subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma- tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.     

Specialization in the behaviour of chromosomes on the meiotic spindle

The kinetic activity of chromosomes is either distributed evenly along the length of the chromosome or localized in a single centromere. Within the group of organisms with evenly distributed (holokinetic) activity great variation occurs, which has its consequences for chromatid segregation. Three major types, with some overlap, can be distinguished. They form a gradient of increasing specialization. Arguments are presented for the hypothesis that the monokinetic chromosomes are nothing but the end point in the same gradient and do not form a fundamentally different category. These arguments include: concentration of kinetic activity at meiosis in some types of holokinetic chromosomes, and atavistic traits in monokinetic chromosomes pointing to a holokinetic origin. Possible reasons for the wide distribution of monokinetic chromosomes in the more evolved taxa are given.     

Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes

We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.     

Mammalian protein SCP1 forms synaptonemal complex-like structures in the absence of meiotic chromosomes

Synaptonemal complexes (SCs) are evolutionary conserved, meiosis-specific structures that play a central role in synapsis of homologous chromosomes, chiasmata distribution, and chromosome segregation. However, it is still for the most part unclear how SCs do assemble during meiotic prophase. Major components of mammalian SCs are the meiosis-specific proteins SCP1, 2, and 3. To investigate the role of SCP1 in SC assembly, we expressed SCP1 in a heterologous system, i.e., in COS-7 cells that normally do not express SC proteins. Notably, under these experimental conditions SCP1 is able to form structures that closely resemble SCs (i.e., polycomplexes). Moreover, we show that mutations that modify the length of the central alpha-helical domain of SCP1 influence the width of polycomplexes. Finally, we demonstrate that deletions of the nonhelical N- or C-termini both affect polycomplex assembly, although in a different manner. We conclude that SCP1 is a primary determinant of SC assembly that plays a key role in synapsis of homologous chromosomes.     

Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.     

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.     

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster. Received: 29 September 1999 / Accepted: 24 November 1999     

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video- enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.     

Separating centrosomes interact in the absence of associated chromosomes during mitosis in cultured vertebrate cells

We detail here how "free" centrosomes, lacking associated chromosomes, behave during mitosis in PtK(2) homokaryons stably expressing GFP-alpha-tubulin. As free centrosomes separate during prometaphase, their associated astral microtubules (Mts) interact to form a spindle-shaped array that is enriched for cytoplasmic dynein and Eg5. Over the next 30 min, these arrays become progressively depleted of Mts until the two centrosomes are linked by a single bundle, containing 10-20 Mts, that persists for > 60 min. The overlapping astral Mts within this bundle are loosely organized, and their plus ends terminate near its midzone, which is enriched for an ill-defined matrix material. At this time, the distance between the centrosomes is not defined by external forces because these organelles remain stationary when the bundle connecting them is severed by laser microsurgery. However, since the centrosomes move towards one another in response to monastrol treatment, the kinesin-like motor protein Eg5 is involved. From these results, we conclude that separating asters interact during prometaphase of mitosis to form a spindle-shaped Mt array, but that in the absence of chromosomes this array is unstable. An analysis of the existing data suggests that the stabilization of spindle Mts during mitosis in vertebrates does not involve the chromatin (i.e., the RCC1/RanGTP pathway), but instead some other chromosomal component, e.g., kinetochores.     

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Meeting the meiotic challenge: Specializations in mammalian oocyte spindle formation

Oocytes uniquely accumulate cytoplasmic constituents to support early embryogenesis. This unique specialization is accompanied by acquisition of a large size and by execution of asymmetric meiotic divisions that preserve precious ooplasm through the expulsion of minimal size polar bodies. While often taken for granted, these basic features of oogenesis necessitate unique specializations of the meiotic apparatus. These include a chromatin‐sourced RanGTP gradient that restricts spindle size by defining a spatial domain where meiotic spindles form, acentriolar centrosomes that rely on microtubule organizing centers to form spindle poles, and an actin‐based mechanism for asymmetric spindle positioning. Additionally, localized protein synthesis to support spindle formation is achieved in the spindle forming region, whilst protein synthesis is reduced elsewhere in the ooplasm. This is achieved through enrichment of spindle‐related mRNAs in the spindle forming region combined with local PLK1‐mediated phosphorylation and inactivation of the translational repressor EIF4EBP1. This allows PLK1 to function as an important regulatory nexus through which endogenous and exogenous signals can impact spindle formation and function, and highlights the important role that PLK1 may have in maintaining oocyte quality and fertility.     

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.     

Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation

We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions. Received: 8 August 1998 / Accepted: 13 September 1998     

Motor activity and mitotic spindle localization of the Drosophila kinesin-like protein KLP61F.

The KLP61F gene product is essential for Drosophila development. Mutations in KLP61F display a mitotic arrest phenotype caused by a failure in the proper separation of duplicated centrosomes (Heck et al., 1993). Sequence analysis of KLP61F identified it as a member of the bimC family of kinesin-like microtubule motor proteins. Here we report that KLP61F is distinct from KRP130, a kinesin-like protein recently purified from Drosophila embryos and suggested to be the product of the KLP61F gene (Cole et al., 1994). We also characterized recombinant KLP61F and found that it possesses microtubule-stimulated ATPase and microtubule translocation activities in vitro. In addition, we have used an affinity-purified, KLP61F-specific antiserum to localize native KLP61F and an epitope-tagged KLP61F fusion protein during various stages of mitosis in Drosophila syncytial blastoderm embryos. From early prophase through anaphase, KLP61F is coincident with the distribution of tubulin. Together these results confirm the existence of multiple bimC-like kinesins in Drosophila and suggest that KLP61F function is intrinsic to the mitotic spindle.     

Construction of centrosomes and spindle poles by molecular motor-driven assembly of protein particles

Centrosomes and other microtubule organizing centers are the largest non-membranous organelles in most cells. This morphologically diverse class of organelles shares a common ability to nucleate and organize microtubules in interphase and participates in the formation of mitotic spindles during cell division. This review summarizes recent evidence suggesting that assembly of centrosomes and mitotic spindle poles require transport of large protein particles along microtubules by the molecular motor cytoplasmic dynein.