Whole-mount immunostaining with anti-Mats antibodies indicated that mats is activated throughout development and ubiquitously expressed at a low level in tissues such as larval eye discs (Lai, 2005).
The directed expression of a dominant negative Trc protein provides a sensitized system for identifying interacting genes (He, 2005a). Deficiencies for each of the fly mob genes enhances the wing hair phenotype that results from driving expression of UAS-trcT453A using either ap-GAL4 or ptc-gal4. The strongest enhancement is seen with deficiencies for mats and Dmob2. These results suggested the possibility that all 4 Dmobs can redundantly interact with Trc, although it is possible that the interactions could be indirect or due to other genes in deleted regions. It is worth noting that such interactions are not common. When the Drosophila deficiency collection was screened for enhancement or suppression of ap-GAL4 UAS-trcT453A, <10% of the Dfs showed an interaction (He, 2005b).
To confirm that the genetic interaction between trc and Df (mats) was due to the reduction in mats dose, two independent alleles were used. One was the null allele described by Lai (2005) (matse235), which is deleted for almost the entire coding region, and the other was a lethal PiggyBac insertion allele of mats [PBac{RB}CG13852e03077] (this allele is referred to as matsPB). Because this later allele has not been well characterized, the insertion was determined to be lethal over a deficiency for the region (Df(3R)Exel6191), and it failed to complement the recessive lethality of matse235 consistent with the lethality being due to the PB insertion. This mutation could be reverted using a source of PiggyBac transposase. It was found that both mats alleles dominantly enhance the trc dominant negative phenotype and this enhancement is lost in the PB revertant. It was also found that over expression of mats from a UAS-mats transgene (Lai, 2005; driven by ptc-Gal4) partially suppresses the multiple hair cell phenotype that results from driving expression of Trc-DN using ptc-Gal4. These dose responses argue that Mats activates Trc. Interestingly, it was found that heterozygosity for a wts mutation also enhances the Trc dominant negative phenotype, although somewhat less strongly (He, 2005b).
Evidence was also obtained for mats functioning with trc and fry using simple loss of function mutations. Wild-type flies or flies heterozygous for either trc, fry, or mats appear normal and only rarely (on fewer than 5% of wings) is even a single multiple hair cell seen. Flies that were heterozygous for two of these genes showed a slightly higher frequency of wings with one or a couple of multiple hair cells (often ~10%) but the increase was not routinely significant. However, almost half of the wings from flies that are heterozygous for all three genes (e.g., fry2 trc1+/+ + matsPB) show a weak multiple hair cell phenotype, a significant increase. This genetic interaction is further support for the hypothesis that trc, fry, and mats function together in regulating wing hair development. In this assay no equivalent interaction with wts3-17 was seen (He, 2005b).
Previous studies established that trc also has a larval denticle phenotype (Geng, 2000). In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of matsPB/matsPB homozygous larvae is also disorganized and contains many split denticles. The number of split denticles is similar in matsPB/Df larvae, suggesting that for this phenotype, matsPB is a strong, near phenotypic null allele. The matse235 also showed a similar denticle phenotype. The phenotype of matsPB homozygotes is slightly less severe than that of trcP/trcP larvae. Notably, trcP matsPB/trcP matsPB double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development (He, 2005b).
Both mats alleles are larval lethals with death typically in the second or early third instar. To examine the phenotype of mats in wing cells, mosaics were generated using FLP/FRT. mats clones on the wing, leg, thorax, and head display two types of phenotype. The most notable is indistinguishable from those produced by clones of wts, suggesting that mats also functions with wts as has been shown by Lai (2005). On the wing, small clones produced bulges that can be seen at low magnification with a stereomicroscope. In mounted wings individual cell outlines are visible in the cuticle and the cells appear to have a bulging apical surface. The hairs are located on an elevated pedestal, a phenotype that is indistinguishable from those seen in wts clones. The hairs were often broader than normal. Particularly in other body regions clones were abnormally pigmented (either darker or lighter than normal, and there were outgrowths of clone tissue. In highly abnormal wing clones, evidence of clustered and split hairs were often seen that were typical of trc mutant clones. Some multiple hair cells were seen in very abnormal wts clones but this phenotype appears less severe (e.g., number of hairs per cell) than that seen with mats or trc. These observations suggest that mats functions with both Trc and Wts (He, 2005b).
matsPB and matse235 clones in pupal wings were examined. Mutant mats cells are able to outcompete their neighbors and end up comprising most of the wing when clones are induced early. As was expected from the morphology of clones in adult wings, the pupal clones produce bulges in the wing and individual cells also often appear bulged. Clone cells stain more brightly for F-actin. This is true both in developing hairs and in the general apical cortex. This phenotype is clear-cut enough that it could be used as a convenient marker of mats mutant cells. These phenotypes were seen with both mats alleles tested. A similar, increase in actin staining was seen in wts clones. A similar, but perhaps less severe increase in staining, is seen in trc clones (He, 2005a). In some, but not all mats clones, large numbers of multiple hair cells can be seen. At later stages a circular pedestal of actin staining could be seen surrounding the base of the hair in mutant cells but not in surrounding wild-type cells. At still later stages the wild-type cells also had a circular pedestal of actin staining, suggesting that the mutant cells might be developmentally more advanced. Consistent with this possibility, in many clones hair initiation and outgrowth appear to be advanced in mats mutant cells compared with neighbors. This is also the case for wts clones, but it is the opposite of trc clones, in which hair development is often delayed (He, 2005a). Cells in mats clones have a smaller cross section so that the array of hairs appears denser, which is also the opposite of what is seen in trc clones, in which there is an increase in cross-sectional area (He, 2005a). Once again the phenotype of the wts clone cells resembles that seen for mats cells. Thus, for several wing phenotypes mats mutant cells resembled wts and not trc cells. Indeed, the mats phenotype is the opposite of trc for both cell area and the timing of hair morphogenesis (He, 2005b).
It has been found that the accumulation of Fry in wing cells is subject to feedback control that is dependent on Trc activity. Hence, in a trc mutant, increased Fry accumulation is found (He, 2005b). Several of the observations described above suggest the hypothesis that mats functions along with trc and is important for Trc activation. From this it is predicted that Fry accumulation would also be elevated in Dmob1 clones. Increased levels of Fry immunostaining were found in Dmob1 clone cells; this is consistent with the hypothesis. This is also seen in wts mutant clones, although the increase appears less dramatic (He, 2005b).
Tumorous overgrowth phenotypes are a consequence of mutations in a number of Drosophila genes. In several cases, such as lethal giant discs overgrown imaginal discs are found in late third instar larvae. To determine whether that is also the case for mats, mats/Df mutant larvae were examined. These larvae grow slowly and after 5 d of growth, when wild-type larvae begin to pupate, mats/Df larvae are the size of early third instar larvae. These larvae routinely die without growing substantially larger. When 5.5-5-d-old mats larvae were dissected, not evidence of tumors or overgrowth of imaginal or other tissues was found. Rather, the imaginal discs were approximately the size of those seen in 4-d larvae. However, the mats homozygous discs did not appear normal, since they were abnormally shaped and more folded than normal discs of this size (He, 2005b).
In a number of experiments involving mats or wts, what appeared to be spontaneous tumors or clones was observed. This was seen most often in flies that also carried reduced doses of Dmob3 and Dmob4. It is thought that these overgrowths are due to spontaneous mitotic recombination, because when the flies were also mutant for trc or fry, evidence of trc or fry clones was seen. The trc and fry clones were seen less frequently. This could be due to these genes being located more proximally on the chromosome than mats or wts, but it might also be due to the competitive advantage of mats and wts clones, resulting in these clones being larger and easier to detect. The basis for these clones is unclear but suggests genomic instability in mats and/or wts mutants (He, 2005b).
A spontaneous lethal mutation was identified in the Drosophila gene mob as tumor suppressor (mats). From mutant clones generated in mats mosaic flies, large tumors can be induced in many organs including the head, notum, eye, wing, leg, antenna, and halteres. Thus, mats appears to function as a general inhibitor of tissue growth. The tumor cells formed unpatterned tissue with many folds on the surface. Using green fluorescent protein (GFP) to positively label mutant cells, it was apparent that mutant cells overproliferated in comparison to wild-type cells. Mosaic larval eye discs with mats mutant clones were apparently larger and folded in many areas. Overproliferation of mats mutant cells could not be explained by change in cell size, because the size of the mutant cells is not significantly different from that of wild-type cells. To directly examine the cell proliferation phenotype, Bromdeoxyuridine (BrdU) was incubated with eye discs to identify cells in the S phase. In wild-type larval eye discs, BrdU incorporation is evident in cells anterior to the morphogenetic furrow (MF) and in the second mitotic wave (SMW), which is a narrow stripe of dividing cells a few rows posterior to the MF. Consistent with an inhibitory role of mats in regulating cell proliferation, BrdU incorporation was elevated in mats mutant clones in eye discs. This phenotype was more evident in mats clones located in the MF and SMW regions. Moreover, immunostaining with anti-phospho histone H3 (PH3) antibody was carried out to identify mitotic cells in eye discs. In wild-type, PH3-positive cells can be found in the anterior and SMW regions, but not in the MF. They were rarely detected in the posterior region. In mats mosaic eye discs, more mitoses were observed in mats clones compared to neighboring wild-type cells, and mitoses can even sometimes occur in the MF. Excess mitoses were also found in mats clones located in the posterior margin. On the basis of these observations, it is concluded that mats is required to restrict cell proliferation and loss of mats function allows cells to divide at a time when they should exit the cell cycle (Lai, 2005).
To characterize the molecular nature of mats gene, deficiency and meiotic mapping experiments were carried out, and mats was localized in the 94A region on the third chromosome. By using a P transposon-mediated site-specific recombination method, mats was further mapped to a 13 kb region at 94A12. Molecular analysis of a candidate gene in this region, designated CG13852, revealed that a 428 bp Roo transposon sequence was inserted behind codon 84 to cause a premature termination. This first mutant allele of CG13852 is named roo in this study. A second mutant allele of this gene, referred to as e235, was generated by mobilizing a P transposon EP(3)3303 inserted approximately 2 kb downstream of CG13852. Like roo, e235 causes homozygous lethality at early second larval stage and induces tumors in somatic clones of mosaic flies. Based on the larval lethal phenotype, e235 failed to complement roo as well as deficiency chromosomes with the 94A12 region deleted. Sequence analysis of e235 has indicated that the second and third exons of CG13852 were deleted. Although the first exon is still intact, it encodes only the first four amino acids of CG13852. Thus, e235 is a null allele of CG13852 (Lai, 2005).
CG13852 encodes a 219-amino acid polypeptide, which is approximately 25 kDa in size. Due to CG13852's apparent homology to the Mob superfamily, it is renamed here Mats (Mob as tumor suppressor). As a member of the Mob superfamily, Mats has no significant homology with any other previously characterized protein domains. To test if CG13852 can rescue defects induced by mats mutations, an in vivo assay was established to allow CG13852 transgene expression only in mutant cells by using the MARCM system. It was found that expression of a full-length CG13852 cDNA in mutant cells suppresses tumor growth and pupal lethality associated with the mosaic flies. Moreover, ubiquitous expression of UAS-CG13852 driven by arm-Gal4 rescues larval lethality of mats homozygous mutants. These results further demonstrated that CG13852 corresponds to mats (Lai, 2005).
Cyclin E, a key regulator for the G1-S transition, is normally expressed in the second mitotic wave (SMW) of larval eye discs. In mats mosaic eye discs, Cyclin E levels are elevated in mutant clones located in the morphogenetic furrow (MF) and SMW regions. Moderate upregulation of Cyclin A and Cyclin B expression is also observed. Thus, an important mechanism for mats to control cell proliferation is to negatively regulate expression of key cell cycle regulators such as Cyclins. Interestingly, Cyclin E expression in mutant cells immediately anterior to the MF is much less elevated than that immediately posterior to the MF, suggesting that mats may use a different mechanism to restrict cell proliferation in cells anterior to the MF. The cell proliferation defects observed in mats mutants are similar to those caused by sav, wts, and hpo mutations (Lai, 2005).
Whether mats plays a role in regulating cellular differentiation in the developing eye was investigated. In larval eye discs, photoreceptors (R) and cone cells in mats clones appear to be specified normally. However, they fail to fully differentiate to generate ommatidia in adult. Defective retinal differentiation occurs at least at mid-pupal stages. Thus, mats is required for cellular differentiation during eye development. In contrast, hpo, sav, and wts do not significantly affect differentiation of retinal cells during eye development (Lai, 2005).
Apoptosis provides an important mechanism for the control of cell number and organ size. To test if mats plays a role in cell death control, expression of DIAP1 in eye discs was examined. DIAP1 is a caspase inhibitor essential for cell survival. Through immunostaining of mats mosaic eye discs, it was found that the level of DIAP1 protein is increased in mats clones. To examine if mats regulates diap1 at the transcriptional level, an enhancer trap line thj5C8 was used, in which a lacZ reporter gene is inserted in diap1 and expression pattern of diap1-lacZ reflect that of the endogenous diap1 gene. It was found that expression of diap1-lacZ was elevated. Thus, mats is required to negatively regulate DIAP1 expression. To directly test the idea that mats promotes apoptosis, mats mutant clones were induced in larval eye discs that overexpress an apoptosis-promoting gene head involution defective (hid) in all cells behind the MF. As expected, expression of hid in a wild-type background increased apoptosis to cause a reduced eye phenotype. Notably, removal of mats function blocks hid-induced cell death and significantly suppresses the small eye phenotype. In these same tissues, developmental cell death is observed in regions anterior to the MF, where expression of the hid transgene is not induced. In these cases, apoptosis occurs only in wild-type tissues but not in mats mutant clones. Thus, mats is also required for developmentally programmed apoptosis. All together, these findings support a model that mats is required to facilitate cell death, and loss of mats’ apoptosis-promoting activity may contribute to tumor development (Lai, 2005).
Through phylogenetic analysis, four major groups of Mob proteins within the Mob superfamily were found. CG13852/Mats and its orthologs from animals and plants form the Mats gene family. Mats proteins are highly conserved. For instance, fly Mats and human Mats1 (also named Mob1A) are 87% identical. Even plant Mats orthologs are over 64% identical to fly Mats. In comparison, fly Mats shares no more than 40%-50% sequence identity with all other non-Mats Mob proteins from species such as yeast, fly, and humans. Such high levels of sequence conservation suggest that function of Mats proteins is evolutionarily conserved. To test functional homology, human Mats1 was introduced into Drosophila; it can effectively suppress tumor growth and rescue pupal lethality of mats mosaic flies. Thus, the growth inhibitory function of Mats has been conserved from insects to humans (Lai, 2005).
To further test the hypothesis that mammalian Mats functions as a tumor suppressor, 89 human and 8 mouse tumor-derived Mats cDNA sequences reported as expressed sequence tags (EST) in GenBank were examined and 2 Mats1 ESTs with disruptions in the coding region were identified, which were subsequently verified by sequencing analysis. In the first case, three nucleotides were deleted in a Mats1 cDNA (hMats1ΔS6/7) derived from a human skin melanoma, which caused deletion of the sixth (or seventh) codon for Ser. This mutation greatly destabilized hMats1 because no hMats1 protein was detectable from hMats1ΔS6/7-transfected human 293T cells. Another Mats1 cDNA (mMats1Δ6-216) derived from a mouse mammary gland carcinoma contains a 38 bp insertion immediately downstream of the fifth codon that causes a premature termination. As expected, no mMats1 protein product was detected. These findings are exciting as they support a model that Mats1 may function as a tumor suppressor in mammals (Lai, 2005).
Reference names in red indicate recommended papers.
Bichsel, S. J., (2004). Mechanism of activation of NDR (Nuclear Dbf2-related) protein kinase by the hMOB1 protein. J. Biol. Chem. 279: 35228-35235. 15197186
Colman-Lerner, A., Chin, T. E., and Brent, R. (2001). Yeast Cbk1 and Mob2 activate daughter-specific genetic programs to induce asymmetric cell fates. Cell 107: 739-750. 11747810
Devroe, E., Erdjument-Bromage, H., Tempst, P. and Silver, P.M. (2004). Human Mob proteins regulate the Ndr1 and Ndr2 serine-threonine kinases. J. Biol. Chem. 279: 24444-24451. 15067004
Geng, W., He, B., Wang, M. and Adler, P. N. (2000). The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase. Genetics 156: 1817-1828. 11102376
Giot, L. et al. (2003). A protein interaction map of Drosophila melanogaster. Science 302: 1727-1736. 14605208
Hay, B. A. and Guo, M. (2003). Coupling cell growth, proliferation, and death. Hippo weighs in. Dev. Cell 5: 361-363. 12967554
He, Y., Fang, X., Emoto, K., Jan, Y. N. and Adler, P. N. (2005a). The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development. Mol. Biol. Cell 16(2): 689-700. 15591127
He, Y., Emoto, K., Fang, X., Ren, N., Tian, X., Jan, Y. N. and Adler, P. N. (2005b). Drosophila Mob family proteins interact with the related tricornered (Trc) and warts (Wts) kinases. Mol. Biol. Cell 16(9): 4139-52. 15975907
Hou, M. C., Wiley, D. J., Verde, F., and McCollum, D. (2003). Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression. J. Cell Sci. 116: 125-135. 12456722
Hou, M. C., Guertin, D. A. and McCollum, D. (2004). Initiation of cytokinesis is controlled through multiple modes of regulation of the Sid2p-Mob1p kinase complex. Mol. Cell. Biol. 24(8): 3262-76. 15060149
Komarnitsky, S. I., et al. (1998). DBF2 protein kinase binds to and acts through the cell cycle regulated MOBI protein. Mol. Cell. Biol. 18: 2100-2107. 9528782
Lai, Z.-C., et al. (2005)
Luca, F. C. and Winey, M. (1998). MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy. Mol. Biol. Cell 9: 29-46. 9436989
Mah, A. S., Jang, J., and Deshaies, R. J. (2001). Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci. 98: 7325-7330. 11404483
Nelson, B., et al. (2003). Ram: A conserved signaling network that regulates ace2p transcriptional activity and polarized morphogenesis. Mol. Biol. Cell 14: 3782-3803. 12972564
Ponchon, L., et al. (2004). NMR solution structure of Mob1, a mitotic exit network protein and its interaction with an NDR kinase peptide. J. Mol. Biol. 337(1): 167-82. 15001360
Rothenberg, M. E. and Jan, Y. N. (2003). Cell biology: the hippo hypothesis. Nature 425: 469-470. 14523431
Ryoo, H. D. and Steller, H. (2003). Hippo and its mission for growth control, Nat. Cell Biol. 5: 853-855. 14523394
Stavridi E. S., et al., (2003). Crystal structure of a human Mob1 protein: toward understanding Mob-regulated cell cycle pathways. Structure 11: 1163-1170. 12962634
Tamaskovic, R., Bichsel. S. J. and Hemmings, B. A. (2003). NDR family of AGC kinases -- essential regulators of the cell cycle and morphogenesis. FEBS Lett. 546: 73-80. 12829239
Weiss, E.L., Kurischko, C., Zhang, C., Shokat, K., Drubin, D.G. and Luca, F.C. (2002). The Saccharomyces cerevisiae Mob2p-Cbk1p kinase complex promotes polarized growth and act with the mitotic exit network to facilitate daughter cell-specific localization of Ace2p transcription factor. J. Cell Biol. 158: 885-900. 12196508
date revised: 21 December 2005
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