NuMA is required for proper spindle assembly and chromosome alignment in prometaphase
© Merdes et al; licensee BioMed Central Ltd. 2009
Received: 03 February 2009
Accepted: 28 April 2009
Published: 28 April 2009
NuMA is a protein that has been previously shown to play a role in focusing microtubules at the mitotic spindle poles. However, most previous work relies on experimental methods that might cause dominant side effects on spindle formation, such as microinjection of antibodies, overexpression of mutant protein, or immunodepletion of NuMA-containing protein complexes.
To circumvent these technical problems, we performed siRNA experiments in which we depleted the majority of NuMA in human cultured cells. Depleted mitotic cells show a prolonged duration of prometaphase, with spindle pole defects and with unattached, unaligned chromosomes.
Our data confirm that NuMA is important for spindle pole formation, and for cohesion of centrosome-derived microtubules with the bulk of spindle microtubules. Our findings of NuMA-dependent defects in chromosome alignment suggest that NuMA is involved in stabilizing kinetochore fibres.
NuMA, the protein of the 'Nucleus and Mitotic Apparatus', is a structural protein in vertebrates of approximately 230 kDa. It localizes to the nucleus during interphase, and accumulates at the spindle poles during mitosis . NuMA has been implicated in the formation of the mitotic spindle, in particular in focusing the spindle poles . Moreover, in recent years it has been shown that part of NuMA localizes to the cell cortex during mitosis where it interacts with the protein LGN/pins [3, 4]. It has been suggested that cortical NuMA participates in spindle orientation, a role that has also been attributed to related proteins in Drosophila and Caenorhabditis elegans, termed Mud or LIN-5, respectively [4–9]. So far, the majority of experiments that tested the role of vertebrate NuMA relied on methods such as antibody microinjection, overexpression of NuMA mutants, or depletion of NuMA from cytoplasmic extracts [10–22]. The cumulative evidence from these experiments pointed towards a function of NuMA in crosslinking microtubules at the spindle poles, enabling the formation and maintenance of the bipolar spindle apparatus. The shortcomings of these experiments were that they could not distinguish between a direct effect on NuMA function, and an indirect effect on interacting proteins: 1) Antibodies are large proteins; therefore, upon microinjection they might sterically hinder the function of neighbouring proteins that are in close contact with NuMA. Moreover, antibodies of the immunoglobulin G type may induce crosslinking of NuMA and produce dominant effects that are unrelated to the normal function of NuMA. 2) Similarly, overexpression of mutant forms of NuMA might produce dominant effects due to unphysiological behaviour of the mutant protein, or due to protein aggregates resulting from the overexpression itself. 3) Although depletion of NuMA from cytoplasmic extracts that form spindles or microtubule asters in vitro seems an interesting experimental alternative, it can be disputed as to how closely these assays reflect the mechanisms of real mitosis in a living cell. Furthermore, it cannot be excluded that during depletion of NuMA, interacting proteins are co-depleted that are themselves essential for regular mitosis. Interestingly, a very recent report documented the properties of a loss-of-function allele of NuMA in mouse cells . This mutant form of NuMA lacked exon 22 and was therefore thought to lack binding to spindle microtubules in mitotic cells. Cells expressing this mutant allele and lacking full-length NuMA displayed spindle pole defects, and showed defects in metaphase chromosome alignment . However, it can't be excluded that the mutant allele produced dominant effects, as already discussed for other NuMA mutants.
In conclusion, our depletion experiments suggest that NuMA is necessary for proper spindle formation in prometaphase, and that NuMA-dependent defects manifest in less efficient formation of kinetochore fibres. Improperly formed kinetochore fibres may in turn be responsible for defects in chromosome alignment and tension at kinetochores. Because we observed that even cells that were only partly depleted of NuMA (45% remaining at the mitotic poles) showed defects in chromosome alignment, we favour a model in which reduced levels of NuMA lead to prolongation of prometaphase due to an active spindle assembly checkpoint, until bipolar attachment and tension at kinetochores is finally achieved. We believe that the fraction of NuMA that localizes to the cell cortex in regular cells, as described by [3, 4], plays only a minor role in mitotic progress in experiments, since we see significant NuMA accumulation at the cortex only in late phases of mitosis, from metaphase/anaphase onwards, i.e. after chromosome alignment has occurred (unpublished observation).
In our RNA silencing experiments we detected smaller amounts of severely malformed spindles as compared to spindle formation assays after NuMA depletion in Xenopus egg extracts . Drastic spindle defects seen in these extracts included loss of focused poles and prolonged length of the mitotic apparatus . The different results may be explained by a higher sensitivity to experimental manipulation of spindles in vitro compared to spindles in intact cells, or by defects from co-depletion of NuMA-associated proteins, as discussed above, or by the presence of low levels of NuMA remaining at the poles after RNA silencing.
The authors would like to thank their colleagues at the Wellcome Trust Centre for Cell Biology, University of Edinburgh, and at the Centre National de la Recherche Scientifique, UMR 2587, Toulouse, for stimulating discussions and technical help. Antibody against BubR1 was kindly provided by Dr Tim Yen, Fox Chase Center, Philadelphia, PA. The work was supported by a Wellcome Trust Senior Research Fellowship to A.M., and in part by salary support from the French 'Centre National de la Recherche Scientifique'.
- Lydersen BK, Pettijohn DE: Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980, 22: 489-499.View ArticlePubMedGoogle Scholar
- Fant X, Merdes A, Haren L: Cell and molecular biology of spindle poles and NuMA. Int Rev Cytol. 2004, 238: 1-57.View ArticlePubMedGoogle Scholar
- Du Q, Stukenberg PT, Macara IG: A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nat Cell Biol. 2001, 3: 1069-1075.View ArticlePubMedGoogle Scholar
- Du Q, Macara IG: Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell. 2004, 119: 503-516.View ArticlePubMedGoogle Scholar
- Bowman SK, Neumüller RA, Novatchkova M, Du Q, Knoblich JA: The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division. Dev Cell. 2006, 10: 731-742.View ArticlePubMedGoogle Scholar
- Izumi Y, Ohta N, Hisata K, Raabe T, Matsuzaki F: Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization. Nat Cell Biol. 2006, 8: 586-593.View ArticlePubMedGoogle Scholar
- Siller KH, Cabernard C, Doe CQ: The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nat Cell Biol. 2006, 8: 594-600.View ArticlePubMedGoogle Scholar
- Srinivasan DG, Fisk RM, Xu H, Heuvel van den S: A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C elegans. Genes Dev. 2003, 17: 1225-1239.PubMed CentralView ArticlePubMedGoogle Scholar
- Park DH, Rose LS: Dynamic localization of LIN-5 and GPR-1/2 to cortical force generation domains during spindle positioning. Dev Biol. 2008, 315: 42-54.PubMed CentralView ArticlePubMedGoogle Scholar
- Price CM, Pettijohn DE: Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein. Exp Cell Res. 1986, 166: 295-311.View ArticlePubMedGoogle Scholar
- Kallajoki M, Weber K, Osborn M: A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies. EMBO J. 1991, 10: 3351-3362.PubMed CentralPubMedGoogle Scholar
- Yang CH, Snyder M: The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus. Mol Biol Cell. 1992, 3: 1259-1267.PubMed CentralView ArticlePubMedGoogle Scholar
- Compton DA, Cleveland DW: NuMA is required for the proper completion of mitosis. J Cell Biol. 1993, 120: 947-957.View ArticlePubMedGoogle Scholar
- Maekawa T, Kuriyama R: Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA. J Cell Sci. 1993, 105: 589-600.PubMedGoogle Scholar
- Compton DA, Luo C: Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis. J Cell Sci. 1995, 108: 621-633.PubMedGoogle Scholar
- Gaglio T, Saredi A, Compton DA: NuMA is required for the organization of microtubules into aster-like mitotic arrays. J Cell Biol. 1995, 131: 693-708.View ArticlePubMedGoogle Scholar
- Gueth-Hallonet C, Weber K, Osborn M: NuMA: a bipartite nuclear location signal and other functional properties of the tail domain. Exp Cell Res. 1996, 225: 207-218.View ArticlePubMedGoogle Scholar
- Gaglio T, Saredi A, Bingham JB, Hasbani MJ, Gill SR, Schroer TA, Compton DA: Opposing motor activities are required for the organization of the mammalian mitotic spindle pole. J Cell Biol. 1996, 135: 399-414.View ArticlePubMedGoogle Scholar
- Merdes A, Ramyar K, Vechio JD, Cleveland DW: A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell. 1996, 87: 447-458.View ArticlePubMedGoogle Scholar
- Gordon MB, Howard L, Compton DA: Chromosome movement in mitosis requires microtubule anchorage at spindle poles. J Cell Biol. 2001, 152: 425-434.PubMed CentralView ArticlePubMedGoogle Scholar
- Levesque AA, Howard L, Gordon MB, Compton DA: A functional relationship between NuMA and kid is involved in both spindle organization and chromosome alignment in vertebrate cells. Mol Biol Cell. 2003, 14: 3541-3552.PubMed CentralView ArticlePubMedGoogle Scholar
- Gehmlich K, Haren L, Merdes A: Cyclin B degradation leads to NuMA release from dynein/dynactin and from spindle poles. EMBO Rep. 2004, 5: 97-103.PubMed CentralView ArticlePubMedGoogle Scholar
- Silk AD, Holland AJ, Cleveland DW: Requirements for NuMA in maintenance and establishment of mammalian spindle poles. J Cell Biol. 2009, 184: 677-690.PubMed CentralView ArticlePubMedGoogle Scholar
- Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K: Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci. 2001, 114: 4557-4565.PubMedGoogle Scholar
- Quintyne NJ, Reing JE, Hoffelder DR, Gollin SM, Saunders WS: Spindle multipolarity is prevented by centrosomal clustering. Science. 2005, 307: 127-129.View ArticlePubMedGoogle Scholar
- Chang W, Dynek JN, Smith S: NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis. Biochem J. 2005, 391: 177-184.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamauchi Y, Kiriyama K, Kimura H, Nishiyama Y: Herpes simplex virus induces extensive modification and dynamic relocalisation of the nuclear mitotic apparatus (NuMA) protein in interphase cells. J Cell Sci. 2008, 121: 2087-2096.View ArticlePubMedGoogle Scholar
- Si H, Verma SC, Lampson MA, Cai Q, Robertson ES: Kaposi's sarcoma-associated herpesvirus-encoded LANA can interact with the nuclear mitotic apparatus protein to regulate genome maintenance and segregation. J Virol. 2008, 82: 6734-6746.PubMed CentralView ArticlePubMedGoogle Scholar
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001, 411: 494-498.View ArticlePubMedGoogle Scholar
- Merdes A, Heald R, Samejima K, Earnshaw WC, Cleveland DW: Formation of spindle poles by dynein/dynactin-dependent transport of NuMA. J Cell Biol. 2000, 149: 851-862.PubMed CentralView ArticlePubMedGoogle Scholar