Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. These results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle (Lorson, 2000).
The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Two closely related proteins, GPR-1 and GPR-2 (G protein regulator), associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galphai/o. A number of proteins in other metazoans contain GoLoco motifs in various numbers, and several GoLoco motif proteins, including mammalian AGS3 and Drosophila Pins, have been shown to interact with Galphai/o subunits of heterotrimeric G proteins. Inactivation of lin-5, gpr-1/gpr-2, or the Galphai/o genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galphai/o signaling (Srinivasan, 2003).
In both Drosophila and C. elegans, a conserved PAR protein complex establishes cell polarity and spindle position but is not required for chromosome movements. This PAR-determined polarity directs spindle positioning possibly through activation of G protein signaling mediated by Pins/Inscuteable (Insc) in Drosophila neuroblasts, Pins/Discs large (Dlg) in Drosophila SOP cells, and GPR/LIN-5 in C. elegans embryos. Although Insc, Dlg, and LIN-5 all act to localize GoLoco proteins, their functions and localizations differ. LIN-5, GPR-1/GPR-2, and Galphai/o interactions appear to be required for cell division and chromosome segregation, whereas no such role has been shown for Drosophila Galphai or Pins. Consistent with a role in chromosome movements, GPR-1/GPR-2 proteins localize to the spindle apparatus, whereas Pins does not. This may indicate that an additional spindle-associated GoLoco protein exists, and/or possibly that in Drosophila multiple Galpha subunits act redundantly in mitosis, as in C. elegans. Consistent with the former hypothesis, a mammalian homolog of Pins, LGN, is required for spindle assembly and localizes to spindle asters (Srinivasan, 2003 and references therein).
Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric G? subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. The Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). The GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. These results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins (Gotta, 2003).
Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, a screen was performed for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5ev571ts, and 68 suppressing mutations were isolated. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5ev571ts. These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5ev571ts mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, these results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5 (Fisk Green, 2004).
Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). A human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. LGN binds the nuclear mitotic apparatus protein NuMA, which tethers spindles at the poles, and this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, a mammalian Pins homolog has been identified as a key regulator of spindle organization during mitosis (Du, 2001).
LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), that is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, it is shown that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. It is therefore proposed that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization (Du, 2002).
During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, it was found that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. It is proposed that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode (Du, 2004).
Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in C. elegans and Drosophila have placed RIC-8 (Ric8a in Drosophila) in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the G alphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of G alphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and G alphai regulate a similar process. Mammalian Ric-8A dissociates G alphai-GDP/LGN/NuMA complexes catalytically, releasing activated G alphai-GTP in vitro. Ric-8A-stimulated activation of G alphai causes concomitant liberation of NuMA from LGN. It is concluded that Ric-8A efficiently utilizes GoLoco/G alphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division (Tall, 2005).
Models are envisioned in which one cellular function of Ric-8A is to dissociate Galphai-GDP/GoLoco complexes by stimulation of nucleotide exchange. G protein control of asymmetric cell division involves cycling of Galphai between its GDP- and GTP-bound forms, as evidenced by the fact that (in C. elegans) both RIC-8 and RGS7 influence the pathway in opposed fashion. It remains speculative whether Galphai-GDP/GoLoco or the production of Galphai-GTP from a GoLoco scaffold activates signaling. It stands to reason that Galphai-GTP must dissociate from GoLoco at some point during signaling. If multiple rounds of cycling between Galphai-GDP/GoLoco and liberated Galphai-GTP are required to complete cell division, then Ric-8A-stimulated dissociation of a Galphai/GoLoco complex could be responsible for either terminating or activating the signal. In either context, RGS-facilitated hydrolysis of GTP by Galpha ensues. The resultant Galphai-GDP could rebind to GoLoco (and not betagamma) to complete one round of the cycle. Rapid cycling of this process may be necessary to regulate the pulling forces on microtubules appropriately during a round of chromosome segregation (supporting information on the PNAS web site, for these proposed models). Regulation of other Galpha or Galpha/GoLoco-mediated signaling pathways by Ric-8A is also worth considering, given the number of distinct Galpha binding partners of mammalian Ric-8A and Ric-8B and the many processes that appear to be regulated by RIC-8 in C. elegans (Tall, 2005).
Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase. PAR is enriched in vertebrate somatic-cell mitotic spindles and a requirement for PAR in the assembly of Xenopus egg extract spindles has been demonstrated. This study performed a knockdown of all characterized PARPs using RNA interference (RNAi), and tankyrase-1 was identified as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed--data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation. Tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. A requirement for tankyrase-1 has been demonstrated in the assembly of bipolar spindles, and the spindle-pole protein NuMA has been identified as a substrate for covalent modification by tankyrase-1 (Chang, 2005).
In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. This study shows that NuMA can bind to microtubules independently of dynein/dynactin. A 100-residue domain located within the C-terminal tail of NuMA has been characterized that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. These results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers (Haren, 2002).
The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. This study used both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, an exogenously expressed green-fluorescent-protein/NuMA fusion was shown to undergo continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, the association was exploited of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, the fate of human NuMA associated with microtubule asters was followed upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is approximately 5 minutes. Using this assay, it was shown that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles (Kisurina-Evgenieva, 2004).
NuMA is a nuclear protein during interphase but redistributes to the spindle poles early in mitosis. To investigate its role during spindle formation, spindle assembly was tested in frog egg extracts from which NuMA was immunodepleted. Immunodepletion revealed that NuMA forms a complex with cytoplasmic dynein and dynactin. The depleted extracts failed to assemble normal mitotic spindles, producing, instead, chromatin-associated irregular arrays of microtubules lacking characteristic spindle poles. A subdomain of the NuMA tail was shown to induce microtubule aster formation by mediating microtubule bundling. These findings suggest that NuMA forms bifunctional complexes with cytoplasmic dynein and dynactin that can tether microtubules at the spindle poles and that are essential for mitotic spindle pole assembly and stabilization (Merdes, 1996).
NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. This process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly (Merdes, 2000).
The protein NuMA localizes to mitotic spindle poles where it contributes to the organization of microtubules. NuMA loses its stable association with the spindle poles after anaphase onset. Using extracts from Xenopus laevis eggs, this study shows that NuMA is dephosphorylated in anaphase and released from dynein and dynactin. In the presence of a nondegradable form of cyclin B (Delta90), NuMA remains phosphorylated and associated with dynein and dynactin, and remains localized to stable spindle poles that fail to disassemble at the end of mitosis. Inhibition of NuMA or dynein allows completion of mitosis, despite inducing spindle pole abnormalities. It is proposed that NuMA functions early in mitosis during the formation of spindle poles, but is released from the spindle after anaphase, to allow spindle disassembly and remodelling of the microtubule network (Gehmlich, 2004).
During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, confocal immunofluorescence microscopy was used to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibits microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promotes MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupts the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA (Becker, 2003).
The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation program to form the barrier. The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells, most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane. This study shows that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. It is further demonstrated that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3-LGN-Inscuteable complex and NuMA-dynactin to align the spindle (Leuchler, 2005).
Acute promyelocytic leukaemia (APL) is uniquely associated with chromosomal translocations that disrupt the gene encoding the retinoic acid receptor, RARA. In more than 99% of cases, this disruption results in the formation of a PML-RARA gene fusion. Two rare variants of APL have been described, in which RARA is fused to one of two other genes, PLZF and NPM. Although RARA dysregulation is evidently important in APL, the role of the various fusion partners remains unclear. A fourth APL gene fusion has been characterized that links exons encoding the retinoic acid and DNA-binding domains of RARA to 5' exons of NuMA, a gene that encodes the nuclear mitotic apparatus protein. The NuMA-RARA fusion protein exists in sheet-like nuclear aggregates with which normal NuMA partly co-localizes. In contrast to t(15;17) APL, the intracellular distribution of PML is normal in these cells. These results suggest that interference with retinoid signalling, and not disruption of PML organization, is essential to the APL phenotype and implicates for the first time an element of the mitotic apparatus in the molecular pathogenesis of human malignancy (Wells, 1997).
Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. This study describe a process of centrosomal clustering that prevents the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein is a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfers with dynein localization, promoting multipolarity (Quintyne, 2005).
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