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The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics

Andrea Serra-Marques, Ronja Houtekamer, Dorine Hintzen, John T. Canty, Ahmet Yildiz, Sophie Dumont

Preprint posted on 3 May 2020 https://www.biorxiv.org/content/10.1101/2020.05.02.070680v1

Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202004202

and

NuMA interaction with chromatin is vital for proper nuclear architecture in human cells

Ashwathi Rajeevan, Riya Keshri, Sachin Kotak

Preprint posted on 3 May 2020 https://www.biorxiv.org/content/10.1101/2020.05.02.073767v1

Article now published in Molecular Biology of the Cell at http://dx.doi.org/10.1091/mbc.E20-06-0415

Mitotic NuMA goes nuclear

Selected by Dey Lab

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.

 

Figure 1, taken from Figure 1 of Rajeevan et al. 2020 under a CC-BY-ND 4.0 Creative Commons License. (A) Domain organization of NuMA with mono FLAG (FL) and AcGFP-tag at the Nterminus (referred to as AcGFP-NuMA). The coiled-coil domain, the region mediating interaction with microtubules (MTs), and the nuclear localization signal (NLS) are depicted. (B) Immunoblot analysis of protein extracts prepared from the mitotically synchronized HeLa Kyoto cells, which are transfected with scrambled siRNAs (Control), siRNAs against NuMA 3′-UTR for 72 hr, or left untreated and stably expressing AcGFP-NuMA. Extracts were probed with antibodies against NuMA and β-actin. Transgenic AcGFPNuMA protein is shown by a blue asterisk that is migrating above the endogenous protein. The molecular mass is indicated in kilodaltons (kDa). (C-I) FRAP analysis of HeLa Kyoto cells that are stably expressing AcGFP-NuMA (C, F), transiently transfected with AcGFP-NuMA(1 2057) (D, G) or AcGFP-NuMA(1-2115m) (E, H) and are depleted for endogenous NuMA. The GFP signal is shown in green, and the time is indicated in seconds (s).

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.

Figure 2. Reproduced from Figure 6 of Serra-Marques et al. 2020 with the authors’ permission. Figure made with BioRender. Model for NuMA’s role in nuclear formation and mechanics, and its long-range structural role over the cell cycle. NuMA (blue) plays a spindle-independent role in nuclear formation (“Mitotic exit”, center) and mechanics (“Interphase”, right). It keeps the chromosome (pink) mass compact at nuclear formation, and is essential to building a single, round and mechanically robust nucleus (“+NuMA”, top). Without NuMA (“-NuMA”, bottom), micronucleation and nuclear shape defects occur. We propose two models for how NuMA, whose C-terminus binds interphase chromosomes (pink arrow), performs its nuclear function. To promote nuclear formation and mechanics, NuMA could crosslink chromosomes(Model A, blue filaments) or regulate nuclear envelope (green) assembly and maturation (Model B, black arrow), either directly or indirectly. At “Mitosis” (left), NuMA plays a critical role in spindle formation and mechanics and its coiled-coil prevents it from binding chromosomes, when these must but segregated instead of kept together. At mitotic exit and interphase, the coiled-coil drives NuMA’s nuclear dynamics and function (blue arrows).

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.

 

Posted on: 11 May 2020

doi: https://doi.org/10.1242/prelights.20394

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Author's response

Andrea Serra-Marques and Sophie Dumont shared about The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics

The nuclear size phenotypes in the two papers are not entirely consistent. Could the authors elaborate on possible reasons for this? 

The systems used in both studies are different. In the Serra-Marques et al study, we perform a loss of function assay (NuMA KO) in cells that go through a biochemical anaphase. In these conditions, we see that the nuclei of NuMA KO cells expand more than those of control cells as they form. This indicates that NuMA helps keep the chromosome mass together, either directly or indirectly, as the nucleus forms. In contrast, Rajeevan et al. look at nuclear expansion in cells that are partially depleted of NuMA and express a mutant NuMA (NuMA1-2057) that does not bind to DNA. In these conditions, the nuclei of cells expressing wild-type NuMA expand more than nuclei expressing NuMA1-2057. In parallel, the authors show that in these conditions expressed NuMA1-2057 incorporates into fibrillar structures that are not observed in cells expressing wild-type NuMA. How the assembly of NuMA1-2057 fibrillar structures impacts chromosome organization, and the rate of nuclear expansion, are not known.

 

The work presented here suggests that NuMA might have had ancestral roles in either interphase or mitosis – perhaps acquiring one of its key functions later. Could you speculate about the evolution of the protein?

We do not know how NuMA’s roles evolved. One hypothesis is that NuMA’s nuclear role evolved after its mitotic one. Indeed, while we are not aware of reports that NuMA is dispensable for mitosis in mammalian cells, some highly differentiated and non-dividing cells do not express NuMA (Merdes and Cleveland 1998; Taimen et al., 2000). Thus, NuMA is not an essential component for the maintenance of all nuclei. Interestingly, non-proliferating cells are the ones that tend to lose NuMA – neither needing NuMA for mitosis nor for nuclear formation. Also, there are systems such as C. elegans where the NuMA homolog is not in the nucleus.

 

How would you interpret the fact that CDK1 is involved in removing NuMA from the chromosomes at mitotic entry, but not involved in readdressing it to the nucleus at mitotic exit?

At mitosis, we show that the coiled-coil inhibits NuMA binding to chromosomes and that CDK1 inhibition is not sufficient to allow NuMA to bind chromosomes. This indicates that another regulatory process controls the re-association of NuMA with chromosomes, working either with or without CDK1. For example, such regulation could occur through another kinase (or phosphatase), or through the association with a regulatory molecular partner not yet identified.

 

What do you think might regulate the coiled-coil dependent shift in nuclear affinity for NuMA between nuclear reformation/interphase and mitosis? Would it be another cell-cycle kinase like Polo or Aurora?

We have not tested a role for Polo and Aurora kinases. These are indeed good candidates. Another idea is that a regulatory binding partner could change NuMA’s affinity for DNA from mitosis to interphase.

 

How would you imagine that limiting deformability of the nucleus would prevent the formation of micronuclei? It seems more intuitive to imagine that a deformable nucleus (ie NuMA KO cells) would be more resistant to breakage. 

While we mechanically perturb established nuclei, we do not probe the deformability of the chromosome mass during nuclear formation. Through live imaging, we show that NuMA helps slow down the expansion of the chromosome mass, directly or indirectly, as the nuclear envelope forms. By keeping the chromosome mass together at nuclear formation, we propose that NuMA helps avoid chromosomes from getting detached from the main chromosome mass – thus reducing the chance of micronucleation.

 

Would it be possible to investigate NuMA networks in vitro to investigate their structure and mechanical properties?

In vitro experiments to test NuMA’s ability to crosslink chromosomes, and define what higher order structures it can and cannot form, are exciting future directions.

 

The paper raises interesting questions about the partitioning between a multifunctional protein’s different roles. In RPE1 and HeLa cells in culture, spindle orientation is not critical. In multicellular tissues where it is – such as the Drosophila notum, or the Xenopus or zebrafish epiderm – is NuMA still as important for nuclear reformation?  

While we do not know the answer to this question, we hypothesize that NuMA is important for both nuclear formation and mechanics in dividing cells that are in a tissue. Testing this hypothesis will require the design of experiments to cleanly decouple NuMA’s roles in the spindle vs nucleus. 

 

Does the amount of NuMA scale with nuclear size (or the nuclear/cell size ratio), across cell types and species? 

We would love to know the answer to this question. While the absence of NuMA in some cell types appears to correlate with non-spherical nuclei (Merdes & Cleveland 1998), we are not aware of work looking at whether and how nuclear size correlates with NuMA levels.

and

Sachin Kotak shared about NuMA interaction with chromatin is vital for proper nuclear architecture in human cells

The nuclear size phenotypes in the two papers are not entirely consistent. Could the authors elaborate on possible reasons for this? 

We believe that the two experimental conditions used in these two papers are very different. Serra-Marques et al., utilized NuMA KO cells, so the complete pool of NuMA protein is absent. Whereas in Rajeevan et al., the N-ter, coiled-coil and a large part of C-ter is intact. These intact part of NuMA may have some additional function in regulating nuclear architecture.

 

The work presented here suggests that NuMA might have had ancestral roles in either interphase or mitosis – perhaps acquiring one of its key functions later. Could you speculate about the evolution of the protein?

The nuclear function of NuMA might have evolved later during evolution, as the best of our knowledge NuMA homolog in C. elegans i.e., LIN-5 is not a nuclear protein. Similarly, Mud , Drosophila homolog of NuMA localizes at the nuclear rim, but not in the nucleus. It may well be that we haven’t analyzed several species for NuMA-like proteins to answer this question with confidence.

 

How would you interpret the fact that CDK1 is involved in removing NuMA from the chromosomes at mitotic entry, but not involved in readdressing it to the nucleus at mitotic exit?

This is a good question! Cdk1 activity peaks upon mitotic entry and it gets gradually inactivated upon anaphase onset as CyclinB1 gets degraded. NuMA re-enters into the nucleus only in late anaphase after nuclear envelope reformation because of the presence of NLS. If Cdk1 is critical in uncoupling NuMA from the chromatin, why does it not localize on the chromatin as soon as Cdk1 gets inactive? We envisage that there could be a minimum of two possibilities: one that another kinase is involved in ensuring NuMA interacts with chromatin only late during mitosis; otherwise, there would be spindle elongation defects as NuMA is required for efficient spindle elongation in anaphase (Kotak et al., 2013 EMBO J). The other possibility is that the phosphorylation site that is phosphorylated by Cdk1 remains active, and it acts upon by a phosphatase only during late anaphase.

 

Would it be possible to investigate NuMA networks in vitro to investigate their structure and mechanical properties?

This is an interesting result, and we are surely looking forward to investigating how these structures form, and also what are the biophysical properties of these structures.

 

The paper raises interesting questions about the partitioning between a multifunctional protein’s different roles. In RPE1 and HeLa cells in culture, spindle orientation is not critical. In multicellular tissues where it is – such as the Drosophila notum, or the Xenopus or zebrafish epiderm – is NuMA still as important for nuclear reformation?  

Studying NuMA nuclear functions in Xenopus, Mouse, or Zebrafish would be critical to link the nuclear function of NuMA with the epithelial morphogenesis. 

 

Does the amount of NuMA scale with nuclear size (or the nuclear/cell size ratio), across cell types and species? 

Several cells with non-spherical nuclei for instance sperm cells, granulocytes, muscle fibers lack NuMA, suggesting that NuMA might be involved in defining the nuclear shape (Merdes and Cleveland, 1998). However, to the best of our knowledge, there is no study that correlates the amount of NuMA with the nuclear size, and exploring this would definitely add some light on the function of this critical molecule.

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