Research Article
4345
The Ste20-like kinase SvkA of Dictyostelium
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: schleicher@lrz.uni-muenchen.de)
Journal of Cell Science
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 genome of the social amoeba Dictyostelium discoideum
encodes ~285 kinases, which represents ~2.6% of the total
genome and suggests a signaling complexity similar to that
of yeasts and humans. The behavior of D. discoideum as an
amoeba and during development relies heavily on fast
rearrangements of the actin cytoskeleton. Here, we
describe the knockout phenotype of the svkA gene encoding
severin kinase, a homolog of the human MST3, MST4 and
YSK1 kinases. SvkA-knockout cells show drastic defects in
cytokinesis, development and directed slug movement. The
defect in cytokinesis is most prominent, leading to
multinucleated cells sometimes with >30 nuclei. The defect
arises from the frequent inability of svkA-knockout cells to
maintain symmetry during formation of the cleavage
furrow and to sever the last cytosolic connection. We
Introduction
Ste20-like kinases are ubiquitous Ser/Thr kinases subdivided
into two families, the p21-activated kinases (PAKs) numbering
six and the germinal centre kinases (GCKs) numbering 27
representatives in humans (Manning et al., 2002). GCKs
harbor the catalytic domain close to the N-terminus, whereas
the PAK family is characterized by a C-terminal kinase domain
and an N-terminal region that facilitates interaction with Rhorelated GTPases by means of PDB or CRIB domains
(Hofmann et al., 2004). By contrast, the role of the highly
diverse C-terminal region of GCKs is not well understood.
There are, however, indications that the C-terminal domain
regulates activity, localization or intermolecular interactions
(Arasada et al., 2006; Dan et al., 2001).
Kinases from both families are believed to be upstream
regulators of mitogen-activated protein kinase (MAPK)
cascades (Raman and Cobb, 2003). In addition, GCKs play
important roles as activators and repressors of apoptosis
(Hipfner and Cohen, 2004; Huang et al., 2005; McCarty et al.,
2005). Well characterized is the role of Hippo, the Drosophila
melanogaster homolog of human MST2, that plays a
fundamental role during the development of the eye (reviewed
in Edgar, 2006). Further functions for GCKs are the regulation
of ion transport and cell volume (reviewed in Gagnon et al.,
2006; Strange et al., 2006) and the response to various cellular
stresses (Lehtinen et al., 2006; Pombo et al., 1996; Taylor et
al., 1996; Tsutsumi et al., 2000). Several GCKs interact with
cytoskeletal proteins (Eichinger et al., 1998; Mitsopoulos et al.,
2003; Nakano et al., 2003; Storbeck et al., 2004). A thoroughly
studied example for the regulation of cytoskeletal components
demonstrate that GFP-SvkA is enriched at the centrosome
and localizes to the midzone during the final stage of cell
division. This distribution is mediated by the C-terminal
half of the kinase, whereas a rescue of the phenotypic
changes requires the active N-terminal kinase domain as
well. The data suggest that SvkA is part of a regulatory
pathway from the centrosome to the midzone, thus
regulating the completion of cell division.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/24/4345/DC1
Key words: Severin kinase, Ste20 kinases, Cytokinesis, Actin
cytoskeleton, Severin, Dictyostelium, Midbody
is the D. melanogaster kinase Misshapen, and its vertebrate
homolog Nck-interacting kinase. These kinases regulate the
formation of filopodia (Ruan et al., 2002) and the localization
of dynein in flies (Houalla et al., 2005), as well as recruitment
of actin and myosin II during fish epiboly (Köppen et al., 2006)
or cell migration in mammalian embryos (Xue et al., 2001).
They are also involved in the formation of lamellipodia upon
stimulation by growth factors (Baumgartner et al., 2006).
The GCK-III (or YSK) subfamily is one of eight GCK
subfamilies and has three representatives in mammals:
YSK1/SOK1 (Osada et al., 1997; Pombo et al., 1996), MST3
(Schinkmann and Blenis, 1997) and MST4 (Lin et al., 2001;
Qian et al., 2001). The role of these kinases in MAPK signaling
cascades is a matter of debate (Lin et al., 2001; Pombo et al.,
1997; Qian et al., 2001; Schinkmann and Blenis, 1997), but
recent data suggest very clearly a regulatory function for MST4
in the ERK pathway (Ma et al., 2007). YSK1/SOK1 was
demonstrated to influence cell migration by phosphorylating
the dimeric adaptor protein 14-3-3␨ (Preisinger et al., 2004).
In addition, YSK1/SOK1 is involved in organization of the
Golgi and shuttles between cytosol and Golgi, modulated by
an interaction with the Golgi matrix protein GM130 (Preisinger
et al., 2004). An RNAi screen in D. melanogaster showed that
the knockdown of the only fly GCK-III representative
(CG5169) causes changes in the actin cytoskeleton (Kiger et
al., 2003). This correlates well with data from Dictyostelium
discoideum, where one member of the GCK-III subfamily was
purified to near homogeneity and shown to phosphorylate the
gelsolin-like F-actin-fragmenting protein severin (Eichinger et
al., 1998). This severin kinase (SvkA) has an overall identity
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of 50% to the stress-dependent human GCK-III kinase
YSK1/SOK1. Furthermore, the kinase domains are 74%
identical.
The social amoeba D. discoideum is a valuable model
organism for investigating the influence of Ste20-like kinases
in cytoskeletal reactions because it is highly mobile as single
cell and during multicellular development, it is amenable to
molecular genetics, and data on the complete genome are
available (Eichinger et al., 2005). The amoeba contains four
PAK proteins, 13 GCK proteins that represent the subfamilies
GCK-II, -III and -VI (Arasada et al., 2006) and several D.
discoideum-specific GCK subfamilies (Goldberg et al., 2006).
An investigation of the Ste20-like kinase Krs1, homologous to
Hippo and MST2, revealed an inhibitory function of the Cterminal domain. Furthermore, Krs1 is necessary for direct cell
migration during chemotaxis (Arasada et al., 2006; Muramoto
et al., 2007).
Here, we characterize the role of the Ste20-like SvkA protein
during cytokinesis in D. discoideum. The disruption of the gene
impairs cell division and leads to multinucleated cells. In
addition, knockout of svkA affects directed slug movement and
the formation of fruiting bodies, suggesting an important role
for this kinase not only in single cells but also during
development.
Results
SvkA knockout and rescue
To examine the cellular activities affected by SvkA, we
disrupted the svkA gene by homologous recombination (Fig.
1A). The svkA gene disruption of several independent clones
was confirmed by PCR and immunoblots (Fig. 1B,C).
Expression of a full-length (FL) kinase fused to GFP under the
control of the actin 15 promoter was used to rescue the svkAnull phenotype (Fig. 1C,D). Despite the good reaction of the
polyclonal antiserum in immunoblots, its specificity in
immunofluorescence was poor. Therefore, we used mainly
GFP-fusion constructs to study the cellular localization of
SvkA. Partially purified GFP-SvkA-FL phosphorylated the
already described in vitro substrates domains 2 and 3 of severin
(DS211C) or myelin basic protein (MyBP) (Eichinger et al.,
1998), which showed that the fusion to GFP did not abolish
SvkA function (Fig. 1D).
Phenotype of the svkA mutant
The svkA-knockout strain had a slight growth defect in shaking
culture and on lawn of Klebsiella aerogenes. Phagocytosis,
adhesion and random motility were normal. The most dramatic
phenotypes were a developmental and a cytokinesis defect, as
discussed below.
In wild-type cells, SvkA is expressed at about equal levels
throughout the developmental cycle (Fig. 2A). Interestingly,
multinucleated svkA-knockout cells (Fig. 2B) separated into
smaller cells within 30-60 minutes after transfer to low-salt
phosphate buffer and remained connected by cytoplasmic
bridges, sometimes for several hours (Fig. 2C,D,
supplementary material Movie 1). A direct result of this
behaviour was an uncoordinated movement towards a cAMP
source and the appearance of multiple leading fronts
(supplementary material Movie 2). Consequently, the
formation of normal streams and early aggregates was delayed
by several hours (Fig. 3A). The fruiting bodies were smaller
and, in particular, the stalks were broader and distorted.
Expression of a full-length kinase restored the normal
phenotype (Fig. 3B), whereas expression of a kinase-dead
mutant could not rescue the phenotype (data not shown).
Mixtures of svkA-knockout and wild-type cells improved the
morphology of the fruiting body gradually, but it required a
composition of >50% wild-type cells for development into
essentially normal fruiting bodies (data not shown).
A transient step in the developmental cycle is the slug stage
in which the strongly polarized multicellular aggregate moves
in a positively thermotactic and phototactic fashion. The
knockout of svkA produced drastic changes in phototactic
motility, and the defect could be rescued again by expression
of full-length SvkA (Fig. 3C). The overall motility of svkAknockout slugs was reduced, but aggregates moved on a
straight path towards the light source. This indicates that
reception of the light signal in mutant slugs is still intact.
Defects in cytokinesis are most pronounced in the
phenotype of the mutant
The loss of SvkA resulted in cells that were up to ten times
larger than average wild-type cells (Fig. 2B), with sometimes
>30 nuclei and a corresponding number of centrosomes (data
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) Blasticidinresistant 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 2⫻105 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).
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SvkA in Dictyostelium
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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 (2⫻105 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
remained multinucleated (Fig. 4A,B). At higher velocity (220
rpm), the number of large cells (more than four nuclei) was
reduced in favor of mono- and binucleated cells (data not
shown). Apparently, an increased shear force helps to rupture
the thin cytoplasmic bridges between incompletely separated
cells. In cultures grown on solid surfaces until confluence or
on a lawn of K. aerogenes, it was noted that the cells tended
to become smaller again, possibly because so-called ‘midwife
cells’ assisted in cell division (supplementary material Movie
3).
Single-cell analysis of cell division
To analyze cell division at higher resolution, we recorded
phase-contrast time-lapse movies of individual svkA-knockout
cells in HL-5 medium. Frequently, dividing cells followed at
first the usual sequence of events: rounding, spreading,
polarization and formation of a cleavage furrow (Fig. 5A). In
10–20% of the observed cell divisions, formation of the
cleavage furrow was asymmetric. Initially, the cleavage furrow
appeared normal, but then further constriction progressed
asymmetrically, leading to a crescent-shaped intermediate. The
final cytoplasmic bridge did not sever and was sometimes still
visible after more than 1 hour. In many cases, the thin
connection allowed a subsequent fusion of the two pseudodaughter cells, thus accounting for the appearance of cells with
increasing numbers of nuclei. The corrupted formation of the
cleavage furrows in binucleated mutant cells led to the
formation of structures reminiscent of a four-part clover leaf
(Fig. 5B) that either divided into mono- and multinuclear cells
or frequently failed to separate altogether.
To mark key events of cell division, we transformed svkAknockout cells with vectors encoding different GFP constructs
(alpha-tubulin, coronin, cortexillin, histone 2B and myosin II).
None of these constructs altered the aberrant cytokinesis, and
the cells remained large and multinucleated. Furthermore,
overexpression of the homologous Ste20-like kinase Krs1 as a
GFP fusion protein (Arasada et al., 2006) or the in vitro
substrate GFP-severin did not alter the phenotype. The
knockout cells progressed normally through the first steps of
cell division as judged by: (1) the separation of chromosomes
(GFP-histone 2B), (2) the assembly and disassembly of a
spindle (GFP-alpha-tubulin), and (3) the first indications of a
cleavage furrow (GFP-cortexillin I). The only mislocalization
in comparison with normal cell division was observed
occasionally in knockout cells expressing GFP-cortexillin I
(Fig. 5C; supplementary material Movie 4).
Immunostaining of fixed svkA-knockout cells showed that
dividing cells became polarized, as judged by the localization
of F-actin (Fig. 6A,B) and coronin (Fig. 6G). Cortexillin I was
enriched in the midzone of the cleavage furrow (Fig. 6C,E),
myosin II was observed at the polar ends and the midzone of
the cell as expected (Fig. 6D), DGAP1 was found in the late
cleavage furrow (Fig. 6F), and alpha-tubulin was visualized at
the centrosome, the mitotic spindle and in the midzone, also as
expected (Fig. 6A-G).
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4348
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
stages of cytokinesis
Fixed cells expressing GFP-SvkA-FL in the knockout
background showed an overall localization of the kinase in the
cytosol, to a lesser extent also in the nuclei and with a
somewhat diffuse, but clear, enrichment around the centrosome
(Fig. 7A). This centrosomal localization was also observed in
fixed wild-type cells expressing the C-terminal half of the
kinase (GFP-SvkA-CT; Fig. 7B). In dividing cells, GFP-SvkAFL formed a cloud around the spindle and nuclei, without clear
colocalization with the microtubules (Fig. 7C).
In an analysis of live cells undergoing cell division, GFPSvkA-FL was enriched in the cleavage furrow during the late
stage of cytokinesis (Fig. 8A), which is the most affected stage
in the svkA knockout (Fig. 5C). The remnants of a midbodylike structure with its enriched GFP-SvkA-FL were observed
for several minutes at the surface of the two daughter cells (Fig.
8A).
The localization of SvkA is a function of the C-terminal half
of the kinase because a GFP-SvkA-CT construct in the svkAnull background, which could not rescue the aberrant
cytokinesis phenotype, still localized at the centrosome (data
not shown) and, during later phases of cytokinesis, localized
to the cleavage furrow (Fig. 8B). The expression of a kinasedead GFP-SvkA-K134A construct could not rescue the
cytokinesis defect, although it localized to the midzone (Fig.
8C). This highlights the combined importance of proper
localization and SvkA kinase activity for cytokinesis.
Discussion
Disruption of the svkA gene in D. discoideum led to defects in
development, phototaxis and, most prominently, in cytokinesis.
SvkA-null cells became multinucleated on solid surfaces as
well as in shaking cultures. The appearance of multinucleated
cells seemed to be the consequence of incomplete cytokinesis
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.
The observed severe phenotype of the svkA mutant cannot
easily be explained by absent phosphorylation of the F-actinfragmenting protein severin. First, the phosphorylation of
severin that coined the name ‘severin kinase’ is based on in
vitro data only (Eichinger et al., 1998). Second, disruption of
the gene encoding severin resulted in mutants with only subtle
defects (André et al., 1989). Thus it is reasonable to conclude
Journal of Cell Science
frequently failed to sever the final connecting bridge between
the two daughter cells. This led to a frequent re-fusion of
daughter cells, resulting in multinucleated cells. The
enrichment of GFP-tagged SvkA at centrosomes and in the late
cleavage furrow suggests that the kinase regulates the final
stages of cytokinesis. All defects could be rescued by
expression of a full-length GFP-SvkA kinase fusion protein.
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).
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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.
that severin is not the primary target of SvkA in a regulatory
cascade during cell division. This would be in agreement with
data from animal cells, where the later stages of cytokinesis
become insensitive to the actin-depolymerizing drug
latrunculin, implying that the plasma membrane is linked to the
midbody by a connection that does not involve actin dynamics
(Eggert et al., 2006).
This raises a question regarding the identity of other proteins
that could interact with, or become phosphorylated by, SvkA.
In mammalian cells, the Golgi matrix protein GM130 interacts
with the SvkA homolog YSK1 and directs the kinase to the
Golgi (Preisinger et al., 2004). There is no clear homolog of
GM130 in D. discoideum, and the svkA-null cells showed no
disruption of the overall Golgi structure (data not shown).
Thus, it is unlikely that the functions of SvkA are similar to
those of YSK1, at least as far as the organization of the Golgi
is concerned. Furthermore, the only known downstream target
for YSK1 is a 14-3-3␨ protein, and 14-3-3 proteins have been
implicated in regulating cell polarity and cytokinesis (Hurd et
al., 2003; Mishra et al., 2005). Another candidate is a nuclear
Dbf2 related (NDR) kinase, which plays a role in cell-cycle
progression and cell morphology (Hergovich et al., 2006). Also
pertinent is the fact that a mammalian Ste20-like kinase
(MST3), which possesses in its catalytic domain 75% sequence
identity to SvkA, phosphorylates NDR2 at a hydrophobic motif
(Stegert et al., 2005).
The localization of D. discoideum SvkA during the late
stages of cytokinesis fits very well to reports on other Ste20like kinases. In the fission yeast Schizosaccharomyces
pombe, the Ste20-like kinase Nak1/Orb3, a member of the
GCK-III family, is required for cell separation and late stages
of cytokinesis (Leonhard and Nurse, 2005). In addition,
Ustilago maydis mutants that lack the Ste20-like kinase Don3
fail to trigger initiation of a secondary septum necessary for
cell separation after mitosis (Sandrock et al., 2006). The Ras
association domain family 1A (RASSF1A) gene product
binds to human MST2 and forms with additional proteins a
complex that controls apoptosis and exit from mitosis (Guo
et al., 2007). This complex is localized at centrosomes,
spindle poles and the midbody. The distribution of SvkA is
very similar, suggestive of the existence of a similar complex
in D. discoideum. But the mammalian and insect signaling
pathway relies on the formation of a complex by means of a
SARAH (Salvador, RASSF1, Hippo) domain that is not
present in SvkA. In D. discoideum, an explicit SARAH
domain is only present in the Ste20-like kinase Krs1 (Arasada
et al., 2006). It is intriguing, however, that the C-terminus of
SvkA is essential for the distribution to the midzone in
dividing cells. However, the inability of the kinase-dead
construct to rescue the aberrant phenotype clearly shows that
phosphorylation of a currently unknown substrate is an
essential function of SvkA.
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SvkA in Dictyostelium
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
defects in cytokinesis (Adachi, 2001; Robinson and Spudich,
2004; Glotzer, 2005) and, according to these data, Uyeda and
Nagasaki divided cytokinesis into four different types (Uyeda
and Nagasaki, 2004): (1) cytokinesis A, the so called ‘purse
string method’, where a ring of actin and myosin II contracts
in the cleavage furrow until the two daughter cells are fully
separated. Besides myosin II, several other proteins were
reported as important for cytokinesis A – for example clathrin
(Gerald et al., 2001; Niswonger and O’Halloran, 1997), LvsA,
a protein implicated in lysosomal membrane processing (Kwak
et al., 1999), possibly PAKa, whose exact function is still
unclear (Chung and Firtel, 1999; Müller-Taubenberger et al.,
2002), RasG (Tuxworth et al., 1997) and talin (Niewohner et
al., 1997). (2) Cytokinesis B was postulated as ‘attachmentassisted mitotic cleavage’. The best examples are myosin-IInull cells, which become multinucleated and burst in shaking
suspension but can divide normally on a solid surface (De
Lozanne and Spudich, 1987; Knecht and Loomis, 1987;
Neujahr et al., 1997a; Neujahr et al., 1997b). The lack of a
contractile ring in these mutants allows only a passive
furrowing that is driven by opposite polar traction forces.
Proteins involved in this myosin-II-independent form of
cytokinesis are, among others, aggregation minus A (AmiA)
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(Nagasaki et al., 2002), coronin (de Hostos et al., 1993),
cortexillin I and II (Faix et al., 1996) and profilin I and II, based
on evidence from gene disruption (Haugwitz et al., 1994). (3)
Cytokinesis C, as classified by Uyeda and Nagasaki (Uyeda
and Nagasaki, 2004), is an extension of this traction-mediated
separation and rather unluckily termed ‘cytokinesis’ as it is
cell-cycle independent. It describes the decreasing volume of
a multinucleated cell that tears itself apart, exploiting adhesive
forces and actin-driven cell migration. The resulting decrease
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
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
Na2HPO4, pH 6.0), resuspended at a density of 1⫻107 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 1⫻108 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
expressing GFP-SvkA-FL accumulate the kinase only after the appearance of
the cleavage furrow. The arrows indicate the localization of SvkA in the
midzone during cell separation. (B) A svkA-knockout cell (confocal section of
live cell, agar overlay) containing four nuclei (stars) and expressing the CTterminal domain of SvkA. GFP-SvkA-CT determines localization to the
midzone but is not sufficient to prevent incomplete cell division (see renewed
fusion of partially separated cells in frame 11⬘). (C) SvkA-knockout cells
expressing the dead kinase show that GFP-SvkA-K134A-FL localizes to the
late cleavage furrow (arrows). (The stars indicate the approximate localization
of the nuclei outside the focus plane. Bars, 5 ␮m.)
Gene knockout, rescue and expression of SvkA
constructs
Gene knockouts were performed as described previously (Faix et al.,
2004), using electroporation and homologous recombination with a
blasticidin S resistance cassette flanked by two homologous fragments of
the kinase gene. Genomic DNA of blasticidin-resistant clones was tested
by PCR with knockout-specific primers. The blasticidin-resistance
cassette was removed using the cre-loxP system, as described previously
(Faix et al., 2004); the knockout mutants did not show any changes in
the phenotype after removal of the resistance cassette. Full-length SvkA
(GFP-SvkA-FL, amino acids 1–478) and the C-terminal domain (GFPSvkA-CT, amino acids 278–478) were amplified from genomic DNA.
The kinase-dead construct (GFP-SvkA-K134A) was generated by
SvkA in Dictyostelium
overlap extension PCR introducing a K134A point mutation. All constructs were
cloned into pDGFP-MCS-Neo (Dumontier et al., 2000). The constructs were
transformed into the knockout and wild-type strains, resulting in expression of Nterminal GFP fusion proteins under the control of the actin 15 promoter.
Fluorescence microscopy
Cells were fixed with picric acid/formaldehyde as described previously (Hagedorn
et al., 2006), F-actin was detected using Alexa-488- or TRITC-labeled phalloidin
(Invitrogen or Sigma). Nuclei were stained with 4,6-diamidino-2-phenylindole
(DAPI, Sigma) or ToPro-3 (Invitrogen). For immunofluorescence, primary
antibodies were visualized with Alexa-488-conjugated (Invitrogen) or Cy3conjugated (Dianova) goat anti-mouse or goat anti-rat IgG. Cells were analyzed on
an Axiovert 200 (Zeiss, Germany) or, for confocal microscopy, on a LSM 510 Meta
(Zeiss, Germany). Live cells expressing GFP-fusion proteins were observed in
phosphate buffer with 4% glucose (with 50 ␮g/ml ampicillin/streptomycin) to
achieve an osmolarity similar to that of the media but without the background
fluorescence. Agar overlay was performed essentially as described previously
(Yumura et al., 1984).
Journal of Cell Science
Miscellaneous
Immunoprecipitations were performed with polyclonal antibodies against GFP as
described previously (Faix et al., 2001). In immunoblots, primary antibodies were
visualized with phosphatase-coupled anti-mouse or anti-rabbit IgG (Dianova,
Hamburg, Germany). Polyclonal antibodies against the C-terminal region of severin
kinase were described recently (Eichinger et al., 1998). Monoclonal antibodies
against D. discoideum actin (act1), comitin (190-340-2) (Weiner et al., 1993),
cortexillin I (241-438-1) (Faix et al., 1996), coronin (176-3-6) (de Hostos et al.,
1993), DGAP1 (216-394-1) (Faix et al., 2001), GFP (K3-184-2) (Noegel et al.,
2004), myosin II (56-396-5) (Pagh and Gerisch, 1986), severin (102-200-1) (André
et al., 1988) and alpha-tubulin (YL1/2 from Chemicon, Hofheim, Germany) were
used for immunostaining. GFP constructs of alpha-tubulin (Rehberg and Gräf,
2002), coronin (Gerisch et al., 1995), cortexillin I and amino acids 352–444 of
cortexillin I (Weber et al., 1999), histone 2B (kindly provided by Günther Gerisch,
MPI für Biochemie, Martinsried, Germany) and severin (M.S., unpublished) were
transformed into the svkA-null cells. Kinase activity was tested using
phosphorylation assays as described previously (Eichinger et al., 1998).
We thank D. Rieger and M. Borath for excellent technical assistance,
J. Faix (Medizinische Hochschule Hannover) and Annette MüllerTaubenberger for antibodies and DNA constructs, and A. A. Noegel,
A. Müller-Taubenberger and J. Faix for fruitful discussions. This work
was supported by grants from the Elite Network of Bavaria (to P.B.)
and by grants from the Deutsche Forschungsgemeinschaft (to M.S.).
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