NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gα_i1‐3 anchor the coiled‐coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gα_i1‐3 are dispensable for NuMA‐dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP₂ (PIP₂) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP₂ prevents NuMA cortical localization during mitosis, and conversely, increasing PIP₂ levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.     

NuMA phosphorylation by CDK1 couples mitotic progression with cortical dynein function

Spindle positioning and spindle elongation are critical for proper cell division. In human cells, an evolutionary conserved ternary complex (NuMA/LGN/Gαi) anchors dynein at the cortex during metaphase, thus ensuring correct spindle positioning. Whether this complex contributes to anaphase spindle elongation is not known. More generally, the mechanisms coupling mitotic progression with spindle behaviour remain elusive. Here, we uncover that levels of cortical dynein markedly increase during anaphase in a NuMA‐dependent manner. We demonstrate that during metaphase, CDK1‐mediated phosphorylation at T2055 negatively regulates NuMA cortical localization and that this phosphorylation is counteracted by PPP2CA phosphatase activity. We establish that this tug of war is essential for proper levels of cortical dynein and thus spindle positioning during metaphase. Moreover, we find that upon CDK1 inactivation in anaphase, the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment, and thus to robust spindle elongation. Our findings uncover a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function.     

Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.     

A CH domain‐containing N terminus in NuMA?

Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding activity for actin-like molecules. We substantiate the primary sequence analysis-based prediction with secondary structure and fold recognition analysis, and we propose the N-terminal CH domain of NuMA as a likely interaction site for actin-related protein 1 (Arp1) protein of the dynactin/dynein complex.     

Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly : Properties of purified NuMA protein

Monoclonal antibodies and human autoimmune sera specific for the nuclear mitotic apparatus protein (NuMA protein) were applied to study the structure of this protein and its intracellular distribution. The NuMA protein was purified using immuno-affinity columns. Studies on this large (250 kD) nuclear protein indicated that it is a highly asymmetric phosphoprotein. It is present in all mammalian cells examined and in those of some non-mammals. Immunofluorescence studies on fixed cells demonstrated that its intracellular distribution is essentially the same in all species at all stages of the cell cycle. Immunoblot (western blot) analysis showed that the size of the NuMA protein varies slightly in different species. At the onset of mitosis the NuMA protein redistributes from the nucleus to two centrosomal structures that later will become part of the mitotic spindle pole. This occurs at the time of nuclear breakdown and eventually leads to an accumulation of the NuMA protein at the polar region of the mitotic spindle. After anaphase the protein redistributes from the spindle polar region into the reforming nucleus and concentrates initially at the site where nuclear lamins and perichomatin have been reported to assemble. Living cells microinjected with fluorescent anti-NuMA antibodies were studied to examine parameters that effect the redistribution of the NuMA protein in vivo. These experiments indicate that microtubule assembly is essential for the NuMA protein to accumulate in the polar region.     

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branchpoints for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism by which alternative promoters can be coordinated with downstream alternative splicing.     

Autoantibodies to components of the mitotic apparatus

Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review. Abbreviations: ATP – adenosine triphosphate; CENP – centromere protein; CREST – calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia; HMG – high mobility group; IB – intercellular bridge; IIF – indirect immunofluorescence; MAPs – microtubule associated proteins; NuMA – nuclear mitotic apparatus; NOR – nucleolar organizer; PBC – primary biliary cirrhosis; PM – polymyositis; Pol I, II, III – RNA polymerases; RA-rheumatoid arthritis; SLE – systemic lupus erythematosus; SS – Sjögren's syndrome; SSc – systemic sclerosis; topo – topoisomerase.     

The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

The C terminus of the nuclear protein NuMA: phylogenetic distribution and structure

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of beta3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily beta fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events.     

The nuclear/mitotic apparatus protein NuMA is a component of the somatodendritic microtubule arrays of the neuron

Neurons are terminally post-mitotic cells that utilize their microrubule arrays for the growth and maintenance of axons and dendrites rather than for the formation of mitotic spindles. Recent studies from our laboratory suggest that the mechanisms that organize the axonal and dendritic microtubule arrays may be variations on the same mechanisms that organize the mitotic spindle in dividing cells. In particular, we have identified molecular motor proteins that serve analogous functions in the establishment of these seemingly very different microtubule arrays. In the present study, we have sought to determine whether a non-motor protein termed NuMA is also a component of both systems. NuMA is a ~230 kDa structural protein that is present exclusively in the nucleus during interphase. During mitosis, NuMA forms aggregates that interact with microtubules and certain motor proteins. As a result of these interactions, NuMA is thought to draw together the minus-ends of microtubules, thereby helping to organize them into a bipolar spindle. In contrast to mitotic cells, post-mitotic neurons display NuMA both in the nucleus and in the cytoplasm. NuMA appears as multiple small particles within the somatodendritic compartment of the neuron, where its levels increase during early dendritic differentation. A partial but not complete colocalization with minus-ends of microtubules is suggested by the distribution of the particles during development and during drug treatments that alter the microtubule array. These observations provide an initial set of clues regarding a potentially important function of NuMA in the organization of microtubules within the somatodendritic compartment of the neuron.     

Oxidation and methylation status determine the effects of arsenic on the mitotic apparatus

We investigated the spindle inhibitory properties of six arsenicals differing in their methylation or oxidation state. Human lymphoblasts were exposed for 6 h to either sodium arsenate (NaAs^V), sodium arsenite (NaAs^III), monomethylarsonic acid (MMA^V), monomethylarsonous acid (MMA^III), dimethylarsinic acid (DMA^V), or dimethylarsinous acid (DMA^III). After exposure slides were prepared, and the mitotic indices (MI) were assessed. We also exposed tubulin directly to each arsenical and spectrophotometrically measured its effect on polymerization. NaAs^V caused a small but significant increase in MI. MMA^V also caused only a slight increase in MI that just reached statistical significance. In contrast, DMA^V caused a significant increase in MI, producing ∼75% the MI of demecolcine and ∼4 times the MI of the control. NaAs^III had no significant effect on MI and was quite toxic. MMA^III induced more than a twofold increase in MI compared to the control, which was about 40% that caused by demecolcine. On a micromolar basis, MMA^III was the most potent of the arsenicals tested. DMA^III gave inconsistent results. None of the pentavalent arsenicals had a substantial effect (either inhibition or enhancement) on GTP-induced polymerization of tubulin. In contrast, NaAs^III inhibited polymerization at concentrations of 1 mM and above and MMA^III and DMA^III at 10 μM and above. Taken together, these results present a complex picture of how arsenicals may affect cells. These studies demonstrate that the metabolites of arsenic are active not only as chromosome breaking and DNA damaging agents but can also interfere with cell division via tubulin disruption.     

Textural analysis of nuclear mitotic apparatus antigen (NuMA) spatial distribution in interphase nuclei from human drug-resistant CEM lymphoblasts.

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.     

Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.