gas2l3, a novel target gene of the dream complex, is required for
TRANSCRIPT
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GAS2L3, A NOVEL TARGET GENE OF THE DREAM COMPLEX, IS REQUIRED
FOR PROPER CYTOKINESIS AND GENOMIC STABILITY
Patrick Wolter1,4, Kathrin Schmitt1,4, Marc Fackler1,4, Heidi Kremling1, Leona Probst1,
Stefanie Hauser1, Oliver J. Gruss2 and Stefan Gaubatz1,3
1Biozentrum, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
and
2Zentrum für Molekulare Biologie der Universität Heidelberg, (ZMBH), DKFZ-ZMBH
Alliance , Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
3Correspondence to:
Stefan Gaubatz
Phone (+49) 931-31-84138
e-mail:[email protected]
4These authors contributed equally to this work
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SUMMARY
The mammalian DREAM complex is key regulator of cell cycle regulated gene transcription
and drives the expression of many gene products required for mitosis and cytokinesis. In this
study we characterized a novel target gene of DREAM, GAS2L3, which belongs to the GAS2
family of proteins with putative actin and microtubule binding domains. We found that
GAS2L3 localizes to the spindle midzone and the midbody during anaphase and cytokinesis,
respectively. Biochemical studies show that GAS2L3 binds to and bundles microtubules as
well as F-actin in vitro. Strikingly, the RNAi-mediated knock-down of GAS2L3 results in
chromosome segregation defects, in multinucleated cells and cells with multi-lobed nuclei.
Likewise, chronic downregulation of GAS2L3 causes chromosome loss and aneuploidy.
Time-lapse video microscopy experiments in GAS2L3 knock-down cells reveal abnormal
oscillation of chromatin and the spindle during cytokinesis. Taken together, our data reveal
novel, important roles of GAS2L3 for faithful cell division. Our work thus contributes to the
understanding of how DREAM regulates cytokinesis.
INTRODUCTION
Correct progression through the cell cycle is essential for normal development and
differentiation. Its deregulation is associated with the loss of genomic integrity and can
contribute to tumorigenesis. E2F transcription factors regulate the expression of a large
number of genes whose products play key roles in cell cycle progression, synthesis of
nucleotides, DNA replication and apoptosis (Dimova and Dyson, 2005; van den Heuvel and
Dyson, 2008). In the G1 phase of the cell cycle, E2F activity is regulated by the binding to
pRB, the product of the retinoblastoma tumor suppressor gene, and by binding to two related
"pocket proteins", p107 and p130 (Burkhart and Sage, 2008; Cobrinik, 2005). Free,
uncomplexed E2F proteins function as transcriptional activators with growth promoting
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activities. In contrast, complexes between E2F and pocket proteins act as active
transcriptional repressors and growth inhibitors (Trimarchi and Lees, 2002). Importantly, it is
believed that the deregulation of the pRB/ E2F pathway is involved in the pathogenesis of
almost all human tumors (Burkhart and Sage, 2008).
DREAM or LINC is a recently identified E2F-pocket protein complex in mammalian cells
that undergoes a cell cycle-dependent switch of subunits (Litovchick et al., 2007; Schmit et al.,
2007; Pilkinton et al., 2007a). DREAM consists of the core subunits LIN9, LIN37, LIN52,
LIN54 and RbAp48. In quiescent cells, the core complex is associated with p130 and E2F4
and contributes to the repression of E2F-regulated genes. In late S phase, the interaction of
DREAM with p130/E2F4 is lost and DREAM now binds to the B-MYB transcription factor.
Genome wide expression studies have shown that DREAM-B-MYB is required for activation
of a cluster of genes required for entry into mitosis, spindle assembly and cytokinesis
(Osterloh et al., 2007; Knight et al., 2009; Reichert et al., 2010; Schmit et al., 2009; Pilkinton
et al., 2007b). Chromatin immunoprecipitation experiments demonstrated that many of these
genes, such as Plk1, Cyclin B1 and Kif20a, are direct target genes of DREAM (Osterloh et al.,
2007; Schmit et al., 2009).
One of the most prominent phenotype after deletion of the LIN9 subunit of DREAM in
mouse embryonic fibroblasts is cytokinesis failure, resulting in binuclear cells (Reichert et al.,
2010). Similarly, in HeLa cells, RNAi mediated silencing of LIN54, another core-subunit of
DREAM, also causes cytokinesis defects (Kittler et al., 2007). Cytokinesis is the final stage of
cell division after duplication and segregation of the genetic material (Eggert et al., 2006). It
is highly regulated to avoid unequal chromosome segregation, which can result in aneuploidy
and tumor formation (Sagona and Stenmark, 2010). During cytokinesis a contractile
actomyosin ring assembles at the cell equator. Constriction of the cell membrane by the
contractile ring results in formation of the cleavage furrow. Subsequent membrane fusion
physically separates the cell into two daughter cells. Formation of the contractile ring is
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controlled by the RhoA GTPase (Narumiya and Yasuda, 2006; Piekny et al., 2005). The
centralspindlin complex, a component of the central spindle that is formed by microtubule
bundles between the segregating chromosomes, recruits the Rho guanine nucleotide exchange
factor (GEF) Ect2 to activate RhoA at the overlying equatorial cortex, which in turn activates
the contractile ring and leads to cleavage furrow ingression. Several mitotic kinases such as
CDKs, PLK1 and Aurora B also regulate RhoA activation and cytokinesis. In addition
scaffolding proteins such as anillin and septins play important roles in stabilizing the cleavage
furrow (Hickson and O'Farrell, 2008; Piekny and Maddox, 2010). Although a large number of
proteins involved in cytokinesis have been identified in RNAi screens and through proteomic
studies, the molecular requirements for positioning and stabilization of the contractile ring are
not fully understood.
In this study we characterized GAS2L3, a novel target gene of DREAM and a member of the
GAS2 protein family. We find that GAS2L3, unlike other members of the GAS2 family is
expressed in mitosis and that it localizes the midbody during cytokinesis. Biochemical studies
indicate that GAS2L3 can bind to and crosslink microtubule and actin filaments. Using RNAi
we find that loss of GAS2L3 leads to abnormal oscillation of chromatin and the spindle
during cytokinesis. Finally, we demonstrate that RNAi-mediated depletion of GAS2L3 leads
to genomic instability. Thus we have identified GAS2L3 as an important target gene of the
DREAM complex that is required for proper cytokinesis.
RESULTS
GAS2L3 is a novel DREAM target gene that is highly expressed in G2 and mitosis
Transcriptional profiling of conditional LIN9 knockout MEFs using Agilent DNA
microarrays identified GAS2L3 as a novel target gene of the mammalian DREAM complex
(Reichert et al., 2010). To verify that GAS2L3 is regulated by DREAM, we depleted LIN9
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and LIN54 in HeLa cells by RNAi and analyzed expression of GAS2L3 by RT-qPCR.
Expression of GAS2L3 was significantly reduced in LIN9 and LIN54 depleted cells,
confirming the microarray data (Figure 1A and B). GAS2L3 expression was also dependent
on LIN9 in untransformed human BJ fibroblasts (Figure 1C). Taken together these
observations indicate that GAS2L3 is expressed in a DREAM dependent manner.
GAS2L3 is an uncharacterized member of the GAS2 protein family that also consists of
GAS2 and the related GAS2L1 and GAS2L2 proteins (Figure 1D). GAS2 proteins contain an
actin-binding CH domain and a GAR domain that mediates binding of GAS2 to tubulin
(Brancolini et al., 1992; Goriounov et al., 2003; Schneider et al., 1988; Zucman-Rossi et al.,
1996).While GAS2 was identified in a screen for genes induced by growth arrest (Brancolini
et al., 1992; Schneider et al., 1988) , little is known about the other family members. Because
many DREAM-regulated genes are expressed in G2/M, we determined the expression of
GAS2L3 during the cell cycle and compared it with the expression of the other family
members. To do so, we used human T98G glioblastoma cells, which were made quiescent by
serum starvation, and, after serum re-stimulation progress synchronously through G1, S and
G2/M (Supplementary Figure S1). GAS2 mRNA expression was low in quiescent cells and
peaked at 6 hours after serum stimulation when cells were still in G1 (Figure 1E). Expression
of GAS2L1 did not significantly change during the cell cycle and expression of GAS2L2
could not be detected in T98G cells (Figure 1E and data not shown). In striking contrast,
GAS2L3 expression was low in quiescent cells but increased after serum-stimulation (Figure
1E). Peak GAS2L3 mRNA levels were detected between 24 and 29 hours after serum
addition when cells entered G2/M. Thus, GAS2 family members are differently expressed
during the cell cycle and GAS2L3 mRNA levels are highest in G2 and mitosis.
To investigate whether protein levels are also regulated during the cell cycle, we established
HeLa cells stably expressing a tetracycline-inducible, HA- and streptavidin binding peptide-
tagged GAS2L3. Upon tetracycline induction, some GAS2L3 protein could be detected in
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asynchronous cells, but GAS2L3 protein levels were much higher in cells that were arrested
in mitosis by nocodazole (Figure 1F). Upon exit from mitosis GAS2L3 protein levels
decreased similar to cyclin B1. We also noted a change in GAS2L3 mobility upon
nocodazole treatment, indicating that GAS2L3 is modified in mitosis, possibly by
phosphorylation. Endogenous GAS2L3 protein could only be detected in lysates of HeLa
cells in G2 and M phase, suggesting that GAS2L3 is degraded after mitosis (Figure 1G).
Localization of GAS2L3 to the mitotic spindle and to the midbody
The subcellular localization of transiently expressed EGFP-GAS2L3 in HeLa cells was
determined by fluorescence microscopy. In interphase cells, EGFP-GAS2L3 was cytoplasmic
and localized to microtubules (Figure 2A). In mitosis EGFP-GAS2L3 was enriched at the
mitotic spindle in prophase and anaphase. In telophase EGFP-GAS2L3 was visible in the
spindle midzone. In cytokinesis EGFP-GAS2L3 was exclusively detected at the midbody, a
dense microtubule-rich region that forms before abscission at the midpoint of the intercellular
bridge. GAS2L3 colocalized with α-tubulin, but not with actin at the midbody (Figure 2B).
Midbody localization was confirmed by co-localization of EGFP-GAS2L3 with Aurora B, a
subunit of the chromosomal passenger complex, which is known to localize to the midbody
(Figure 2C). Confocal fluorescence microscopy of HA-tagged mouse GAS2L3 revealed that
GAS2L3 flanks the midbody on both sides but is excluded from the central region of the
midbody (Figure 2D). Live cell imaging of EGFP-GAS2L3 in HeLa cells stably expressing
mCherry-α-tubulin confirmed the localization of GAS2L3 observed in fixed cells, indicating
that localization of GAS2L3 is not an artifact of fixation (Figure 2E; Movie 1; Supplementary
Figure S2).
The localization of endogenous GAS2L3 in relationship to mCherry-α-tubulin and MKLP1, a
subunit of centralspindlin (Matuliene and Kuriyama, 2002; Mishima et al., 2002), was
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determined by staining with a monoclonal antibody directed at GAS2L3 (Figure 2F,
Supplementary Figures S3). Endogenous GAS2L3 localized to the spindle midzone and
midbodies in cytokinesis in HeLa and U2OS cells. In some cells also GAS2L3 localized to
the midbody-ring that forms as a remnant of the contractile ring around the midbody
microtubules during cytokinesis (Supplementary Figure S4) (Gromley et al., 2005; Pohl and
Jentsch, 2008). Furthermore, in some interphase cells, GAS2L3 was detected at midbody
remnants in ring-like structures (Supplementary Figure S4). Co-staining with α-tubulin
showed that these ring-like structures are rich in microtubules. Although spindle localization
of endogenous GAS2L3 was not observed, this could be due to the difficulty in detecting low
amounts of the protein with the available reagents.
Domains required for localization of GAS2L3 In order to investigate the role of the CH and GAR domains in subcellular localization of the
protein, we generated a set of GAS2L3 deletion constructs (Figure 3A). Deletion constructs
were transiently expressed as EGFP-fusion proteins in HeLa cells and their localization in
interphase, mitosis and cytokinesis was determined (Figure 3B).
We found that mutants containing only the CH domain (mutant A) or the CH and the GAR
domain (mutant B) or the GAR domain (mutant C) failed to colocalize with interphase
microtubule, mitotic spindles or spindle midzones. Instead, as evidenced by Phalloidin co-
staining, mutants with the CH domain prominently induced and colocalized with actin stress
fibers (Supplementary Figure S5), suggesting that the CH domain can indeed function as an
actin-interacting domain. Mutants that contain the C-terminus but lack the CH domain
(mutant D) or the CH and the GAR domain (mutant E) colocalized with microtubules in
interphase and to metaphase spindles, similar to full-length EGFP-GAS2L3 and consistent
with a recent study by Stroud et al. who showed that the C-terminus of GAS2L3 localizes to
microtubules in interphase. These mutants also still localized to the spindle midzone in
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cytokinesis, however they were not as sharply concentrated at the midbody as full length
GAS2L3. Mutant F, which only contains part of the C-terminus, weakly associated with
mitotic spindles and with the central spindle but showed no colocalization with microtubules
in interphase. The smallest mutant (mutant G) showed no overlap with microtubules.
Taken together, these results indicate that the C-terminus of GAS2L3 is sufficient for
microtubule colocalization and localization to the spindle midzone while the CH and GAR
domains are required for efficient midbody localization of GAS2L3.
GAS2L3 stabilizes and bundles microtubule
EGFP-GAS2L3 appeared to bundle microtubules into rings around the nucleus indicative of
hyperstable, buckling microtubules (Figure 4A). A similar rearrangement of microtubules into
perinuclear rings has been observed before, after overexpression of microtubule-associated
proteins such as PRC1 and Lis1 (Mollinari et al., 2002; Smith et al., 2000). To further confirm
that GAS2L3 can stabilize microtubules in intact cells, we expressed EGFP-GAS2L3 in HeLa
cells and treated them with low doses of nocodazole to depolymerize microtubules (Figure
4B). Strikingly, in cells that express high levels of EGFP-GAS2L3, microbtubules were
resistant to depolymerization, indicating that GAS2L3 can stabilize microtubules in vivo.
Next, to investigate whether GAS2L3 has microtubule-bundling activity, taxol-stabilized
microtubules were incubated in vitro with GST or GST-GAS2L3 and investigated by
microscopy. In the presence of only GST, microtubules were short and unbundled, as
expected (Figure 4C). In stark contrast, GST-GAS2L3 induced a high degree of microtubule-
bundling.
GAS2L3 is a microtubule- and actin-associated protein
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We next determined the domains of GAS2L3 that mediate direct binding to microtubules. To
do so, we performed co-sedimentation assays with in vitro polymerized microtubules and
purified recombinant GST-GAS2L3 wildtype or deletion mutants. GST-GAS2L3, but not
GST alone, co-sedimented with microtubules indicating that GAS2L3 can directly associate
with microtubules (Figure 5A). While a N-terminal construct containing the CH and GAR
domain (mutant B) did not bind to microtubules, the C-terminus (mutant E) was sufficient for
this binding, consistent with the localization data and with a previous study (Stroud et al.,
2011). The C-terminal domain of GAS2L3 contains several clusters of positively charged
amino acids that could mediate binding to the negatively charged microtubules. Consistent
with this observation, two non-overlapping constructs which both contain basic clusters
(mutant F and mutant H) independently associated with microtubules. In contrast, a smaller
construct (mutant G) that contains fewer basic residues only very weakly associated with
microtubules. Therefore, whereas the GAR domain appears not to function as microtubule
binding domain in this protein, GAS2L3 appears to have multiple C-terminal microtubules
binding domains.
Since GAS2L3 also has a putative actin-binding CH domain, we performed F-actin co-
sedimentation assays. GST alone did not co-sediment with actin. In the absence of actin,
GST-GAS2L3 also remained in the supernatant (Figure 5B). However, when GST-GAS2L3
was mixed with actin, it was found in the pellet fraction, indicating that it can directly bind to
F-actin. As expected the N-terminus of GAS2L3 containing the CH domain (mutant B) was
sufficient for binding to F-actin. Surprisingly, the C-terminus lacking the CH domain (mutant
E) efficiently associated with actin as did the two non-overlapping C-terminal parts tested
(mutants F and H). Only the small C-terminal mutant G associated with F-actin with reduced
affinity. We conclude that GAS2L3 has several actin-binding domains - the CH domain and
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at least two different domains in the C-terminus. The C-terminal F-actin binding sites cannot
be clearly separated from the microtubule binding domains.
Since our data indicate that GAS2L3 has multiple microtubule and F-actin binding domains,
we next asked whether GAS2L3 can simultaneously bind to both microtubules and F-actin.
This possibility was addressed by low-speed centrifugation. When microtubules and F-actin
were centrifuged together, actin remained in the supernatant after low-speed centrifugation
(Figure 5C). When GAS2L3 was mixed with actin alone, a small fraction of F-actin was
found in the pellet, suggesting that GAS2L3 has some actin-bundling activity. Importantly,
when wildtype GAS2L3, microtubules and F-actin were mixed, actin bundles efficiently co-
sedimented along with microtubules, indicating that GAS2L3 can cross-link microtubules and
F-actin in vitro (Figure 5C, Supplementary Figure S8). Because the N-terminus of GAS2L3
(mutant B) does not bind to microtubules, it was unable to crosslink F-actin and microtubules,
although it appeared to have similar F-actin bundling activity as full length GAS2L3.
Interestingly, however, the C-terminus of GAS2L3 (mutant E) was sufficient to crosslink
microtubules and F-actin, consistent with the observation that it contains multiple F-actin and
microtubule binding-domains.
Taken together our data indicate that GAS2L3 binds to and bundles F-actin and microtubules.
GAS2L3 is required for the completion of cytokinesis
The expression and localization of GAS2L3 suggests a role in mitosis or cytokinesis. To
assess the in vivo role of GAS2L3, we depleted GAS2L3 by RNA interference (RNAi) using
three different siRNAs. GAS2L3 mRNA levels were strongly reduced by all three siRNAs as
determined by RT-qPCR (Figure 6A). We confirmed depletion of GAS2L3 to undetectable
levels by immunofluorescence on a single cell basis (Figure 6B). All three siRNAs caused a
significant increase in multinucleated cells and cells with multilobed nuclei (Figure 6C,D),
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suggesting defects in chromosome segregation and/or cytokinesis. The majority of abnormal
nuclei in GAS2L3 depleted cells were multilobed and a small fraction of about 5% of nuclei
were binuclear. To confirm that depletion of GAS2L3 caused the multilobed phenotype, we
performed rescue experiments with a siRNA-resistant GAS2L3 construct that was created by
introducing silent mutations into the recognition sequence for siRNA#2. GAS2L3 siRNA#2
reduced expression of wildtype EGFP-GAS2L3 to undetectable levels while the EGFP-signal
of the mutated construct was not reduced, confirming the resistance (Supplementary Figure
S10). HeLa cells stably expressing RNAi-resistant GAS2L3 were cultured in presence or
absence of tetracycline and then transfected with either control siRNA or GAS2L3 specific
siRNA#2. In the presence of tetracycline all three proteins were expressed (Figure 6E).
Expression of the RNAi-resistant GAS2L3 partially rescued the multilobed phenotype,
indicating that the phenotype is indeed due to depletion of GAS2L3 and is not an off-target
effect (Figure 6F). Neither the N-terminus (mutant B) nor the C-terminus (mutant E) of
GAS2L3 were able to rescue the multilobed phenotype. We conclude that both the N-
terminus and the C-terminal domains of GAS2L3 are crucial for proper function of GAS2L3.
To investigate the role of GAS2L3 in mitosis and cytokinesis in more detail, we performed
time-lapse video microscopy in HeLa cells engineered to stably express H2B-EGFP and α-
tubulin-mRFP as markers for chromosomes and microtubules, respectively. Cells transfected
with a control siRNA progressed normally through mitosis and cytokinesis, as expected
(Figure 7A, Supplementary Movie 2). Cells depleted of endogenous GAS2L3 with either
siRNA#1 or siRNA#2 formed a normal mitotic spindle and progressed normally through
metaphase and anaphase (Figure 7B,C; Supplementary Movies 3 and 4). In telophase,
however, GAS2L3-depleted cells exhibited a spindle rocking phenotype similar to the
phenotype that has for example been reported for cells depleted of anillin or containing
hyperstabilized astral microtubules (Rankin and Wordeman, 2010; Zhao and Fang, 2005).
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GAS2L3 depleted cells displayed an oscillation of the entire spindle and the chromatin
between the proto-daughter cells. Oscillations started just at the beginning of cytokinesis,
approximately 8 minutes after anaphase onset. In oscillating cells the chromatin and the
spindle moved back and forth several times over a period of approximately 20 minutes before
the separated sets of chromatin fell back into a single mass. The oscillation phenotype was
observed independently with siRNA#1 in 45 of 214 cells (17.4%) and with siRNA#2 in 24 of
143 cells (16.8%) but only in 1 out of 93 control-depleted cells (1.1%). In addition,
substantive membrane blebbing was observed during anaphase in approximately 20% of
GAS2L3 depleted cells while less than 2% of control cells showed this phenotype (see Movie
3 and 4 and Figure 7D). In cells with large blebs, RhoA localization was not restricted to the
equator but was detected at the blebs (Figure 7D). Myosin IIA also accumulated at the cortex
in GAS2L3 depleted cells. Thus, GAS2L3 is required to restrict RhoA and Myosin II to the
cleavage furrow.
GAS2L3 protects cells from aneuploidy
Because of the role of GAS2L3 in cytokinesis, we next asked whether GAS2L3 is required to
maintain long-term genomic stability. To test this possibility, we stably expressed a GAS2L3
specific shRNA in immortalized human BJ fibroblasts. After 26 generations in culture, we
prepared metaphase spreads of GAS2L3 depleted cells and control cells and determined
chromosome numbers (Figure 8A,B). About 20% of metaphases from GAS2L3 depleted BJ-
cells showed polyploidy or aneuploidy whereas control cells had normal or nearly normal
karyotypes. We next investigated chromosomally stable HCT116 cells, a near-diploid colon
cancer cell line (Lengauer et al., 1997). Single cell clones of cells derived from cells stably
expressing a GAS2L3-specific shRNA or a control shRNA were isolated and passaged for 5
generations. Chromosome numbers were quantified by FISH using chromosome-specific
satellite enumeration probes in interphase nuclei. Control transfected cells were
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chromosomally stable, as judged by a small fraction of about 3% of cells with deviation from
the modal value (Figure 8C,D). In contrast, 8% - 9% of GAS2L3 shRNA expressing cells
exhibited gains and losses of chromosome 7 and 8. These data indicate that depletion of
GAS2L3 is sufficient to induce low levels of aneuploidy. We next asked whether a reduction
in GAS2L3 enhances the effect of MAD2 heterozygosity, which is known to result in
genomic instability because of spindle checkpoint defects (Michel et al., 2001). Stable clones
of HCT116 MAD2+/- cells transfected with a control shRNA or a GAS2L3 specific shRNA
were analyzed by FISH. HCT116 MAD2+/- cells showed higher levels of aneuploidy
compared to HCT116 wildtype cells, as expected. 12% to 13% of GAS2L3 depleted HCT116
MAD2+/- showed aneuploidy for chromosome 7 and 8 (Figure 8D). Thus aneuploidy is
further enhanced by combined inhibition of MAD2 and GAS2L3.
DISCUSSION
In this study we investigated GAS2L3, a novel target gene of the DREAM complex. GAS2L3
belongs to the GAS2 proteins, which are characterized by an actin-binding CH domain and a
GAS2-related (GAR) domain. Previous studies have shown roles for GAS2 proteins in
interphase cells (Brancolini et al., 1992; Goriounov et al., 2003; Stroud et al., 2011; Zucman-
Rossi et al., 1996). We now report that unlike the other members of the GAS2 family,
GAS2L3 is expressed in M-phase, localizes to spindles, the spindle midzone and the midbody,
and is required for cytokinesis.
GAS2L3 is cell cycle regulated at several levels. First, GAS2L3 mRNA levels are low during
G1, increase in S-phase and peak in G2 and mitosis. Transcriptional activation of GAS2L3 is
dependent on the DREAM complex, a master regulator of mitotic genes. Secondly, GAS2L3
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is also regulated at the protein level. GAS2L3 protein levels decrease when cells exit from
mitosis. The degradation of many unstable proteins at the end of mitosis is mediated by the
anaphase-promoting complex (APC). Interestingly, GAS2L3 contains a consensus D-box, a
known recognition sequence for the APC. Future mutational analysis will have to reveal
whether degradation of GAS2L3 depends on this D-box motif.
We performed co-sedimentation assays to analyze the interaction of GAS2L3 with F-actin
and microtubules in vitro. Similar to a recent study, we find that the C-terminus but not the
GAR domain mediates the interaction with microtubules (Stroud et al., 2011). Further
experiments revealed that there are at least two non-overlapping microtubule-binding domains
in the C-terminus. In addition, GAS2L3 has several actin-binding domains - the N-terminal
CH domain and at least two different domains in the C-terminus that cannot clearly separated
from the microtubule-binding sites. The unstructured C-terminal domain of GAS2L3 contains
several clusters of positively charged amino acids that could mediate binding to the negatively
charged microtubules and actin. GAS2L3 not only binds to microtubule but also has
microtubule bundling activity in vitro and, when overexpressed it can protect microtubule
from nocodazole-induced depolymerisation. Bundling activity of GAS2L3 can be mediated
by a single molecule since we found at least two non-overlapping microtubule binding sites in
the C-terminus. Our analysis also revealed that GAS2L3 can simultaneously bind to F-actin
and microtubule, suggesting that may function to coordinate the actin and microtubule
cytoskeletons.
The ability of GAS2L3 to localize to the spindle and spindle midzone correlates well its
ability to bind to microtubules in vitro (Figure 3). Even a small C-terminal fragment with only
one of the two microtubule binding domains displays some overlap with the spindle and
spindle midzone. However, only the full length GAS2L3 shows the same midbody
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localization as endogenous GAS2L3 indicating that the N-terminal CH and GAR domains in
addition to the C-terminal actin- and microtubule- binding domains are required to localize
GAS2L3 to the midbody. This might be due to the additional actin binding activity of the CH
domain. Alternatively, it is possible that these domains mediate interaction with other proteins
that contribute to the localization of GAS2L3 to the midbody.
So far, no loss-of function studies of mammalian GAS2 proteins have been reported. We
found that depletion of GAS2L3 by RNAi results in an increase in multinuclear cells and cells
with multi-lobed nuclei, indicating defects in cytokinesis, consistent with its expression and
localization during the cell cycle. Time-lapse video microscopy revealed that multinucleation
was not due to a failure to ingress the furrow. Instead, it was caused by abnormal contractions
and oscillations that started at the beginning of cytokinesis. During these oscillations the
chromatin and the spindle were moved several times back and forth between the future
daughter cells. Furrows eventually collapsed and cells became multinucleated and multilobed.
We also observed increased membrane blebbing after depletion of GAS2L3 and found
abnormal localization of Myosin IIA and RhoA at the polar cortex. Interestingly, this
phenotype of GAS2L3 depleted cells is very similar to the oscillating phenotype that has been
reported after deletion of anillin, the formin mDia2, the kinesin MCAK or the motor protein
HSET that is required for spindle midzone organization (Cai et al., 2010; Piekny and Glotzer,
2008; Straight et al., 2005; Watanabe et al., 2008; Zhao and Fang, 2005). MCAK depletion
leads to an increase in the length of astral microtubules that invade and increase the size of
membrane blebs (Rankin and Wordeman, 2010). Oscillations are then triggered by Rho-
dependent myosin contractions. Anillin or mDia2 depletion induce oscillations because these
proteins act as scaffolds that stabilize the position of the contractile ring during cytokinesis
(Straight et al., 2005; Watanabe et al., 2008). Given the similar phenotypes, it is likely that
GAS2L3 is also involved in regulating components of the contractile ring. Because GAS2L3
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can simultaneously bind to microtubules and actin filaments in vitro, GAS2L3 could function
as a scaffolding protein in the cleavage furrow to stabilize interactions between midbody
microtubules and the actomyosin-ring. Consistent with such a function, RhoA and Myosin
were not restricted to the cleavage furrow after GAS2L3 depletion but also aberrantly
localized at the sites of large membrane blebs, where it may induce abnormal cortical
oscillation. Although these observations support a role for GAS2L3 in stabilization of
components of the contractile ring, we cannot exclude the possibility that other mechanisms,
such as changes in astral microtubules contribute to the observed phenotype, although there is
no evidence that GAS2L3 can localize to astral microtubules. To elucidate the precise
function of GAS2L3 in cytokinesis further studies such as the identification of interacting
proteins will be needed.
In this study we also found that the long-term depletion of GAS2L3 resulted in aneuploidy in
human BJ fibroblasts and HCT116 cells, indicating that GAS2L3 is required to maintain
genomic stability (Figure 8). Given that genomic instability is a hallmark of most cancers
(Holland and Cleveland, 2009), the deregulation of GAS2L3 could therefore contribute to
tumorigenesis. Interestingly, a search of the Oncomine database revealed significant
deregulation of GAS2L3 mRNA in certain tumor types. For example, an upregulation of
GAS2L3 in glioblastoma has been reported in two recent studies (Sun et al., 2006; Bredel et
al., 2005). Conversely, GAS2L3 is significantly downregulated in childhood T-cell ALL
(Andersson et al., 2007). In the future it will be interesting to investigate whether GAS2L3
plays a role in human cancer.
In conclusion, our data establish GAS2L3 as a novel target gene of the DREAM complex that
is required for proper cytokinesis and for maintenance of genomic stability. Downregulation
of GAS2L3 likely contributes to the cytokinesis defects after inactivation of DREAM.
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MATERIALS AND METHODS
Tissue culture
All cells were cultured in DMEM (Invitrogen, Darmstadt, Germany) supplemented with 10%
FCS (Invitrogen, Darmstadt, Germany). HeLa cells stably expressing H2B-EGFP and α-
tubulin-RFP were a kind gift of Patrick Meraldi (Toso et al., 2009). HCT116 MAD2+/- cells
were a kind gift of Robert Benezra. (Michel et al., 2001). HeLa FlpIn-TRex cells were a kind
gift from Stephen Taylor (Tighe et al., 2008). HeLa cells stably expressing mCherry-
α−tubulin were generated by stable transfection with the plasmid pmCherry-α-tubulin-
IRESpuro2 (Steigemann et al., 2009).
Plasmids, siRNA and transfections
Human GAS2L3 cDNA was obtained by RT-PCR from HeLa mRNA and inserted into
pCDNA3-EGFP and pGEX-4T-2 expression vectors. Mouse GAS2L3 cDNA was obtained by
RT-PCR from NIH-3T3 cells and inserted into pCDNA3-HA. To generate stable HeLa cells
expressing double-tagged GAS2L3, the GAS2L3 cDNA was inserted into
pcDNA5/FRT/TO/nHASt-TAP encoding for a N-terminal HA-tag and a streptavidin-binding
peptide (Wyler et al., 2011). The plasmid (0.1 µg) was cotransfected together with the flp-
recombinase expression vector pOG44 (0.9 µg) into HeLa Flp-In TRex cells (Tighe et al.,
2008). Cells were selected for two weeks with 100 mg/ml hygromycin.
The following GAS2L3-specific siRNAs were used:
siRNA#1: 5' GGGAUACUCUUCAAGGAUU 3',
siRNA#2: 5' CUAUGUCAGUCCGUUCUAA 3',
siRNA#3: 5' CAUUAAAUCCAGUAGGUAA 3'.
siRNAs were purchased from MWG-Biotech (Ebersberg, Germany) and were transfected
with Lipofectamine 2000 (Invitrogen, Darmstadt, Germany) or Metafectene Pro (Biontex,
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Martinsried) according to the manufacturers protocol. A retroviral GAS2L3 shRNA construct
was generated by insertion of an shRNA corresponding to the siRNA#2 into the retroviral
vector pMSCV. Retroviral infections with shRNA viruses were performed as described
(Gagrica et al., 2004). A siRNA-resistant GAS2L3 was generated by introducing silent point
mutations that impairs siRNA-mediated knockdown. Using site-directed mutagenesis,
nucleotides 1337-1339 of GAS2L3 encoding for serine 371 were changed from TCT to AGC.
The mutated cDNA was inserted into pcDNA5/FRT/TO/nHASt-TAP and stable HeLa cells
using the HeLa Flp-In TRex cells were generated as described above.
Antibodies
The following primary antibodies were used: GAS2L3 (Abnova, 1D4 and 1C8), α-tubulin
(Sigma, T6074), Actin (Santa Cruz, sc-47778), Aurora B (Abcam, ab2254), Cyclin B (Santa
Cruz, sc-245), HA (Covance, HA.11), MKLP1 (Santa Cruz, sc-867), phospho-Histone H3
(Upstate, 06-570), RhoA (Santa Cruz, sc-418), Myosin IIA (Sigma, M8064).
Immunofluorescence
Cells grown on coverslips were either fixed for 4 minutes at -20°C with methanol or for 10
min at room temperature with PFA [PBS, 3% paraformaldehyde, 2 % sucrose]. PFA-fixed
cells were permeabilized for 5 min with 0.2 % Triton-X-100 in PBS and washed with PBST
[0.1 % Triton-X-100 in PBS]. Slides were blocked for 20 min - 60 min with 2-3 % BSA in
PBS, washed 3 times in PBS and incubated with primary antibodies. Coverslips were washed
3 times with PBS and incubated with secondary antibody (Invitrogen, Darmstadt, Germany)
in PBST for 30 minutes. Nuclei were stained with Hoechst 33258 (Sigma, Munich, Germany).
For the detection of RhoA and Myosin IIA, cells were fixed for 15 minutes in 10% TCA and
processed as described above. For F-actin staining, cells were fixed with 4% PFA in PBS,
permeabilized, and stained with Alexa Fluor 594 phalloidin (Molecular Probes).
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Recombinant proteins
Expression of recombinant GST-GAS2L3 in BL21(DE3) pLysS or Rosetta (DE3) E. coli was
induced by addition of 1mM IPTG at 15°C overnight. The recombinant protein was purified
on glutathione-linked sepharose beads according to standard protocols. GST-GAS2L3 was
dialyzed against BRB80 buffer [80mM PIPES, pH 6.8, 1mM MgCl2 and 1mM EGTA]
overnight.
Microtubules and F-actin co-sedimentation assay
Tubulin was prepared from porcine brain following the protocol described in (Mitchison and
Kirschner, 1984). Tubulin was polymerized at 37°C for 30 min by addition of 0.5 volumes of
glycerol and 1mM GTP. The polymerized tubulin was diluted to 10μM with 20μM taxol
containing BRB80 buffer. For co-sedimentation assays 20 μl of polymerized tubulin was
mixed with 1μM GST-GAS2l3 or GST protein and incubated for 15 min at room temperature.
Mixtures were pelleted by centrifugation at 30.000 g for 30 min at 30°C over a 30% glycerol
cushion. The supernatant and pellet fractions were recovered and separated by SDS-PAGE.
Gels were stained with Coomassie blue.
F-actin co-sedimentation assay was performed using the Non-Muscle Actin Binding Protein
Biochem Kit (Cytoskeleton, Denver, USA). Actin was polymerized at room temperature for
1h according to the manufacturers protocol. For co-sedimentation assays 40 μl F-actin was
mixed with 1μM GST-GAS2l3 or GST protein and incubated for 30 min at room temperature.
Mixtures were pelleted by centrifugation at 120.000 g for 60 min at 24°C. Supernatant and
pellet fractions were recovered and separated by SDS-PAGE. Gels were stained with
Coomassie blue.
Microtubule bundling assay
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5μM tubulin and 0.4μM Cy5-labelled tubulin were mixed and polymerized by addition of
20μM taxol and 1mM GTP in BRB80 at 37°C for 30 min. After addition of 500nM GST-
GAS2L3 or GST, the mixtures were incubated for an additional 30 min at 37°C. 1.5 μl
aliquots were pipetted onto slides, covered with 1.5 μl fixing solution [BRB80 buffer, 50%
glycerol and 8% formaldehyde] and analyzed via fluorescence microscopy.
Microtubules / F-actin crosslinking assay
The microtubules / F-actin crosslinking assay was performed as described previously (Miller
et al., 2004). Briefly, 1μM GST-GAS2l3 protein was incubated with 5μM polymerized
tubulin in tubulin polymerization buffer and incubated for 15 min at room temperature. After
addition of 5μM F-actin, the mixture was incubated for an additional 15 min at room
temperature and centrifuged at 5.000 g for 10 min at 24°C. Supernatant (S) and pellet (P)
fractions were recovered, separated by SDS-PAGE and stained with Coomassie blue. The
fraction of actin in the pellet was quantified in ImageJ using the method outlined at
http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ü
Time-lapse microscopy
HeLa cells stably expressing H2B-EGFP and α-tubulin-mRFP were transfected with control
siRNA or GAS2L3 specific siRNA in 35 mm µ-dish chambers (Ibidi, Munich, Germany).
HeLa cells stably expressing mCherry-α-tubulin were transiently transfected with EGFP-
GAS2L3. Live cell imaging was performed using a Leica heating insert with attached
incubator S-2. Images were captured every 2 minutes using Leica Application Suite.
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RT-PCR
Total RNA was isolated with Trizol (Invitrogen, Darmstadt, Germany), reverse transcribed
with 0.5 units M-MLV-RT Transcriptase (Thermo Scientific, Epsom, UK) and analyzed with
quantitative real-time PCR with SYBR Green reagents from Thermo Scientific using the
Mx3000 (Agilent technologies, Waldbronn, Germany) detection system. Expression
differences were calculated relative to GAPDH as described before (Schmit et al., 2007).
Immunoblotting
Cells were lysed in TNN [50 mmol/L Tris (pH 7.5), 120 mmol/L NaCl, 5 mmol/L EDTA,
0.5% NP40, 10 mmol/L Na4P2O7, 2 mmol/L Na3VO4, 100 mmol/L NaF, 10 mg/mL
phenylmethylsulfonyl fluoride, protease inhibitors (Sigma, Munich, Germany)]. Proteins were
separated by SDS-PAGE, transferred to PVDF membrane and detected by immunoblotting.
Karyotype analysis and FISH
To prepare metaphase spreads, cells were treated with 10 ng/ml colcemid (Sigma, Munich,
Germany) for 2 h (HCT116 cells) or 5 h (BJ cells) at 37°C. Cells were collected by
trypsinization, washed with DMEM and suspended in 0.8% sodium citrate at 37°C for 30-40
minutes. Next, cells were fixed in methanol/acetic acid (3:1) over night at -20°C, dropped
onto slides and stained with 5% Giemsa solution for 5 minutes. Metaphase spreads were
observed under light microscopy and chromosome numbers determined.
For FISH in interphase cells, poseidon chromosome 7 and 8 satellite enumeration probes
(Kreatech diagnostics, Amsterdam, Netherlands) were used according to the manufacturer.
DNA was counterstained with Hoechst 33258. Fluorescence signals of at least 500 nuclei
were counted.
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ACKNOWLEDGMENTS
We thank all members of the laboratory for their suggestions and critical reading of the
manuscript. We thank Susanne Spahr and Adelgunde Wolpert for their excellent technical
assistance and Meik Kunz for help with the time-lapse analysis. We thank Patrick Meraldi,
Stephen Taylor, Robert Benezra, Holger Bastians, Daniel Gerlich and Ulrike Kutay for
providing cell lines and plasmids. We thank Olaf Stemmann for helpful discussions. We
thank Claus Steinlein, Michael Schmid and Clemens Grimm for their help with karyotype
analysis and recombinant protein expression. This work was supported by grants from the
DFG (575/6-1 and TR17-B1) towards SG.
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FIGURE LEGENDS
Figure 1: GA2SL3 is a novel DREAM target gene that is expressed in mitosis.
Expression of GAS2L3 mRNA was analyzed by RT-qPCR in HeLa cells transfected with a
control siRNA, a LIN9-specific siRNA (A) or a LIN54-specific siRNA (B). (C) GAS2L3
mRNA expression in BJ cells transfected with a control siRNA or a LIN9-specific siRNA was
analyzed by RT-qPCR. (D) Schematic comparison of GAS2 proteins. The conserved CH and
GAR domains are shown. (E) Expression of GAS2, GAS2L1 and GAS2L3 during the cell
cycle was analyzed in synchronized T98G cells by RT-qPCR. (F) Expression of GAS2L3 in
HeLa cells stably expressing a tetracycline-inducible and HA- and streptavidin-binding-
petide-tagged-GAS2L3 was analyzed by immunoblotting. The expression of the transgene
was induced by tetracycline addition as indicated. Cells were arrested in metaphase by
nocodazole-treatment and released for 2 hours as indicated. (G) Protein levels of endogenous
GAS2L3 in cells synchronized in G2, M and G1 phase were determined by
immunoprecipitation with a monoclonal antibody followed by immunoblotting with
polyclonal GAS2L3 antiserum. Synchronization was verified by detection of phosphorylated
histone H3, a marker for G2 and M. as: asynchronous. Expression of a GAS2L3 specific
shRNA strongly reduced the intensity of the band, confirming the identity of the endogenous
protein.
Figure 2: GAS2L3 localizes to the mitotic spindle and to the midbody.
(A) Cells transfected with EGFP-GAS2L3 were fixed with methanol and stained for α-tubulin
(red) and DNA (blue). (B)
(C) Midbody localization of EGFP-GAS2L3 was confirmed by co-staining with antibodies
against Aurora B in methanol fixed cells (red). (D) Confocal microscopy shows that HA-
GAS2L3 flanks the midbody region. Cells were fixed with PFA (E) Selected frames from
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time-lapse analysis of EGFP-GAS2L3 in HeLa cells stably expressing mCherry-α-tubulin.
The full movie is provided in Supplementary Material (Movie 1). The separate red and green
channels are provided in Supplementary Figure S2. Bar: 10 µm. Time is in hrs:min.
Figure 3: Domains that mediate localization of GAS2L3
(A) Schematic overview of the deletion constructs used and summary of the experiments
shown in Figure 3 B and Figure 5. n.d.: not determined (B) HeLa cells transiently transfected
with the indicated GAS2L3 deletion constructs fused to EGFP were fixed with methanol and
stained for α-tubulin (red) and DNA (blue). Localization in interphase, mitosis and
cytokinesis was determined. Bar: 10 µm.
Figure 4: GAS2L3 bundles microtubule
(A) EGFP-GAS2L3 was expressed in HeLa cells. Cells were fixed with PFA and stained for
α-tubulin (red) and DNA (blue). EGFP-GAS2L3 colocalizes with and bundles microtubules
in interphase cells. (B) Untransfected HeLa cells or HeLa cells transfected with EGFP-
GAS2L3 were treated with nocodazole as indicated. Microtubules were stained with α-
tubulin antibodies (red). MTs were disrupted in untransfected cells by nocodazole. In cells
expressing EGFP-GAS2L3, MTs remained bundled in the presence of nocodazole. (C) GST
or GST-GAS2L3 was incubated with taxol-stabilized Cy5-labelled microtubules. Reaction
mixes were applied to slides and analyzed by fluorescence microscopy. Incubation with GST-
GAS2L3 resulted in microtubule bundling. Bar: 10 µm.
Figure 5: GAS2L3 binds to and crosslinks microtubule and F-actin
(A) Purified GST, GST-GAS2L3 or the indicated deletion mutants were incubated in absence
or presence of taxol-stabilized microtubules in a co-sedimentation assay. Supernatants (S) and
pellet (P) were recovered and analyzed by SDS-PAGE and Coomassie staining. (B) Purified
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GST or GST-GAS2L3 or indicated deletion mutants were incubated with F-actin and
subjected to high-speed centrifugation. Supernatant (S) and pellet (P) were analyzed by SDS-
PAGE and Coomassie staining. (C) GAS2L3 crosslinks F-actin and microtubules. Purified
GST-GAS2L3 or mutants E and B were incubated with microtubules and F-actin as indicated.
Complexes were recovered by low-speed centrifugation and analyzed by SDS-PAGE
followed by Coomassie-staining. S: supernatant; P: pellet. A quantification of the fraction of
actin found in the pellet relative to the supernatant is provided in Supplementary Figure S9.
Full scans of the gels are provided in Supplementary Figure S6-S8. (D) Cartoon depicting the
actin and microtubule binding domains of GAS2L3 identified. See Figure 3A for a schematic
overview of the mutants and summary of the results.
Figure 6: Phenotype of GAS2L3 depleted cells
HeLa cells were transfected with a control siRNA (ctrl.) or GAS2L3-specific siRNAs#1-#3.
Depletion of GAS2L3 was verified by RT-qPCR (A) and by immunostaining (B). (C)
Representative images of cells transfected with control siRNA or GAS2L3 specific siRNAs
demonstrate the presence of multinucleated cells and cells with multilobed nuclei after
depletion of GAS2L3. Cells were fixed with PFA and stained for tubulin (red) and DNA
(blue). Bar: 10 µm (D) The percentage of multinucleated cells and cells with multilobed
nuclei was quantified. The differences were statistically significant (p<0.005, students t-test,
two-tailed). More than 300 cells were counted, and results are from three independent
experiments. (E) Western blot showing expression of the resistant GAS2L3 (full length and
mutants B and E) after induction with tetracycline. Lysates were immunoprecipitated with
HA antibodies and immunoblotted with GAS2L3 antibodies. (F) HeLa cells stably expressing
tet-inducible RNAi-resistant GAS2L3 (wildtype or mutant B or E) were treated with
tetracycline before siRNA transfection as indicated. The percentage of cells with abnormal
nuclei was quantified. The differences between cells expressing full length GAS2L3 were
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statistically significant (p=0.007; students t-test, two-tailed). Results are from 3 different
experiment and more than 300 cells were counted in each experiment.
Figure 7: GAS2L3 is required for cytokinesis.
(A-C) Selected frames from time-lapse movies of HeLa cells stably expressing H2B-EGFP
and mRFP-α-tubulin and transfected with a control (A) or GAS2L3–specific siRNA#1 (B) or
siRNA#2 (C). Time relative to nuclear envelope breakdown is shown in hrs:min. Movies are
provided in supplementary material (Movies 2-4). (D) Control and GAS2L3 knockdown cells
were fixed with TCA and stained for RhoA (red) and DNA (blue) or Myosin IIA (red) and
DNA (blue). Bar:10 µm. Arrows indicate abnormal cortical localization of RhoA or Myosin
IIA.
Figure 8: GAS2L3 is required to maintain genomic stability.
(A) Metaphase spreads of BJ cells stably expressing a control shRNA or a GAS2L3 specific-
shRNA. (B) GAS2L3 depleted BJ cells show increased aneuploidy. Chromosome numbers of
100 metaphase spreads were counted. (C) Interphase FISH analysis using centromere-specific,
fluorescently labeled probes for chromosome 7 (red) and 8 (green). Nuclei were stained with
Hoechst 33258 (blue). (D) Quantification of FISH signals for chromosomes 7 and 8 in control
and GAS2L3 depleted HCT116 and HCT116 MAD2+/- clones. The table shows the number
of cells carrying the indicated FISH signals. Also shown is the total percentage of cells
diverging from the modal position (cells off the mode) for chromosomes 7 and 8. In all cases,
the differences between control-depleted cells and GAS2L3 depleted cells were statistically
significant (p<0.0025; Fisher's exact test).
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LIN9 GAS2L3
0
0.2
0.4
0.6
0.8
1
1.2
real
tive
expr
essi
on
0
0.2
0.4
0.6
0.8
1
1.2
siRNA ctrl. LIN9 ctrl. LIN9
real
tive
expr
essi
on
siRNA ctrl. LIN54 ctrl. LIN54
LIN54 GAS2L3A B
0
1
2
3
4
5
6
7
8
0 6 12 18 24 29 35
GAS2 GAS2L1
0
2
4
6
8
10
12
14
0 6 12 18 24 29 35
GAS2L3
real
tive
expr
essi
on
G1G0 S G2/M G1
C
E
0
0.2
0.4
0.6
0.8
1
1.2 LIN9 GAS2L3
siRNA ctrl. LIN9 ctrl. LIN9
real
tive
expr
essi
on
D
0
1
2
3
4
5
6
7
8
0 6 12 18 24 29 35
real
tive
expr
essi
on
real
tive
expr
essi
on
G1G0 S G2/M G1 G1G0 S G2/M G1time [h]
CH GAR
CH GAR1 681
1 694GAS2L3
GAS2L1
CH GAR1 313
GAS2
CH GAR1 880
GAS2L2
Figure 1
F
Cyclin B
Tubulin
+ Noc.release- 0h 2h-
- + + + Tet
GAS2L3
Tubulin
Phospho-H3
GAS2L3 as G2 M G1
G
GAS2L3
shRNA- +
Tubulin
95
55
55
kDa kDa kDa
17
95
55
95
55
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Met
apha
seA
naph
ase
Telo
phas
eC
ytok
ines
is
EGFP-GAS2L3 DNAα-tubulin merge
A
Inte
rpha
seP
ro-
met
apha
se
B C
Figure 2
F
DNAAurora B merge
Met
apha
seA
naph
ase
Telo
phas
eC
ytok
ines
is
GAS2L3mCherry-α-tubulin DNA merge
Telo
phas
eC
ytok
ines
is
EGFP-GAS2L3
00:00 00:12 00:38 00:40 00:4200:28 00:44
00:46 00:48 00:54 01:02 01:0800:50 01:14
E
DEGFP-GAS2L3
ActinDNA
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Figure 3
mut D
mut E
GAS2L3
mut A
mut B
mut C
CH GAR
GAR
CH
CH GAR
GAR
694
455
375
mut F
mut G
303
303
303
694
694
170
1
1
1
176
309
170
309
mut H456 694
midbody/central spindle
+
n.d
+
-
-
-
-
+/-
+
n.d
-
-
+
-
-
-
+
+
n.d
-
+/-
+
-
-
-
+
+
MT actin
+-
+
+
-
n.d
n.d
n.d
+
++/-
+
+
+
n.d
n.d
n.d
+
in vitro bindinglocalizationinterphasemicrotubule
metaphasespindle
A
B
mut A
mut B
wt
mut C
mut D
mut E
mut F
mut G
mitosiscytokinesis interphaseEGFP-GAS2L3 merge EGFP-GAS2L3 merge EGFP-GAS2L3 merge
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Figure 4
AEGFP-GAS2L3 DNA merge
ctrl- Noc.
ctrl+ Noc.
EGFP-GAS2L3+ Noc.
DNAα-tubulin merge
EGFP-GAS2L3
B
α-tubulin
Buffer GST GST-GAS2L3C
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Figure 5
GST-GAS2L3
Tubulin
GST
Tubulin
+ MT - MTS P S P
GST
f.l.
GST-Mut E
TubulinMut E
GST-Mut F
TubulinMut F
GST-Mut HTubulin
Mut H
GST-Mut B
TubulinMut B
Mut GGST-Mut G
TubulinGST
Actin
+ F-actin - F-actinS P S P
GST-GAS2L3
Actinf.l.
Mut F
Mut H
GST
GST-Mut FActin
GST-Mut H
Actin
Mut EGST-Mut E
Actin
Mut GGST-Mut G
Actin
Mut BGST-Mut B
Actin
Actin
GST-GAS2L3
Tubulin
S P S P S P S P
GST-GAS2L3MTsActin
--+ + +
-+ +
++++
Actin
GST-GAS2L3-mut E
Tubulin
Actin
GST-GAS2L3-mut B
Tubulin
GST-GAS2L3
GST-GAS2L3Mut E
GST-GAS2L3Mut B
A B
C
CH GAR
Actin
MT+
Actin
MT+
Actin
midbody and spindle localization
D
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- + tet
GAS2L3(f.l.)
GAS2L3(mut E)
GAS2L3(mut B)
ctrl
si#1
si#2
si#3
0
5
10
15
20
siRNA
mul
tinuc
elat
ed c
ells
or c
ells
w
ith m
ulti-
lobe
d nu
clei
[%]
real
tive
expr
essi
on
A GAS2L3
DNAAurora B
detail
B
C D
ctrl
si#1
si#2
si#3
0
20
40
60
80
100
siC
trlsi
GA
S2L
3#2
E
F
mut E
GAS2L3
mut B
CH GAR
CH GAR
mul
ltilo
bed
cells
(per
cent
age)
Tet: - + - + - +
GAS2L3: f.l. mut B mut E
0
20
40
60
80
100
120
siC
trlsi
GA
S2L
3#2
siG
AS
2L3#
1si
GA
S2L
3#3
DNA DNAα-tubulin
Figure 6
95
43
55
kDa
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00:00 00:08 00:32 00:34 00:38 00:40 00:42
00:44 00:46 00:48 00:50 00:52 01:00 01:54
GAS2L3 siRNA #1
GAS2L3 siRNA #200:00 00:14 00:46 00:48 00:50 00:54 00:56
00:58 01:00 01:02 01:04 01:06 01:10 01:28 02:12
ctrl siRNA00:00 00:08 00:54 00:56 01:00 01:02 01:04
01:06 01:08 01:10 01:12 01:14 01:16 01:30 01:46
00:22
00:44
00:34
00:58
Figure 7A
B
C
Dctrl siRNA GAS2L3 siRNA #2 GAS2L3 siRNA #2
RhoADNA
MyosinDNA
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chromosomeanalyzed
number of FISH signals per cell cells off the mode (%)
78
1 2 3 4 >4
2.993.04
12 520 2 2 07 510 4 5 0
HCT116ctrl. shRNA clone 1
78
2.973.12
15 555 2 0 09 528 5 3 0
HCT116ctrl. shRNA clone 2
78
8.367.08
26 515 7 13 116 525 16 7 1
HCT116GAS2L3 shRNA clone 1
78
9.578.77
28 510 9 15 223 520 17 10 0
HCT116GAS2L3 shRNA clone 2
78
6.825.19
23 519 10 5 09 518 12 17 0
HCT116 MAD2+/-ctrl. shRNA clone 1
78
5.124.58
14 500 5 8 015 500 4 4 0
HCT116 MAD2+/-ctrl. shRNA clone 2
78
13.1612.82
21 508 25 31 328 503 10 27 9
HCT116 MAD2+/-GAS2L3 shRNA clone 1
78
13.2611.71
23 530 20 28 1020 520 12 29 9
HCT116 MAD2+/-GAS2L3 shRNA clone 2
41-43 44-46 47-49 50-79 >80
kary
otyo
pe (%
) 80
60
40
20
0
100
ctrl. shRNAGAS2L3 shRNA
BJ-ET
A B
C
D
HCT116ctrl. shRNA cl. 2
HCT116GAS2L3 shRNA cl. 2
HCT116 MAD2+/-ctrl.shRNA cl. 1
HCT116 MAD2+/-GAS2l3 shRNA cl. 1
Figure 8
Chromosome number
ctrl. shRNA GAS2L3 shRNA
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