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Originally published In Press as doi:10.1074/jbc.M608562200 on May 3, 2007

J. Biol. Chem., Vol. 282, Issue 26, 19259-19271, June 29, 2007
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Lats2 Is an Essential Mitotic Regulator Required for the Coordination of Cell Division*Formula

Norikazu Yabuta{ddagger}, Nobuhiro Okada{ddagger}, Akihiko Ito§, Toshiya Hosomi{ddagger}, Souichi Nishihara{ddagger}, Yuya Sasayama{ddagger}, Azumi Fujimori{ddagger}, Daisuke Okuzaki{ddagger}, Hanjun Zhao{ddagger}, Masahito Ikawa, Masaru Okabe, and Hiroshi Nojima{ddagger}1

From the {ddagger}Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan, the §Division of Surgical Pathology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan, and the Genome Information Research Center, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan

Received for publication, September 5, 2006 , and in revised form, May 1, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 REFERENCES
 
Many human malignant tumors exhibit mitotic defects such as centrosome aberration, abnormal spindle formation, chromosome missegregation, and defective cytokinesis. Chromosome instability phenotypes, or aneuploidies, are the major causative factors of spontaneous abortion, perinatal defects, and malignant tumor progression. Therefore, mitosis is stringently controlled by various regulators and the checkpoint machinery (1-5). In particular, failure of cytokinesis causes severe cell division defects such as tetraploidy, with nuclear enlargement and multinucleation. In contrast to organisms such as yeasts and nematodes, in mammalian cells the molecular mechanisms underlying exit of mitosis and cytokinesis remains unclear (6-9).

Lats2 (large tumor suppressor)/WARTS (Wts) was originally identified as a serine/threonine kinase tumor suppressor in Drosophila as its inactivation resulted in overproliferation phenotypes and developmental defects in mosaic animals (10, 11). Drosophila Lats coordinates cell proliferation and cell death with the protein kinase Hippo (Hpo) and the scaffold protein Salvador (Sav) by regulating levels of cyclin E or DIAP1, required for cell cycle progression and apoptotic inhibition, respectively (12-17). Members of the Dbf2 kinase family such as Saccharomyces cerevisiae Dbf2 and Dbf20, Schizosaccharomyces pombe Sid2, Caenorhabditis elegans Lats, and mammalian Lats1 and Lats2, have been highly conserved across species (18-23). S. cerevisiae Dbf2 localizes to the spindle pole body (SPB; centrosome in yeast) in anaphase and the bud neck (contractile ring in yeast) in cytokinesis, functioning as an integral component of the mitotic exit network (MEN) that supervises inactivation of cyclin-dependent kinases (Cdks) and cytokinesis; Dbf20 is also likely a component of the MEN (24, 25). S. pombe Sid2 also localizes to the SPB throughout mitosis and is a component of the cytokinesis checkpoint and the septation initiation network (SIN), which is equivalent to the MEN machinery of S. cerevisiae (6, 26). In contrast, the Ndr (nuclear Dbf2-related) kinase family represents a similar but distinct subfamily of the Dbf2 family. The Ndr kinase family includes S. cerevisiae Cbk1, S. pombe Orb6, C. elegans Sax-1, Drosophila Trc, Neurospora Cot-1, and the mammalian Ndr1 and Ndr2 kinases, which are involved in cellular morphogenesis (27).

Furthermore, the kinase activation of the Dbf2 and Ndr families require a highly conserved co-activator, the Mob protein family (28). In yeast, Mob1 acts as a co-activator of the Dbf2 family, whereas another Mob, Mob2, acts as co-activator of the Ndr family (29-32). The mammalian Mob family consists of at least seven proteins, namely, Mob1A (also called Mob1beta and Mats1), Mob1B (also called Mob1{alpha} and Mats2), Mob1C, Mob1D (also called Mob3{alpha}), Mob2, Mob3 (also called Phocein) and Mob3beta (33-36). Human Mob1A binds to and activates Ndr2 kinase (37), whereas Mob1B and Mob2 interact with Ndr1 and Ndr2 kinases (23, 34). More recently, Lats1 has been reported to interact with its activator, Mob1A, and function as a putative component of the MEN in mammalian cells (38). These activities suggest similarities to interactions reported for Lats1 in S. cerevisiae Dbf2, S. pombe Sid2 and Drosophila Lats, which interact with S. cerevisiae Mob1, S. pombe Mob1 and Drosophila Mats, respectively (38, 39).

Two mammalian Lats kinases have been identified: Lats1/WARTS and Lats2. Lats1-deficient mice develop pituitary hyperplasia, soft-tissue sarcomas, and ovarian stromal cell tumors (40). Human Lats1 is phosphorylated during mitosis and associates with Cdc2 and the actin filament assembly factor Zyxin, co-localizing to the centrosomes and mitotic spindle. Overexpression of Lats1 leads to G2/M arrest as Lats1 acts as a negative regulator of Cdc2 kinase by associating with Cdc2 (21, 41, 42). Additionally, overexpression of Lats1 results in up-regulation of the pro-apoptotic proteins Bax and caspase-3, or activation of Omi protease, whereas Lats1 is proteolysed by Omi (43-46). Recently, Lats1 has been reported to affect cytokinesis by directly inhibiting the kinase activity of LIMK1 (47).

We identified the other mammalian Lats kinase, Lats2 (22), which is also phosphorylated during mitosis (48, 49). Human Lats2 and Lats1 share 85% sequence identity in the kinase domain at the C terminus and 52% overall sequence (22). Similar to Lats1 and Drosophila Lats, Lats2 also appears to coordinate cell proliferation and cell death: overexpression of Lats2/Kpm results in G2/M arrest via inhibition of Cdc2 kinase activity, inhibition of G1/S transition via down-regulation of cyclin E/Cdk2 kinase activity, and induction of apoptosis via down-regulation of apoptotic inhibitors such as Bcl-2 and Bcl-xL (50-52). Moreover, Lats2 is a centrosomal protein that is phosphorylated by the centrosomal kinase Aurora-A; this phosphorylation is important for the centrosomal localization of Lats2 (49). Lats2 binds Mdm2, inhibits its E3 ligase activity and activates p53, while p53 rapidly and selectively up-regulates Lats2 expression in G2/M cells, thereby defining a positive feedback loop which constitutes a novel checkpoint pathway critical for the maintenance of proper chromosome number (53). On the other hand, two miRNAs, miRNA-372 and-373, function as potential novel oncogenes in testicular germ cell tumors by inhibition of LATS2 expression, which suggests that Lats2 is an important tumor suppressor (54). However, the mitotic functions of Lats2, including its role in the centrosome, remain unclear. Recently, knock-out mice in which the LATS2 gene was partially disrupted, leaving its N-terminal amino acid sequence (amino acids 1-370) intact, showed embryonic lethality attributed to defects in cardiac growth (55). Mouse embryonic fibroblasts (MEFs) derived from this knock-out mouse exhibited defects in growth control and centrosome duplication. However, Lats2 kinase activity and two Lats Conserved Domains (LCD1 and LCD2; highly conserved sequences in the N-terminal region between Lats1 and Lats2), are required for Lats2 to suppress tumorigenicity and inhibit cell proliferation (51). We have reported that the N terminus of Lats2 (amino acids 1-393) interacts with Aurora-A kinase and that phosphorylation of Ser83 in LCD1 by Aurora-A plays a role in its centrosomal targeting (49). Therefore, the short N-terminal peptide, expressed in the partially LATS2-deficient mice, may be performing biological functions that mask the true physiological role of Lats2. Thus, we have generated complete LATS2-deficient mice, which do not express any short peptides derived from the LATS2 locus. Here, we demonstrate that completely LATS2-deficient mice show similar but distinct phenotypes from partially LATS2-deficient mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 REFERENCES
 
Lats2 Is Essential for Embryonic Development—To elucidate the biological function of Lats2, we disrupted the entire coding region of mouse LATS2 using a gene-targeting vector. We replaced the first coding exon (E1) with the neomycin selection cassette PGK-neo, flanked by 1.6 and 6.0 kb of sequence homologous to LATS2, resulting in deletion of the translational start site. The herpes simplex virus thymidine kinase (HSV-tk) gene was used as a negative selection cassette (Fig. 1A). After electroporation of the linearized targeting vector into D3 ES cells, the resultant clones were analyzed with Southern blots using probes A and B. One of 92 ES clones was identified as having the correct targeting (Fig. 1B, clone no. 22), and was injected into blastocysts which were transferred to pseudopregnant females to generate chimeras. This process yielded 7 chimeric mice that produced offspring with germline transmission of the disrupted Lats2 gene. Germline transmission of the targeted Lats2 gene in heterozygous mice was confirmed by Southern blot analysis (Fig. 1C).

Lats2 heterozygous (Lats2+/-) mice were born healthy and fertile. However, we found no homozygous (Lats2-/-) mice among the 453 live-born offspring from intercrossing heterozygous mice. The ratio of wild-type (Lats2+/+), Lats2+/-, and Lats2-/- genotypes was 178:275:0 (Table S1). This suggests that Lats2 deficiency results in embryonic lethality. Unexpectedly, the ratio between Lats2+/+ and Lats2+/- genotypes was ~2:3, indicating a non-Mendelian distribution, and suggests that Lats2 heterozygosity may also cause embryonic lethality. Interestingly, the ratio between Lats2+/+ and Lats2+/- genotypes was ~1:1.5 in males, whereas the ratio was ~1:1.8 in females, indicating that Lats2+/- males tend to cause embryonic lethality rather than Lats2+/- females (supplemental Table S1).

To examine the timing of developmental arrest in Lats2-/- offspring, the genotypes of embryos generated from Lats2+/- intercross matings were examined by PCR using the primers, KO.con5, E1-AS6, and KS.conB. The 1.9-kb band was amplified by the primers KO.con5 and E1-AS6, indicating the wild-type locus; the 1.6-kb band was amplified by the primers KO.con5 and KS.conB, indicating the Lats2-targeted allele (Fig. 1A). We obtained 53 Lats2-/- embryos (e.g. nos. 7-2, 9-10, and 10-3; Fig. 1D). Using total extracts from Lats2-deficient mouse embryo fibroblasts (MEFs), absence of the full-length mRNA and Lats2 protein was confirmed by RT-PCR and Western blot analysis (Fig. 6, B and A, respectively). These results confirmed that our targeting strategy produced a null allele. Notably, levels of Lats2 protein were indistinguishable between Lats2+/- and Lats2+/+ MEFs. It is possible that Lats2+/- MEFs increased Lats2 protein levels by an as-yet unidentified feedback pathway. In kidney extracts, the levels of Lats2 were also indistinguishable between Lats2+/- and Lats2+/+ mice.3 We monitored life span and tumor formation in Lats2+/- and Lats2+/+ mice exposed to {gamma}-irradiation (dosages of 6 or 8 Gy) over a period of 469 or 40 days, respectively. There were no differences in survival rates (supplemental Fig. S1), distinguishable phenotypes or spontaneous tumor development between Lats2+/- and Lats2+/+ mice.3


Figure 1
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  		FIGURE 1.
Targeted disruption of the complete mouse LATS2. A, schematic representation of WT Lats2 locus, targeting vector, and targeted locus. In the targeting vector and targeted locus, the neomycin selection cassette replaces the first coding exon (E1), to disrupt the complete LATS2 gene. On the short arm, an ApaLI digest generates a 4.2-kb fragment from the WT allele and a 5.5-kb fragment from the targeted allele. On the long arm, a BanI/EcoRI double digest generates a 7.1-kb fragment from the WT allele and an 8.8-kb fragment from the targeted allele. Short and long arm fragments are recognized by probes A and B, respectively. B, Southern blot analysis of genomic DNA from ES clones (D3, 2, 22, 26, 27, and 28). Genomic DNA was digested with ApaLI (left panel) and BanI/EcoRI (right panel) and hybridized with probes A and B, respectively. The WT (4.2 and 7.1 kb) and mutated fragments (5.5 and 8.8 kb) are indicated. D3 is a negative control. C, Southern blot analysis of genomic DNA from the tails of offspring obtained from heterozygote intercrosses. Genomic DNA was digested with ApaLI, and probe A was used for hybridization of the blot. Fragments corresponding to WT and mutated alleles (4.2 and 5.5 kb, respectively) are indicated. D, PCR analysis of genomic DNA of embryos obtained from heterozygote intercrosses. Amplification products corresponding to WT and mutated (KO, knock-out) alleles (1.9 and 1.6 kb, respectively) are indicated. Genomic DNA from tails of mice nos. 10 and 11 in C were used as control templates.

 
We analyzed the morphology of embryos at various stages and found 30 and 3 viable Lats2-/- embryos at embryonic days 10.5 (E10.5) and E11.5, respectively (supplemental Table S2). However, we found no viable Lats2-/- embryos at E12.5 or beyond, and all the Lats2-/- embryos were smaller than their Lats2+/+ and Lats2+/- counterparts (supplemental Fig. S2). Moreover, abnormal dead Lats2-/- embryos were also observed at E10.5, E11.5, E12.5, E13.5, E15.5, and E16.5 and these exhibited severely retarded growth (supplemental Table S2 and Fig. S2). At E16, one Lats2-/- embryo presented an abnormal gross morphology whereby it remained enveloped in amnion or deciduas.3 These results suggest that the lethality of Lats2-null embryos may occur before E12.5 because Lats2 is essential for embryonic development.

Although Lats2 deficiency leads to embryonic lethality, the cause of death before E12.5 remains unclear. Therefore, to clarify the role of Lats2 in embryonic development and cell growth, we performed histological analyses of Lats2-/- embryos at various stages of development. At E13.5, histological examination revealed developmental retardation of multiple organs (Fig. 2, A and B). Notably, although the heart was small, the degree of developmental retardation was modest and muscular trabeculation was well-developed in the ventricle (Fig. 2, C and D). In contrast, the development of the nervous system was severely impaired. In the neuroepithelial layer of the fourth ventricle, a dramatic reduction in the number of nerve cells was observed, as well as infiltration by many inflammatory cells (Fig. 2, E and F). In the spinal cord, both the number of nerve cells and density of nerve fibers were greatly reduced (Fig. 2, G and H). Similar effects were observed as early as E10.5 (Fig. 2, I-N), and although these embryos appeared to be alive, developmental retardation was evident in a number of organs, including the branchial arches and skeletal muscle. Notably, development of the heart was relatively normal: the ventricle had a well-developed muscular trabeculation and the atrium had a wide cavity (Fig. 2, I-L). In contrast, the nervous system exhibited some abnormalities: the neuroepithelial layer of the fourth ventricle was thinner and contained many pyknotic nuclei (Fig. 2, M and N). These results suggest that in the nervous system, a deficiency in Lats2 results in growth arrest or cell death.


Figure 2
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  		FIGURE 2.
Lats2-deficient embryos exhibit severe nervous system developmental defects. A-H, histological analyses of E13.5 embryos obtained from heterozygote intercrosses. H&E-stained sections from wild-type (Lats2+/+) and Lats2-/- whole embryos (A and B), hearts (C and D), neuroepithelium of the fourth ventricles (E and F), and spinal cords (G and H). I-N, H&E stained sections of hearts (I-L) and neuroepithelia of the fourth ventricle (M and N), from Lats2+/+ and Lats2-/- embryos at E10.5. I and J indicate lower magnification. O and P, TUNEL assay of spinal cord sections from Lats2+/+ and Lats2-/- embryos at E13.5. Embryo sections were stained with propidium iodide (PI, red) and Fluorescein-12-dUTP (green); merged yellow signals reflect TUNEL-positive apoptotic cells. a, atrium; bv, brain ventricle; ec, endocardiac cushion; sc, spinal cord; v, ventricle.

 
To confirm whether the cell death observed in Lats2-/- embryos could be attributed to apoptosis, we performed a TdT-mediated dUTP nick-end labeling (TUNEL) assay with sections from Lats2-/- embryos at E13.5. TUNEL-positive apoptotic cells were observed in the spinal cord (Fig. 2, O and P) but not the heart, although they were visible in its general proximity (supplemental Fig. S3). Taken together, these results suggest that Lats2 deficiency results in apoptosis primarily in the nervous system. These phenotypes are not necessarily similar to those of partial LATS2-knock-out mice (55).


Figure 3
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  		FIGURE 3.
Loss of Lats2 leads to centrosome fragmentation. A, immunofluorescence analysis of Lats2-/- MEFs that shows the increased number of centrosomes during interphase (upper panels). In contrast, normal centrosome numbers were observed in wild-type (Lats2+/+) MEFs (bottom panels). The cells were fixed in 4% formaldehyde, stained with anti-{gamma}-tubulin (green) and anti-{alpha}-tubulin antibodies (red), and counterstained with Hoechst 33258 for DNA (blue). The inset in A is the same image at higher magnification. Arrows indicate the increased centrosomes. B, frequency of the increased centrosomes in primary and immortalized Lats2+/+ and Lats2-/- MEFs. The cells were immunostained with anti-{gamma}-tubulin antibody as described above and those with more than two centrosomes per cell were counted. The data shown are averages of three independent experiments. In each experiment, over 200 cells were counted. The error bars represent standard deviations. C, immunofluorescence analysis of Lats2-/- MEFs that shows colocalization of {gamma}-tubulin and Centrin in the amplified centrosomes (I, II, and III, see the text in detail). The centrosomes in wild-type MEFs were normal (upper panels). The cells were fixed in cold-methanol/acetone (1:1), stained with anti-{gamma}-tubulin (green) and anti-Centrin antibodies (red) and counterstained with Hoechst (blue). The white arrows indicate the increased {gamma}-tubulin spots that did not overlap with Centrin spots. The insets in II and III are the same images of the centrosomes at higher magnification. Scale bars, 10 µm. D, frequency of colocalization between {gamma}-tubulin spots and Centrin spots in Lats2+/+ and Lats2-/- MEFs. Blue columns and red columns indicate the ratio of cells with and without the colocalization of {gamma}-tubulin and Centrin, respectively. I, II, and III indicate the ratio of the cells with centrosomal aspects shown in C. {gamma}-Tub and Nuc indicate the numbers of {gamma}-tubulin spots and nuclei per a cell, respectively. In each experiment, over 30 mononucleated cells and over 20 multinucleated cells were counted. The data shown are averages of three independent experiments. E, frequency of centrosome fragmentation on spindle poles during metaphase/anaphase in Lats2+/+ and Lats2-/- MEFs. The data shown are averages of three independent experiments. In each experiment, over 30 mitotic cells were counted. The error bars represent the standard deviations. The inset picture shows a Lats2-/- MEF immunostained with anti-{gamma}-tubulin (green) and anti-{alpha}-tubulin antibodies (red) and counterstained with Hoechst (blue). The arrows indicate the fragmentation of {gamma}-tubulin spots in Lats2-/- MEF during anaphase. Scale bars, 10 µm.

 
Loss of Lats2 Leads to Centrosome Fragmentation—We have reported previously that Lats2 is a centrosomal protein, phosphorylated by Aurora-A kinase (49). To elucidate the function of Lats2 in centrosome regulation, we examined and quantified the status of the centrosome in early-passaged (PDL7) Lats2-/- MEFs during interphase, followed by indirect immunofluorescence analysis using anti-{gamma}-tubulin and anti-{alpha}-tubulin antibodies for visualization of centrosomes and microtubules, respectively. Increased number of centrosome was induced in Lats2-deficient MEFs, which contained >2 centrosomes per cell (Fig. 3A, top panels). In contrast, wild-type MEFs contained a normal number of centrosomes (Fig. 3B, bottom panels). We evaluated centrosome numbers in Lats2-/- and wild-type MEFs (Fig. 3B). In primary cultures (early passage), cells with multiple numbers of centrosomes (i.e. >2 per cell) were observed frequently in Lats2-deficient MEFs (24.0%), but rarely in wild-type derived from litters (7.6%). In immortalized Lats2-/- MEFs, the multiple numbers of centrosomes also increased ~2.9-fold compared with wild-type (Fig. 3B). Next, to examine whether the increased number of centrosome is associated with amplification of centriole, Lats2-deficient MEFs were stained with immunofluorescent antibodies against centrin, a component of centriolar structure (56), and {gamma}-tubulin, then counterstained with Hoechst to visualize the DNA. As shown in Fig. 3C, the pattern of multiple centrosomes by loss of Lats2 was classified into three subtypes: type I, multiple {gamma}-tubulin spots (>2) were not associated with all amplified centrin spots in a cell that have one nucleus (second panel from top, white arrows); type II, multiple {gamma}-tubulin spots (>2) were associated with all amplified centrin spots in a cell that have one nucleus (third panel from top, insets); type III, multiple {gamma}-tubulin spots (>= 2) were associated with all amplified centrin spots in a cell that have two or more nuclei (bottom panel, insets). When we counted the number of cells in each subtype of Lats2-/- cells, we found that the majority (78.3%) of type I cells was mononuclear (Fig. 3, C-I and D-I). The percentage of type I cells was ~22.4% of total Lats2-/- cells (supplemental Table S3). Moreover, we investigated the structure of centrioles in thin sections from Lats2+/+ and Lats2-/- embryos using electron microscopy, but we could observe no structural abnormalities in centrioles of Lats2-/- embryo (data not shown). These results suggest that loss of Lats2 causes centrosome fragmentation (fragmentation of the pericentriolar material) because this multiple centrosomes are not associated with amplification of centriole. Indeed, we also observed a cluster of multiple {gamma}-tubulin spots on a mitotic spindle pole in mitotic Lats2-/- cells (63.0%, Fig. 3E, white arrows in an inset). Furthermore, to address whether these extra {gamma}-tubulin foci possess microtubule nucleating activity, we performed the standard in vivo microtubule regrowth assay. Lats2+/+ and Lats2-/- MEFs were treated with cold medium containing nocodazole to depolymerize microtubules, followed by replacement of fresh warm medium without nocodazole to allow regrowth of microtubules. Cells were fixed and immunostained with anti-{gamma}-tubulin and anti-{alpha}-tubulin antibodies. Two mature centrosomes of Lats2+/+ and Lats2-/- MEFs successfully formed two significant asters (supplemental Fig. S4, C and F, yellow arrows). However, the extra {gamma}-tubulin foci could not form any asters in Lats2-/- MEFs (Fig. S4F, white arrows). In the case of Lats2-/- MEFs with four mature centrosomes, these mature centrosomes could form four perfect asters (supplemental Fig. S4G). These results suggest that the extra {gamma}-tubulin foci in Lats2-/- MEFs do not possess the microtubule nucleating activity. On the other hand, the majority (98.5%) of type III cells was multinucleated, namely, most of the multiple centrosomes were associated with amplification of centriole (Fig. 3, C-III and D-III). This result suggests that loss of Lats2 also causes the increased number of centrosome that originates in cytokinesis failure. Taken together, it is likely that the increased number of centrosome in Lats2-deficient cells could be caused by both centrosome fragmentation and as a consequence of cytokinesis defects, but not overduplication of centrosomes.

Aberrant amplification of centrosomes is expected to cause abnormal spindle formation during mitosis (2). Abnormal amplification of centrosome number also occurs in failure to arrest at aG1 tetraploidy checkpoint after missegregation of the chromosome in mitosis, and is followed by a clustering of centrosomes at a single pole in subsequent tetraploid or aneuploid mitosis (2, 57, 58). Therefore, we examined whether absence of Lats2 leads to aberrant mitotic spindle formation, misaligned chromosomes and abnormal telophase including a cluster of centrosomes at a single pole of telophase cells. Lats2-deficient MEFs were fixed at various mitotic stages and stained with immunofluorescent anti-{gamma}-tubulin and anti-{alpha}-tubulin antibodies, then counter-stained with Hoechst to visualize the DNA (supplemental Fig. S5, insets). In mitotic Lats2-deficient MEFs, we observed misaligned chromosomes at the metaphase plate, despite condensation (supplemental Fig. S5A, white arrows in inset). Such severe misalignment of chromosomes positioned close to the poles is similar to a previously reported phenotype in HeLa cells down-regulated by Aurora-A siRNA (59). To assess the percentage of mitotic cells with chromosome misalignments, we counted such abnormal mitotic cells in Lats2+/+ and Lats2-/- MEFs (supplemental Fig. S5A, bar graph). The percentage of aberrant mitotic cells with chromosome misalignment was significantly increased ~2-fold in Lats2-/- MEFs compared with wild-type (29.4% of total mitotic Lats2-/- cells). In addition, the percentages of Lats2-/- cells with the symmetry and the asymmetry of the misaligned chromosomes were 10.1 and 16.5% of total mitotic Lats2-/- cells, respectively. It is unlikely that loss of Lats2 aggressively causes premature sister chromatid dissociation as opposed to metaphase misalignment. On the other hand, we were not able to observe a remarkable increase in the mitotic cells with abnormal spindles such as multi-polar spindle in Lats2-deficient MEFs (supplemental Fig. S5B). Moreover, during telophase, we could also find clustering of centrosomes at a single pole in dividing daughter cells (supplemental Fig. S5C, white arrows in inset). The percentage of these abnormal telophase cells was also increased ~3.3-fold in Lats2-/- MEFs (14.4%) compared with wild-type (supplemental Fig. S5C, bar graph). These results indicate that loss of Lats2 leads to aberrant mitosis with misaligned chromosomes in addition to centrosome fragmentation, but not to multipolar formation.

Loss of Lats2 Results in Nuclear Enlargement and Multinucleation—In malignant tumor cells, abnormal nuclear morphologies, such as enlarged polyploid nuclei, multinuclei, and micronuclei, are observed as frequently as centrosome abnormalities (60, 61). Therefore, to examine the abnormal nuclear morphologies in Lats2-/-, Lats2+/+ and Lats2-/- MEFs derived from littermates were fixed and Hoechst stained for visualization of the nucleus. We observed frequent enlargement of nuclei in Lats2-/- MEFs (Fig. 4A, bottom panels) and their average size was noticeably larger than those of wild-type cells. We analyzed the frequency of nuclear enlargement quantitatively for Lats2-/- and Lats2+/+ MEFs. Enlargement was observed in 15.1 and 8.5% of Lats2-/- cells at primary and immortalized cultures, respectively. In contrast, enlargement was only observed in 1.7 and 1.6% of wild-type cells at primary and immortalized culture, respectively (Fig. 4B). Furthermore, multinuclei were also observed more frequently in Lats2-/- than Lats2+/+ MEFs at both primary and immortalized cultures (Fig. 4C). Lats2-/- MEFs with multinuclei increased ~3.4- and 3.3-fold compared with wild-type cells at primary and immortalized cultures, respectively (21.4 and 11.6% in Lats2-/- MEFs). Interestingly, cells with over 3 nuclei were rarely identified in Lats2-/- MEFs (1.7%, data not shown). Moreover, to rule out the possibility that the nuclear morphologic defects observed in Lats2-/- MEFs are because of additional mutations during the derivation of these cell lines, we performed Lats2 siRNA in wild-type MEFs and examined whether a similar phenotype develops following the acute knockdown of this protein. Indeed, we could observe a similar multinucleated phenotype in Lats2-depleted MEFs as well as in Lats2-/- MEFs, although a rate of multinucleated cells in GL2-introduced cells, as negative control cells, was increased slightly than that in intact wild-type MEFs (supplemental Fig. S6 and Fig. 4C). Thus, it is likely that the multinucleated phenotype in Lats2-/- MEFs is caused by loss of Lats2 protein. On the other hand, to address that increased nuclear size or number in Lats2-/- MEFs correlates with polyploidy, we calculated the nuclear area and intensity of Hoechst-staining nuclear DNA in Lats2+/+ and Lats2-/- MEFs and evaluated the integrated and average intensity of nuclear DNA to their nuclear area (supplemental Fig. S7, A and B, respectively). As a result, the integrated intensity of DNA in Lats2-/- MEFs was elevated in proportion to the increase in the nuclear area, whereas the average intensity of DNA in Lats2-/- MEFs kept a constant range of value in no inverse proportion to the increase in the nuclear area. These results suggest that the increased nuclear size or number in Lats2-/- MEFs were associated with polyploidy. Therefore, it is likely that loss of Lats2 results in nuclear enlargement, multinucleation, and polyploidy. Taken together with previous reports that multinucleation causes subsequent failure of cytokinesis (62), our data suggest that Lats2 also regulates these processes.

Loss of Lats2 Leads to Cytokinesis Failure—To determine whether Lats2 regulates cytokinesis, the fate of Lats2-deficient MEFs was monitored by time-lapse video microscopy, where differential interference contrast (DIC) images were captured at 1-min intervals. As expected, the wild-type MEFs underwent normal cell cleavage and the two daughter cells showed complete separation within 108 min after metaphase (Fig. 4D and supplemental Movie S1). Like the wild-type MEFs, the Lats2-deficient cells entered mitosis normally without G2 arrest. However, during late mitosis, some of the Lats2-deficient cells failed to complete cytokinesis and instead underwent furrow regression that resulted in binucleate cells (Fig. 4E and supplemental Movie S2). As shown in Fig. 4F, the frequency of Lats2-/- MEFs that failed to complete cytokinesis exceeded the frequency of cytokinesis-deficient wild-type MEFs in primary cultures by ~3.3-fold, which is consistent with the frequency of the multinucleated cells in Lats2-/- MEFs as shown in Fig. 4C. The multinucleate Lats2-/- MEFs also possessed multiple centrosomes (Fig. 3C, type III). Thus, the loss of Lats2 function leads to abnormal mitotic progression and subsequently inhibits cytokinesis. This suggests that Lats2 specifically regulates mitosis.


Figure 4
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  		FIGURE 4.
Lats2 is required for cytokinesis as its absence causes nuclear enlargement and multinucleation. A, nuclear enlargement in Lats2-/- MEFs. The cells were fixed with 4% formaldehyde at passage 7 (PDL7) and stained with Hoechst 33258 (blue), anti-{gamma}-tubulin (green) and anti-{alpha}-tubulin (red) antibodies. The nuclear enlargement in Lats2-/- MEFs is indicated by the white arrows. Scale bars, 10 µm. B, frequency of enlarged nuclei (>30 µm in diameter) in Lats2+/+ and Lats2-/- MEFs cells in primary and immortalized cultures. The data shown are the averages of three independent experiments. In each experiment, more than 450 cells were counted. The error bars represent the standard deviations. C, frequency of multinuclei in Lats2+/+ and Lats2-/- MEFs at primary and immortalized cultures. The data shown are the averages of five independent experiments. In each experiment, more than 200 cells were counted. The error bars represent the standard deviations. The cells were fixed and stained as described in A. Hoechst staining revealed binucleated Lats2-/- MEFs (blue in the inset). Scale bars, 10 µm. D and E, time-lapse DIC images of primary Lats2+/+ (D) and Lats2-/- MEFs (E) undergoing mitotic exit. These images only show a subset of the DIC images that were captured every 1 min. The Lats2-deficient MEFs failed to complete cytokinesis and instead formed multinucleated cells. Time in minutes is included in each panel. Scale bars, 10 µm. F, the frequency of Lats2+/+ and Lats2-/- MEFs that failed to complete cytokinesis (red bars). n, total number of cells counted.

 
In contrast, Lats1 has been reported to regulate cytokinesis by inhibiting LIMK1 activity and by acting as a MEN kinase by binding Mob1A (38, 47) and to be activated by membrane-targeted Mob1A and B but not Mob2 (28). However, it remains unclear whether Lats2 interacts with the Mob family. Mob1A and Mob1B share a high degree of sequence similarity (~95%) but both show less homology to Mob2 (~40%) (33). Because it is known that Ndr1, but not Lats1, interacts with Mob2 (38), we speculated that Lats2 may interact with Mob1A or Mob1B. To test this, we co-transfected 293T cells with GFP (green fluorescent protein)-tagged human Lats2 and 3Flag-tagged Mob1A or Mob1B. Immunoprecipitation experiments with anti-GFP antibody revealed that Lats2 associates with both Mob1A and Mob1B (supplemental Fig. S8, A and B). Furthermore, we found that the autophosphorylation activity of Lats2 was markedly enhanced by Mob1A; this was revealed by subjecting Lats2 proteins immunoprecipitated from 293T cells that co-expressed Lats2 with Mob1A or Mob1B to in vitro kinase assays in the presence of [32P]ATP (supplemental Fig. S8B, upper panel). Interestingly, the kinase activity of Lats2 also appeared to be strongly stimulated when the Lats2-expressing cells were treated with the protein phosphatase inhibitor okadaic acid. Furthermore, this okadaic acid-induced elevation in Lats2 autophosphorylation was further enhanced by the binding of Mob1A (supplemental Fig. S8B). This effect of Mob1A increased in proportion to its expression levels (supplemental Fig. S8C). Thus, like Lats1, which acts as a MEN kinase upon binding to Mob1A (38), Lats2 may also act as a MEN kinase by interacting with Mob1A. That the activation of Lats2 requires not only the binding of Mob1A but also phosphatase inhibition suggests Lats2 may form an activation complex that is similar to other Lats-Mob complexes such as Lats1-Mob1A, Dbf2-Mob1, Sid2-Mob1, and Lats-Mats (26, 38, 39, 63).


Figure 5
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  		FIGURE 5.
Loss of Lats2 leads to acceleration of the exit from mitosis. A, growth curves of primary Lats2+/+ and Lats2-/- MEFs at PDL2. Cells were plated at 105 cells per 35-mm dish. Means and standard deviations were derived from three individual Lats2+/+ and Lats2-/- MEF lines. B, time length of each part of divided mitosis (part I, II, III, and IV as shown in C) in Lats2+/+ and Lats2-/- MEFs. A blue dot and a red dot represent a single cell of Lats2+/+ and Lats2-/-, respectively. Each time length of 15 Lats2+/+ and 19 Lats2-/- cells were measured. C, the morphological classification of mitosis in a MEF. The mitotic progression of MEFs was conveniently divided into four parts as follows: part I, from initiation of mitosis to round morphology; part II, from round morphology to ingression of cleavage furrow; part III, from ingression to the completion of furrow formation; part IV, from cleavage furrow formation to cell division. The indicated DIC images are mitotic progression of a Lats2-/- MEF.

 


Figure 6
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  		FIGURE 6.
Loss of Lats2 leads to down-regulation of mitotic regulators. A, Western blot analysis of the expression levels of Aurora-B, Plk1, cyclin B1, Cdc2, phosphorylated tyrosine 15 on Cdc2 (pTyr15-Cdc2), cyclin D1, cyclin E, E2F1, Cdc25A, Cdc25B, Cdc25C, NPM (Nucleophosmin/B23), and Cdc14A. Lysates (100 µg per lane) from Lats2+/+, Lats2+/-, and Lats2-/- MEFs were resolved by SDS-PAGE, followed by Western blotting with antibodies specific to the proteins indicated. Anti-GAPDH antibody was used as loading control. Asterisks indicate nonspecific bands. B, RT-PCR analysis of mRNA expression level of Aurora-B in Lats2+/+, Lats2+/-, and Lats2-/- MEFs. The indicated amounts of total RNA from each MEF were reverse-transcribed and amplified by PCR using specific primer pairs for the genes indicated (Lats2, Aurora-B, and GAPDH). C, immunoprecipitates obtained from Lats2+/+ and Lats2-/- MEF extracts by using the anti-cyclin B1 antibody were resolved by SDS-PAGE, followed by Western blotting with anti-cyclin B1 antibody. As a negative control, immunoprecipitation with normal mouse IgG (NMG) was performed. As a positive control, extracts from asynchronous HeLa S3 cells (AS) were used. The asterisk indicates nonspecific bands. D, Western blot analysis of the stability of Aurora-B, Plk1, and cyclin B1 proteins after CHX treatment (50 µg/ml) for the indicated time points (hours). GAPDH is a loading control. The ratio of each band is shown relative to the amount of each protein in absence of CHX (CHX(-)). Quantification of the intensity of each band was performed by scanning the blots with the fluorescent image analyzer FLA-7000 and analysis with the Multi Gauge software (FUJIFILM, Tokyo, Japan). E, the ratio of Aurora-B, Plk1 and cyclin B1 proteins obtained from D were normalized by the ratio of GAPDH. Aurora-B, Plk1, and cyclin B1 are shown as circles, squares, and diamonds, respectively. Black, blue, and green colors, Lats2+/+ MEFs; Red, pink, and orange colors, Lats2-/- MEFs. F and G, restoration of the levels of Aurora-B, Plk1, and cyclin B1 proteins by knockdown of Cdh1 in Lats2-/- MEFs. The siRNA duplex targeted for mouse Cdh1 (siCdh1) or firefly luciferase as a negative control (GL2) were introduced into primary (F) and immortalized (G) Lats2-/- MEFs by electroporation. Lysates (70 µg per lane) from the siRNA-introduced Lats2-/- MEFs were resolved by SDS-PAGE, followed by Western blotting with antibodies specific to the proteins indicated. Anti-GAPDH antibody was used as a loading control. As controls, lysates (100 µg per lane) from intact primary Lats2+/+ and Lats2-/- MEFs were used.

 
Loss of Lats2 Results in an Acceleration of Exit from Mitosis—When the proliferation rate of cells that completely lacked LATS2 expression was compared with that of wild-type cells derived from littermates, the former were found to grow faster (Fig. 5A). MEFs that are partially deficient in LATS2 expression have a similar phenotype (55). Thus, Lats2-/- cells grow faster regardless of apparent mitotic defects such as centrosome fragmentation, chromosome misalignment, and cytokinesis failure, suggesting that MEFs that completely lack LATS2 expression have an impairment in a checkpoint machinery that governs the mitotic arrest. To address which mitotic stage is sped up in Lats2-/- MEFs, we divided the mitotic progression into four parts based on the morphology of growing cells: part I, from initiation of mitosis to round morphology; part II, from round morphology to ingression of cleavage furrow (which almost corresponds from prometaphase to late anaphase); part III, from ingression to the completion of furrow formation (which almost corresponds from late anaphase to telophase); part IV, from cleavage furrow formation to cell division (Fig. 5C) and measured the time span of each part in Lats2-/- and Lats2+/+ MEFs. As shown in Fig. 5B, part IV in Lats2-/- cells was more dramatically sped up than that in wild-type cells, whereas no remarkable difference was detected between part I and part II. In addition, part III in Lats2-/- cells was also slightly sped up compared with wild-type cells. Moreover, to address whether Lats2-/- cells are defective in mitotic spindle assembly check-point signaling, Lats2-/- and Lats2+/+ MEFs were exposed to spindle damage reagent, nocodazole (an inhibitor of microtubule polymerizing), and then fixed at the indicated time after release (supplemental Fig. S9). For identification of mitotic cells, these cells were stained with anti-phosphorylated histone H3 antibody. The measurements of these mitotic cells showed that Lats2-/- MEFs could be arrested by nocodazole treatment as well as wild-type MEFs (supplemental Fig. S9, 0h). This result suggests that mitotic arrest induced by at least the spindle damage is not defective in Lats2-deficient cells. Therefore, these observations indicate that loss of Lats2 function results in an acceleration of exit from mitosis.

Loss of Lats2 Results in Down-regulation of Mitotic Regulators—It has been shown previously that when mitotic regulators such as Aurora and polo-like kinases are down-regulated by siRNA or the injection of specific antibodies, highly defective mitosis ensues (5, 59, 64-66). Because the loss of Lats2 induces various typical mitotic defects, including fragmentation of centrosomes, chromosome misalignment, and cytokinesis failure (as mentioned above), we examined the protein levels of various mitotic regulators in Lats2-deficient cells. We found that the protein levels of major mitotic regulators such as Aurora-B, Plk1, and cyclin B1 were all markedly decreased in Lats2-/- MEFs (Fig. 6A). To test whether these Lats2-/--specific reductions are because of post-translational degradation, we subjected the total RNAs of Lats2+/+, Lats2+/-, and Lats2-/- MEFs to RT-PCR analysis employing a series of specific primers (Fig. 6B). Because the Aurora-B mRNA level was identical between the three MEF types, it appears that these proteins were present at reduced levels in Lats2-/- MEFs because of post-translational degradation. Notably, while the Western blot analysis seemed to indicate the complete absence of cyclin B1 protein in Lats2-/- cells, small amounts of this protein could be immunoprecipitated from these cells with anti-cyclin B1 antibody (Fig. 6C). Thus, endogenous cyclin B1 protein is present in Lats2-/- cells at dramatically decreased levels. To address the relative stabilities of Aurora-B, Plk1, and cyclin B1 in Lats2+/+ and Lats2-/- MEFs, both cells were exposed to a protein synthesis inhibitor, cycloheximide (CHX), and collected at the indicated time (Fig. 6D). When these cell extracts were subjected to Western blotting with anti-Aurora-B, Plk1, and cyclin B1, the levels of these mitotic proteins were markedly decreased after CHX treatment in Lats2-/- more than Lats2+/+ MEFs, although the levels of Aurora-B and cyclin B1 were also gradually decreased in wild-type MEFs. To estimate the relative amount of these mitotic proteins in Lats2+/+ and Lats2-/- MEFs at each indicated time after CHX treatment, the amounts of these proteins were quantified by densitometry, and the ratio of each protein was normalized by the ratio of GAPDH, a loading control (Fig. 6E). Notably, the normalized ratio of Aurora-B and Plk1 were markedly reduced in Lats2-/- MEFs (red circles and pink squares, respectively), whereas the ratio of cyclin B1 that decreased in Lats2-/- MEFs was slightly different compared with that of cyclin B1 in Lats2+/+ MEFs (orange and green diamonds, respectively). These results suggest that Lats2 is more important for stabilization of Aurora-B and Plk1 than cyclin B1. It has been shown that, along with Cdc20, CENP-E, and Aurora-A, Aurora-B, Plk1, and cyclin B1 appear to be degraded by the proteasome pathway at distinct points during mitotic exit or G1 phase (67). This suggests that Lats2 is involved in stabilization of Aurora-B, Plk1, and Cyclin B1. Since these mitotic proteins are regulated spatially, we examined how the loss of Lats2 affects their subcellular distributions (supplemental Fig. S10). We found that Aurora-B localized normally to the chromosome during G2 and metaphase and at the midbody during cytokinesis in Lats2-/- as well as Lats2+/+ MEFs (supplemental Fig. S10A). In addition, we could not observe any change in kinetochore-like localization of Aurora-B in Lats2-/- MEFs (supplemental Fig. S11). We could also observe the normal centrosomal and midbody localizations of Plk1 in Lats2-/- as well as Lats2+/+ MEFs (supplemental Fig. S10B). Moreover, there was no influence in the nuclear transport and centrosomal localization of cyclin B1 in Lats2-/- MEFs (supplemental Fig. S10C). Thus, it is unlikely that loss of Lats2 affects the subcellular localization of Aurora-B, Plk1, and cyclin B1. On the other hand, the E2F1 protein levels were also slightly reduced in homozygous MEFs (Fig. 6A). In contrast, the levels of cyclin D1 (a G1-cyclin) were increased in Lats2+/- and Lats2-/- MEFs. Notably, the Lats2-/- MEFs showed decreased levels of the inhibited form of Cdc2 (whose tyrosine 15 is phosphorylated) but normal levels of the active form. This suggests that Lats2-/- cells contain more active Cdc2 than Lats2+/+ or Lats2+/- MEFs. Thus, it appears that even though their Cdc2 kinase proteins remain activated, Lats2-deficient cells are forced to exit mitosis without completing the process; this forced exit may be caused by the irregular degradation of major mitotic regulators. This suggests that loss of Lats2 leads to defective mitotic exit and subsequently induces a premature M/G1 phase transition.

It has been reported that critical mitotic regulators including Aurora-B, Plk1, and cyclin B1 are tightly regulated by their protein degradation with the anaphase-promoting complex/cyclosome (APC/C) that is activated by Cdh1 at the M-to-G1 transition (67-69). Therefore, to examine whether inactivation of the APC/C-Cdh1 restores the levels of the reduced mitotic regulators in Lats2-/- MEFs, we knocked down endogenous Cdh1 in Lats2-/- MEFs by siRNA. As shown in Fig. 6F, the levels of Aurora-B, Plk1, and cyclin B1 proteins were successfully restored in primary Lats2-/- MEFs by introduction of Cdh1 siRNA, whereas negative control GL2 did not restore any protein levels. Moreover, we also obtained the similar results in immortalized Lats2-/- MEFs to the primary cells (Fig. 6G). These results indicate that reduction of mitotic proteins in Lats2-null cells depends on the Cdh1-mediated APC/C proteasome pathway, suggesting that Lats2 could prevent a premature mitotic exit by the negative regulation of this pathway.

Taken together, our data suggest that Lats2 is required for a wide variety of critical mitotic events, namely, centrosome regulation, chromosome alignment, precise progression of mitotic exit, and completion of cytokinesis. They also suggest that Lats2 may govern the stability of Aurora-B, Plk1, and cyclin B1 by a negative regulation of the APC/C-Cdh1 pathway. However, it remains unclear whether Lats2 directly regulates the protein levels of these mitotic regulators.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
REFERENCES
 
Embryonic Lethality of a Total LATS2 Knockout in Mouse Is Caused by Disrupted Development of the Nervous System—In this study, we have demonstrated that Lats2-deficient embryos, with complete disruption of Lats2, developed an abnormality in the nervous system that was ultimately lethal at E11.5. A recent report showed that a partial LATS2-knock-out mouse, in which the N-terminal region of Lats2 remained intact, displayed cardiac disruption with invasion of irregular supernumerary cells into the atrial chamber, as well as defective trabeculae in the ventricular chamber, resulting in embryonic lethality before E12.5 (55). However, in our complete LATS2-knock-out mice we observed no defects in cardiac growth at any embryonic developmental stage (Fig. 2). In brief, the hearts developed normally, without any trabeculation defects or thinner myocardium, but were smaller than those of the wild-type.

Instead, we found that our complete LATS2-knock-out embryos exhibited severe abnormalities in the nervous system, as well as nerve cell death. These results appear contradictory to previous data showing that overexpression of Lats2 induces apoptosis in human cancer cells (50, 52). However, Hou et al. (26) reported that Sid2 forms homodimers in its inactive form, whereas SIN-dependent phosphorylated Sid2 associates with Mob1 as an active complex. In a yeast two-hybrid assay, the N terminus of Lats1 was shown to associate with its own C terminus (21), and using a pull-down assay, we found recently that Lats2 can associate with Lats1.3 Thus, in the normal developmental processes of the mouse nervous system, Lats2 is predicted to associate with Lats1, which is prominently expressed in tissues of ectodermal origin, such as the neural tube and head fold neuroepithelium. It might also be predicted to inhibit irregular induction of apoptosis by excess Lats1, via a mechanism similar to Sid2 self-association. Thus, loss of Lats2 may cause inappropriate activation of Lats1 and lead to irregular apoptosis in the nervous system, as observed in the spinal cord of our complete LATS2-knock-out embryos.

Moreover, unlike Lats1, which is expressed mainly in tissues of ectodermal origin during embryonic developmental stages E8.5-E10.5, Lats2 is prominently expressed in tissues of mesodermal origin (55). However, it remains unclear whether or not Lats2 is expressed in tissues of ectodermal origin (including the nervous system) after E10.5. Since mouse and human Lats2 may be expressed in the brain of normal adult mice, and the brain and spinal cord of humans, respectively (22), it is likely that Lats2 is expressed and functions in the nervous system during embryogenesis. Further studies are required to establish the relationship between Lats2 and Lats1 in respect to embryonic development.

Mechanism of Centrosomal Regulation by Lats2—Centrosome dysregulation, which leads to centrosome overduplication, is associated with tumor progression in most human cancers such as the breast, prostate, and colon (2, 4). The centrosome checkpoint ensures that centrosomes duplicate only once during the cell cycle. Loss of centrosome checkpoint function leads to centrosome amplification including centriole overduplication and centrosome fragmentation, resulting in aberrant mitotic spindle formation followed by genomic instability and aneuploidy. Inactivation of Lats2 caused centrosome abnormality but not aberrant mitotic spindle formation such as multipolar spindle formation. How does centrosome abnormality occur in Lats2-deficient cells? Recently suggested models may provide insight into the mechanism of centrosome abnormality: one such model proposes that direct deregulation of centrosome duplication may bypass the cell division cycle, leading to centrosome overduplication, and the centrosome amplification so-called (70); another suggests that failure of cytokinesis results in tetraploidy, resulting in more than one centrosome per cell (57, 71). It is thought that cyclin E/Cdk2 kinase plays a key role in the regulation of centrosome duplication through phosphorylation of the nucleolar proteins Nucleophosmin (NPM/B23), a centrosomal kinase (Mps1) and a centrosome duplication-promoting factor CP110 (72-75). Recent publications have indicated that inactivation of p53, Skp2, E2F3, or Pml in mice causes aberrant centrosome amplification that is related to activation of cyclin E/Cdk2 kinase (76-79). However, centrosome abnormality in Lats2-deficient cells might not be accompanied by activation of cyclin E/Cdk2, as the protein level of cyclin E was unaffected between Lats2-deficient and wild-type MEFs (Fig. 6A). Moreover, our results demonstrated that Lats2-deficient cells aborted cytokinesis, and that this was followed by binucleation, suggesting that failures of cell division may contribute to the increased number of centrosomes in Lats2-deficient cells (Fig. 3, C and D, type III). However, some Lats2-deficient cells possessed >4 centrosomes per cell (e.g. 7 per cell, data not shown), a finding which does not correspond to the idea of centrosome number being a consequence of defects in cell division. In fact, we demonstrated that loss of Lats2 leads to the fragmentation of pericentriolar material (Fig. 3, C and D, type I). In addition, a recent report has shown that depletion of Lats2 in HeLa cells by siRNA treatment blocks the recruitment of {gamma}-tubulin to centrosomes; this effect was also observed when Ajuba, a centrosomal LIM protein, was depleted (80). Taken together, these results suggest that centrosome abnormality in Lats2-deficient cells may be responsible for both the centrosome fragmentation and the observed defects in cytokinesis. Conversely, it is also possible that the centrosome abnormality is caused by the dysfunction of mitotic spindle as a collapsed spindles, by which cytokinesis could be disturbed.

Regulation of Cytokinesis by Lats2—Recently, it has been reported that a number of novel centrosomal proteins such as Cep55 and Su48 regulate cytokinesis in mammalian cells (81, 82). We have shown here that, like these centrosomal proteins, the centrosomal protein Lats2 localizes at the midbody during late mitosis and plays a role in cytokinesis. Although a previous study has revealed that partial LATS2-knock-out MEFs show increased retention of the cytoplasmic bridge (55), our results indicate that this does not occur in MEFs that completely lack LATS2 expression. This suggests that the cell division cycle in the latter cells may progress without telophase arrest because of cytokinesis failure. Supporting this is that the Lats2-deficient cells proliferated faster than the wild-type cells derived from littermates (Fig. 5A). The partial LATS2-knock-out MEFs also showed increased growth rates (55). In addition, we demonstrated that Lats2-null cells cause an acceleration of exit from mitosis (Fig. 5B). Notably, compared with Lats2-null cells, wild-type MEFs take longer and more variable time span to complete cytokinesis (Fig. 5B, part IV, blue circles). It is possible that normal MEFs possess an unknown machinery that insures a precise timing of exit from mitosis by arresting cell cycle until cytokinesis is normally completed, if cells encounter a crisis that may cause cytokinesis failure. MEFs that completely lack LATS2 expression may have an impairment in this machinery that governs arrest during cytokinesis; this impairment may cause the cells to undergo an incomplete exit from mitosis to G1 phase and uncouple the onset of cytokinesis.

Why do Lats2-deficient cells grow rapidly despite their cytokinetic defects? It is possible that Lats1 protein at least partially complements the intrinsic biological functions of Lats2 in cytokinesis. Consequently, both Lats2-deficient cells and the subsequent tetraploid cells are able to continue duplicating and proliferating. Thus, to further clarify the biological function of Lats2 in mitotic exit and cytokinesis, it will be necessary to generate and analyze double mutant cells that lack both Lats1 and Lats2.

Is Lats2 an Essential Regulator of Mitotic Exit?—Recent publications have shown that in mammalian cells, Lats1 inhibits the impairment of cytokinesis induced by LIMK1; moreover, as a component of the MEN, it functions by interacting with the Lats1 activator Mob1A (38, 47). It is possible that Lats2 also functions as a MEN kinase since we found that Lats2 can interact with both Mob1A and Mob1B (supplemental Fig. S8). Interestingly, the recruitments of the Lats1-Mob1A and the Lats1-Mob1B complexes to the plasma membrane are required for their activations (28), whereas our data revealed that the Lats2-Mob1A complex is activated without such recruitment (supplemental Fig. S8). These results suggest that the biological function of the Lats2-Mob1A is not identical to that of the Lats1-Mob1A/B. To clarify whether Lats2 acts as a bona fide MEN kinase in mitotic exit, further studies will be required. In particular, it will be of interest to determine whether Lats2 is activated by Mob1A and Mob1B during mitotic exit and how Lats2 helps to regulate mitotic exit and cytokinesis. Identification of the phosphorylation target of Lats2 kinase (e.g. Cdc14A or Cdh1) will also be informative. Moreover, it may be of interest to determine whether Lats2 is involved in the activation of separase, which is a sister chromatid-separating protease. This is because the activation of separase in budding yeast, which induces the release of Cdc14 by down-regulating protein phosphatase 2A and Cdc55, initiates mitotic exit (83).

We have also demonstrated that the protein levels of major mitotic regulators such as Aurora-B, Plk1, and cyclin B1 are markedly decreased in Lats2-/- MEFs (Fig. 6). It will be of interest to determine whether Lats2 regulates these mitotic regulators directly or indirectly. We have reported previously that Lats2 co-localizes with Aurora-A at the centrosome, and other recent reports have indicated that Aurora-A, Plk1, and cyclin B1 localize at the centrosome (1, 66, 84). Thus, it is likely that Lats2 forms a functional complex with these mitotic proteins. However, it remains unclear whether Lats2 can directly phosphorylate these proteins in vitro or in vivo.

It has been demonstrated that the proteins Aurora-B, Plk1, and cyclin B1 are rapidly degraded by the proteasome pathway at distinct times during mitotic exit (67). Therefore, the degradation of these proteins may be responsible for the rapid progression of the M/G1 transition induced by loss of Lats2 function. In fact, we have shown a possibility that the dramatic reduction of these mitotic proteins in Lats2-/- MEFs is provoked by a profound dysregulation of the APC/C-Cdh1 proteasome pathway and the timing of exit from mitosis is consequently accelerated. In budding yeast, the Cdh1/Hct1 has been reported to be dephosphorylated by Cdc14 phosphatase and be subsequently activated in order to initiate the destruction of B-type cyclin in late mitosis (85), while Dbf2-Mob1 complex has been shown to sustain Cdc14 release from the nucleolus to keep its phosphatase activity under the MEN signaling (86). Thus, it is likely that Dbf2 is a component of the budding yeast MEN and is a positive regulator of mitotic exit and cytokinesis. However, in mammalian cells, Lats2 is required for cytokinesis but negatively regulates mitotic exit because our presented data suggest that Lats2 delays mitotic exit through inactivation of Cdh1 (Figs. 5 and 6). In contrast to the MEN, the fission yeast SIN primarily regulates cytokinesis and is dispensable for mitotic exit. The SIN mutants containing sid2 cause cytokinesis errors and results in multinucleated cells. In fact, Lats2-deficient cells also cause cytokinesis failure and results in multinucleated cells (Fig. 4). In addition, these cells lead to the instability of important mitotic kinases in undergoing cytokinesis, Plk1 and Aurora-B (Fig. 6). Therefore, the signaling network to which Lats2 belongs appears to behave more like the SIN than the MEN. Furthermore, there was a decrease in the inhibitory phosphorylation of tyrosine 15 on Cdc2, in Lats2-/- MEFs, indicating enhanced activation of Cdc2 compared with wild-type MEFs. These results are consistent with a previous report that Plk1 depletion results in elevated Cdc2 protein kinase activity and incomplete separation of sister chromatids (64). In light of the above results, we propose that Lats2 is one of the critical mitotic regulators of centrosomal integrity and completion of cytokinesis, that operates via stabilization of mitotic regulators such as Aurora-B, Plk1, and cyclin B1.

In summary, we have demonstrated that completely LATS2-deficient mice show partly similar, but essentially distinct phenotypes, from partially LATS2-deficient mice. We also report here novel phenotypes of LATS2-deficient cells that were not reported previously (55). These distinct phenotypes and novel findings are as follows: First, embryonic lethality of the complete LATS2-knock-out mouse due to severe developmental defects in the nervous system, but not in cardiac growth or structures; second, MEFs from complete LATS2-knock-out embryos exhibit a wide variety of mitotic defects such as centrosome fragmentation and misaligned chromosomes; third, complete LATS2-knock-out MEFs exhibit cytokinetic defects without telophase arrest, followed by tetraploidy formation, nuclear enlargement and multinucleation. Our results reported here suggest that Lats2 is required for cytokinesis. In addition, we have also demonstrated that complete LATS2-knock-out MEFs exhibit an acceleration of exit from mitosis and down-regulation of critical mitotic regulators, suggesting that Lats2 is an essential mitotic regulator that governs the stabilization of other mitotic regulators.


   FOOTNOTES
 
* This work was supported in part by Grants-In-Aid from Innovation Plaza Osaka of Japan Science and Technology Agency (JST), the Bio-Medical Cluster Project In Saito, and Scientific Research on Priority Areas, Scientific Research (S), Exploratory Research and Science, and the Technology Incubation Program in Advanced Regions of the Ministry of Education, Science, Sports and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains detailed experimental procedures, Figs. S1-S11, Tables S1-S3, and Movies S1 and S2. Back

1 To whom correspondence should be addressed: Dept. of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan. Tel.: 81-6-6875-3980; Fax: 81-6-6875-5192; E-mail: snj-0212{at}biken.osaka-u.ac.jp.

2 The abbreviations used are: Lats, large tumor suppressor; cdk, cyclin-dependent kinase; WT, wild type; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; CHX, cycloheximide; miRNA, microRNA; siRNA, small interfering RNA; PGK-neo, phosphoglycerate kinase-neomycin; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; LCD, Lats conserved domains; GFP, green fluorescent protein; MEN, mitotic exit network; SIN, septation initiation network. Back

3 N. Yabuta and H. Nojima, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Hughes for critically reading the manuscript. We also thank A. Kawai, Y. Maruyama (Genome Info. Res. Center, Osaka University) and M. Sugimoto (R. I. M. D., Osaka University) for technical assistance.



   REFERENCES
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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