series of projections calculated from stacks of confocal images with GFP-cortexillin I expressed in svkA-knockout cells. The dividing cell shows an asymmetric cleavage furrow and a remaining cytoplasmic bridge that eventually ruptures ~8 minutes after the onset of cytokinesis (bar, 5 ␮m). 4350 Journal of Cell Science 120 (24)

Fig. 6. Analysis of cytokinesis with marker proteins. (A-G) Confocal sections of fixed svkA-knockout cells representing different stages and the symmetrical population of SvkA-null cell division. All cells are stained for alpha-tubulin and in addition for F-actin (A,B) (the arrow in A indicates the dividing cell), cortexillin I (C,E), myosin II (D), DGAP1 (F) or coronin (G). Bars, 5 ␮m. Journal of Cell Science that severin is not the primary target of SvkA in a regulatory The localization of D. discoideum SvkA during the late cascade during cell division. This would be in agreement with stages of cytokinesis fits very well to reports on other Ste20- data from animal cells, where the later stages of cytokinesis like kinases. In the fission yeast Schizosaccharomyces become insensitive to the actin-depolymerizing drug pombe, the Ste20-like kinase Nak1/Orb3, a member of the latrunculin, implying that the plasma membrane is linked to the GCK-III family, is required for cell separation and late stages midbody by a connection that does not involve actin dynamics of cytokinesis (Leonhard and Nurse, 2005). In addition, (Eggert et al., 2006). Ustilago maydis mutants that lack the Ste20-like kinase Don3 This raises a question regarding the identity of other proteins fail to trigger initiation of a secondary septum necessary for that could interact with, or become phosphorylated by, SvkA. cell separation after (Sandrock et al., 2006). The Ras In mammalian cells, the Golgi matrix protein GM130 interacts association domain family 1A (RASSF1A) gene product with the SvkA homolog YSK1 and directs the kinase to the binds to human MST2 and forms with additional proteins a Golgi (Preisinger et al., 2004). There is no clear homolog of complex that controls apoptosis and exit from mitosis (Guo GM130 in D. discoideum, and the svkA-null cells showed no et al., 2007). This complex is localized at centrosomes, disruption of the overall Golgi structure (data not shown). spindle poles and the midbody. The distribution of SvkA is Thus, it is unlikely that the functions of SvkA are similar to very similar, suggestive of the existence of a similar complex those of YSK1, at least as far as the organization of the Golgi in D. discoideum. But the mammalian and insect signaling is concerned. Furthermore, the only known downstream target pathway relies on the formation of a complex by means of a for YSK1 is a 14-3-3␨ protein, and 14-3-3 proteins have been SARAH (Salvador, RASSF1, Hippo) domain that is not implicated in regulating cell polarity and cytokinesis (Hurd et present in SvkA. In D. discoideum, an explicit SARAH al., 2003; Mishra et al., 2005). Another candidate is a nuclear domain is only present in the Ste20-like kinase Krs1 (Arasada Dbf2 related (NDR) kinase, which plays a role in cell-cycle et al., 2006). It is intriguing, however, that the C-terminus of progression and cell morphology (Hergovich et al., 2006). Also SvkA is essential for the distribution to the midzone in pertinent is the fact that a mammalian Ste20-like kinase dividing cells. However, the inability of the kinase-dead (MST3), which possesses in its catalytic domain 75% sequence construct to rescue the aberrant phenotype clearly shows that identity to SvkA, phosphorylates NDR2 at a hydrophobic motif phosphorylation of a currently unknown substrate is an (Stegert et al., 2005). essential function of SvkA. SvkA in Dictyostelium 4351 Journal of Cell Science

Fig. 7. Enrichment of SvkA around the centrosome, the spindle and the midzone. (A) SvkA-knockout cells expressing GFP-SvkA-FL were fixed and stained with antibodies against alpha-tubulin. Three confocal sections are shown. GFP-SvkA-FL is enriched diffusely around the centrosome of interphase cells (open arrow) and around the spindle in mitotic cells (closed arrows). (B) Confocal section of fixed wild-type cells expressing the C-terminal domain construct GFP-SvkA-CT. The arrow indicates the localization of GFP-SvkA-CT around the centrosome. (C) A confocal section of fixed wild-type cells expressing GFP-SvkA-FL shows colocalization with the DNA (upper panel) and, at later stages, around the spindle (lower panel). Bars, 5 ␮m.

A variety of gene disruptions in D. discoideum lead to unclear (Chung and Firtel, 1999; Müller-Taubenberger et al., defects in cytokinesis (Adachi, 2001; Robinson and Spudich, 2002), RasG (Tuxworth et al., 1997) and talin (Niewohner et 2004; Glotzer, 2005) and, according to these data, Uyeda and al., 1997). (2) Cytokinesis B was postulated as ‘attachment- Nagasaki divided cytokinesis into four different types (Uyeda assisted mitotic cleavage’. The best examples are myosin-II- and Nagasaki, 2004): (1) cytokinesis A, the so called ‘purse null cells, which become multinucleated and burst in shaking string method’, where a ring of actin and myosin II contracts suspension but can divide normally on a solid surface (De in the cleavage furrow until the two daughter cells are fully Lozanne and Spudich, 1987; Knecht and Loomis, 1987; separated. Besides myosin II, several other proteins were Neujahr et al., 1997a; Neujahr et al., 1997b). The lack of a reported as important for cytokinesis A – for example clathrin contractile ring in these mutants allows only a passive (Gerald et al., 2001; Niswonger and O’Halloran, 1997), LvsA, furrowing that is driven by opposite polar traction forces. a protein implicated in lysosomal membrane processing (Kwak Proteins involved in this myosin-II-independent form of et al., 1999), possibly PAKa, whose exact function is still cytokinesis are, among others, aggregation minus A (AmiA) 4352 Journal of Cell Science 120 (24)

(Nagasaki et al., 2002), coronin (de Hostos et al., 1993), separation and rather unluckily termed ‘cytokinesis’ as it is cortexillin I and II (Faix et al., 1996) and profilin I and II, based cell-cycle independent. It describes the decreasing volume of on evidence from gene disruption (Haugwitz et al., 1994). (3) a multinucleated cell that tears itself apart, exploiting adhesive Cytokinesis C, as classified by Uyeda and Nagasaki (Uyeda forces and actin-driven cell migration. The resulting decrease and Nagasaki, 2004), is an extension of this traction-mediated in the number of nuclei occurs also in large and multinucleated wild-type cells that have been generated by forced cell fusion (Neujahr et al., 1997b). Thus, this type of cell separation is better described as cytofission. In the svkA-knockout cells, we observed a similar effect when we reduced the osmolarity of the medium and by this way induced cell separation. (4) Uyeda and Nagasaki (Uyeda and Nagasaki, 2004) classified cytokinesis D as a cell-cycle-dependent form of division that needs the help of midwife cells that, attracted by a stimulus, forcefully sever the remaining connection between the two emerging daughter cells (Insall et al., 2001). For Entamoeba invades, it was shown that 30% of division events rely on the activities of midwife cells (Biron et al., 2001). In svkA-null mutants, we also observed this type of cytokinesis, especially in dense cultures (supplementary material Movie 3). However, we stress the point that the remaining strings between daughter cells are the primary defect in cytokinesis D and that the mechanical separation by cells that move across these bridges is a secondary effect. Under these assumptions, the defective cytokinesis in svkA-knockout cells can best be described as defective cytokinesis D because the essential feature of this type of cell division is the lack of final cleavage. The localization of SvkA to the midzone underscores the importance of this kinase for the regulation of this crucial step during cytokinesis.

Materials and Methods

Journal of Cell Science D. discoideum strains, growth conditions, development and mutant analysis D. discoideum strain AX2 (referred to as wild type) and transformants derived from this strain were cultivated in suspension culture with HL-5 medium [1% proteose peptone, 0.5% yeast extract (both Oxoid), 1% glucose, 3.6 mM Na2HPO4, 3.5 mM KH2PO4, pH 7.5] at 21°C, essentially as described previously (Urushihara, 2006). For development, cells were washed in phosphate buffer (14.6 mM KH2PO4, 2 mM 7 Na2HPO4, pH 6.0), resuspended at a density of 1ϫ10 cells/ml, starved for at least 6 hours, and the movement towards a capillary filled with cAMP was observed, essentially as described previously (Mendoza and Firtel, 2006). For development on a solid substratum, growing cells were harvested, washed and transferred onto phosphate agar plates (9 cm) at a density of 1ϫ108 cells per plate (Dormann and Weijer, 2006). To analyze phototaxis of slugs, cell aggregates from the edge of a colony growing into a lawn of Klebsiella aerogenes were placed on water agar plates (9 cm) (Fisher and Annesley, 2006). Slugs were allowed to form and migrate towards light for 2 to 3 days before slugs and slime trails were transferred to nitrocellulose filters and visualized with antibodies against actin.

Fig. 8. Live-cell recordings show SvkA in the midzone. (A) Wild-type cells Gene knockout, rescue and expression of SvkA expressing GFP-SvkA-FL accumulate the kinase only after the appearance of constructs Gene knockouts were performed as described previously (Faix et al., the cleavage furrow. The arrows indicate the localization of SvkA in the 2004), using electroporation and homologous recombination with a midzone during cell separation. (B) A svkA-knockout cell (confocal section of blasticidin S resistance cassette flanked by two homologous fragments of live cell, agar overlay) containing four nuclei (stars) and expressing the CT- the kinase gene. Genomic DNA of blasticidin-resistant clones was tested terminal domain of SvkA. GFP-SvkA-CT determines localization to the by PCR with knockout-specific primers. The blasticidin-resistance midzone but is not sufficient to prevent incomplete cell division (see renewed cassette was removed using the cre-loxP system, as described previously fusion of partially separated cells in frame 11Ј). (C) SvkA-knockout cells (Faix et al., 2004); the knockout mutants did not show any changes in the phenotype after removal of the resistance cassette. Full-length SvkA expressing the dead kinase show that GFP-SvkA-K134A-FL localizes to the (GFP-SvkA-FL, amino acids 1–478) and the C-terminal domain (GFP- late cleavage furrow (arrows). (The stars indicate the approximate localization SvkA-CT, amino acids 278–478) were amplified from genomic DNA. of the nuclei outside the focus plane. Bars, 5 ␮m.) The kinase-dead construct (GFP-SvkA-K134A) was generated by SvkA in Dictyostelium 4353

overlap extension PCR introducing a K134A point mutation. All constructs were Edgar, B. A. (2006). From cell structure to transcription: Hippo forges a new path. Cell cloned into pDGFP-MCS-Neo (Dumontier et al., 2000). The constructs were 124, 267-273. transformed into the knockout and wild-type strains, resulting in expression of N- Eggert, U. S., Mitchison, T. J. and Field, C. M. (2006). Animal cytokinesis: from parts terminal GFP fusion proteins under the control of the actin 15 promoter. list to mechanisms. Annu. Rev. Biochem. 75, 543-566. Eichinger, L., Bahler, M., Dietz, M., Eckerskorn, C. and Schleicher, M. (1998). Fluorescence microscopy Characterization and cloning of a Dictyostelium Ste20-like protein kinase that Cells were fixed with picric acid/formaldehyde as described previously (Hagedorn phosphorylates the actin-binding protein severin. J. Biol. 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(Zeiss, Germany). Live cells expressing GFP-fusion proteins were observed in ␮ Faix, J., Weber, I., Mintert, U., Kohler, J., Lottspeich, F. and Marriott, G. (2001). phosphate buffer with 4% glucose (with 50 g/ml ampicillin/streptomycin) to Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP- achieve an osmolarity similar to that of the media but without the background related proteins. EMBO J. 20, 3705-3715. fluorescence. Agar overlay was performed essentially as described previously Faix, J., Kreppel, L., Shaulsky, G., Schleicher, M. and Kimmel, A. R. (2004). A rapid (Yumura et al., 1984). and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Miscellaneous Res. 32, e143. Immunoprecipitations were performed with polyclonal antibodies against GFP as Fisher, P. R. and Annesley, S. J. (2006). Slug phototaxis, thermotaxis, and spontaneous described previously (Faix et al., 2001). In immunoblots, primary antibodies were turning behavior. Methods Mol. Biol. 346, 137-170. visualized with phosphatase-coupled anti-mouse or anti-rabbit IgG (Dianova, Gagnon, K. B., England, R. and Delpire, E. (2006). Characterization of SPAK and Hamburg, Germany). Polyclonal antibodies against the C-terminal region of severin OSR1, regulatory kinases of the Na-K-2Cl cotransporter. Mol. Cell. Biol. 26, 689-698. kinase were described recently (Eichinger et al., 1998). Monoclonal antibodies Gerald, N. J., Damer, C. K., O’Halloran, T. J. and De Lozanne, A. (2001). Cytokinesis against D. discoideum actin (act1), comitin (190-340-2) (Weiner et al., 1993), failure in clathrin-minus cells is caused by cleavage furrow instability. Cell Motil. cortexillin I (241-438-1) (Faix et al., 1996), coronin (176-3-6) (de Hostos et al., Cytoskeleton 48, 213-223. 1993), DGAP1 (216-394-1) (Faix et al., 2001), GFP (K3-184-2) (Noegel et al., Gerisch, G., Albrecht, R., Heizer, C., Hodgkinson, S. and Maniak, M. (1995). 2004), myosin II (56-396-5) (Pagh and Gerisch, 1986), severin (102-200-1) (André Chemoattractant-controlled accumulation of coronin at the leading edge of et al., 1988) and alpha-tubulin (YL1/2 from Chemicon, Hofheim, Germany) were Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein. used for immunostaining. GFP constructs of alpha-tubulin (Rehberg and Gräf, Curr. Biol. 5, 1280-1285. 2002), coronin (Gerisch et al., 1995), cortexillin I and amino acids 352–444 of Glotzer, M. (2005). The molecular requirements for cytokinesis. Science 307, 1735-1739. cortexillin I (Weber et al., 1999), histone 2B (kindly provided by Günther Gerisch, Goldberg, J. M., Manning, G., Liu, A., Fey, P., Pilcher, K. E., Xu, Y. and Smith, J. L. (2006). The Dictyostelium kinome – analysis of the protein kinases from a simple MPI für Biochemie, Martinsried, Germany) and severin (M.S., unpublished) were . PLoS Genet. 2, e38. transformed into the svkA-null cells. Kinase activity was tested using Guo, C., Tommasi, S., Liu, L., Yee, J. K., Dammann, R. and Pfeifer, G. P. (2007). phosphorylation assays as described previously (Eichinger et al., 1998). RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor- suppressor network. Curr. Biol. 17, 700-705. We thank D. Rieger and M. Borath for excellent technical assistance, Hagedorn, M., Neuhaus, E. M. and Soldati, T. (2006). Optimized fixation and J. Faix (Medizinische Hochschule Hannover) and Annette Müller- immunofluorescence staining methods for Dictyostelium cells. Methods Mol. Biol. 346, Taubenberger for antibodies and DNA constructs, and A. A. Noegel, 327-338. Haugwitz, M., Noegel, A. A., Karakesisoglou, J. and Schleicher, M. (1994). A. Müller-Taubenberger and J. Faix for fruitful discussions. This work Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin was supported by grants from the Elite Network of Bavaria (to P.B.) content, cytokinesis, and development. Cell 79, 303-314. and by grants from the Deutsche Forschungsgemeinschaft (to M.S.). Hergovich, A., Stegert, M. R., Schmitz, D. and Hemmings, B. A. (2006). NDR kinases

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