The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics
Posted on: 11 May 2020
Preprint posted on 3 May 2020
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202004202
NuMA interaction with chromatin is vital for proper nuclear architecture in human cells
Posted on:
Preprint posted on 3 May 2020
Article now published in Molecular Biology of the Cell at http://dx.doi.org/10.1091/mbc.E20-06-0415
Categories: biochemistry, cell biology, molecular biology
Agathe Chaigne and Gautam Dey
Background
The cell division community has had a long-standing interest in the protein NuMA (Nuclear Mitotic Apparatus protein), for its dual role in organising and positioning the mitotic spindle. NuMA is a long coiled-coil protein with 2 globular domains at its C and N-termini. NuMA interacts with minus ends of spindle microtubules through its C-terminal domain (Haren and Merdes, 2002), which allows it to cluster spindle poles of centrosomal (Silk et al., 2009) as well as acentrosomal spindles (Kolano et al., 2012). Moreover, NuMA participates in a trimeric complex at the cell cortex (including LGN and Gɑi in mammals) via its C-terminus; the N-terminus can recruit the microtubule motor dynein, which in turn slides on astral microtubules and orients the mitotic spindle in metaphase and anaphase in cells and tissues (Bosveld et al., 2016; Kiyomitsu and Cheeseman, 2012; Kotak et al., 2012; Woodard et al., 2010).
The literature, however, hints at a role for NuMA beyond the spindle. NuMA is highly abundant in the interphase nucleus, where it is recruited right as the nuclear envelope forms. Defects in NuMA have long been implicated in micronucleation at mitotic exit (Compton and Cleveland, 1993; Kallajoki et al., 1991, 1993), and NuMA has been proposed to form a nuclear mesh-like structure in the nucleus (Harborth, 1999; Zeng et al., 1994). Critically, potential roles in nuclear reformation after mitosis have been difficult to disentangle from NuMA’s other mitotic functions.
Now, a pair of preprints have tackled these long-standing questions about NuMA function in nuclear organisation using complementary cell biology, biophysical and biochemical approaches (1: Serra-Marques et al., 2: Rajeevan et al.). Using a clever mix of tools to study functional domains in isolation, they elegantly uncouple the role of NuMA in nuclear reformation from its role in mitosis.
Key results
Serra-Marques et al. knock NuMA out via inducible CRISPR/Cas9 in RPE1 cells and force the cells through a spindle-less mitosis via nocodazole treatment and inhibition of the spindle assembly checkpoint. This leads to defects in nuclear reformation, the generation of multiple micronuclei, and overexpansion of the chromosome mass as the nuclear envelope attempts to reform. In Rajeevan et al, they first identify the region of the protein responsible for interacting with DNA (the 58 C-terminal amino acids) and then investigate the role of different constructs in an NuMA-depleted background. Interestingly, they find that cells lacking DNA-binding NuMA have smaller nuclei than controls, somewhat contradicting the findings outlined in Serra-Marques et al. They also note nuclear shape defects in cells lacking the DNA-binding region.
Next, the authors seek to understand which part of the protein could be involved in this role at mitotic exit. Using FRAP (Figure 1) and different NuMA truncation constructs (1, 2), Serra-Marques et al find that a central coiled-coil domain is important for the stability of NuMA in the nucleus and further show that the region is required for the reformation of an intact nucleus. Both teams identify the C-terminus part of NuMA as interacting with DNA in vivo, and Rajeevan et al showed that the 58 last amino-acid of the C-terminus domain of NuMA is necessary and sufficient to target NuMA at the DNA during all stages of mitosis, and regulate nuclear stability. Finally, they demonstrate that in vitro, the C-terminus of NuMA directly binds DNA (1 and 2).
What then prevents NuMA from interacting with DNA during mitosis, while it is restricted to the cortex and the spindle poles? The authors investigate the localisation of different truncation mutants and show that the coiled-coil domain prevents NuMA from localising to the chromosomes during mitosis – although, given that CDK1 does not seem to be involved (1), the mechanism for the transition between anaphase and nuclear reformation is not clear. Rajeevan et al further show that NuMA localizes to the DNA and in particular at chromosomes as late as prophase, where it colocalises with RCC1, although RCC1 is not responsible for this localisation (2). Interestingly, they show that the relocation of NuMA from prometaphase chromosomes to the spindle and cortex depends on CDK1, which suggests that the removal of NuMA from the chromosomes at mitotic entry and the addressing of NuMA at the nucleus at mitotic exit could be mediated by different pathways.
How does NuMA ensure nuclear integrity at mitotic exit (Figure 2)? Serra-Marques et al show that the deformed NuMAΔ nuclei are more susceptible to mechanical stress, suggesting that NuMA might act to limit deformation which in turn prevents fragmentation. The authors propose that NuMA could prevent nuclear membrane reformation between chromosomes, similarly to BAF (Samwer et al., 2017), or promote nuclear membrane recruitment, although how NuMA KO would lead to fragmentation of the nucleus is not clear. Rajeevan et al show that a NuMA unable to bind DNA forms stable punctate and fibrillar structures in the nucleus which correlate with the presence of nuclear deformations. The authors conclude that the binding of NuMA to DNA prevents it from destabilising nuclear architecture.
A discussion with the authors
These two papers have really cracked open a part of the field that has long proven difficult to tackle. Read on below as both sets of authors respond to our questions!
References and further reading
Bosveld, F., Markova, O., Guirao, B., Martin, C., Wang, Z., Pierre, A., Balakireva, M., Gaugue, I., Ainslie, A., Christophorou, N., et al. (2016). Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis. Nature 530, 495–498.
Compton, D.A., and Cleveland, D.W. (1993). NuMA is required for the proper completion of mitosis. The Journal of Cell Biology 120, 947–957.
Harborth, J. (1999). Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice. The EMBO Journal 18, 1689–1700.
Haren, L., and Merdes, A. (2002). Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules. J. Cell. Sci. 115, 1815–1824.
Kallajoki, M., Weber, K., and Osborn, M. (1991). A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies. EMBO J. 10, 3351–3362.
Kallajoki, M., Harborth, J., Weber, K., and Osborn, M. (1993). Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells. J. Cell. Sci. 104 ( Pt 1), 139–150.
Kiyomitsu, T., and Cheeseman, I.M. (2012). Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation. Nature Cell Biology 14, 311–317.
Kolano, A., Brunet, S., Silk, A.D., Cleveland, D.W., and Verlhac, M.-H. (2012). Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension. Proceedings of the National Academy of Sciences 109, E1858–E1867.
Kotak, S., Busso, C., and Gönczy, P. (2012). Cortical dynein is critical for proper spindle positioning in human cells. The Journal of Cell Biology 199, 97–110.
Kotak, S., Busso, C., and Gönczy, P. (2013). NuMA phosphorylation by CDK1 couples mitotic progression with cortical dynein function. EMBO J. 32, 2517–2529
Merdes, A., and Cleveland, D.W. (1998). The role of NuMA in the interphase nucleus. J. Cell. Sci. 111 ( Pt 1), 71–79.
Rajeevan, A., Keshri, R., and Kotak, S. (2020). NuMA interaction with chromatin is vital for proper nuclear architecture in human cells (Cell Biology).
Samwer, M., Schneider, M.W.G., Hoefler, R., Schmalhorst, P.S., Jude, J.G., Zuber, J., and Gerlich, D.W. (2017). DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes. Cell 170, 956-972.e23.
Serra-Marques, A., Houtekamer, R., Hintzen, D., Canty, J.T., Yildiz, A., and Dumont, S. (2020). The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics (Cell Biology).
Silk, A.D., Holland, A.J., and Cleveland, D.W. (2009). Requirements for NuMA in maintenance and establishment of mammalian spindle poles. Journal of Cell Biology 184, 677–690.
Woodard, G.E., Huang, N.-N., Cho, H., Miki, T., Tall, G.G., and Kehrl, J.H. (2010). Ric-8A and Giα Recruit LGN, NuMA, and Dynein to the Cell Cortex To Help Orient the Mitotic Spindle. Molecular and Cellular Biology 30, 3519–3530.
Zeng, C., He, D., and Brinkley, B.R. (1994). Localization of NuMA protein isoforms in the nuclear matrix of mammalian cells. Cell Motility and the Cytoskeleton 29, 167–176.
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