Half Spindles Research Articles

Effect of a cooling unit on high-speed motorized spindle temperature with a scaling factor

Forced cooling, as an efficient way of heat dissipation, significantly affects the spindle temperature. Although a full cooling passage was factored into the finite element analyses by some scholars, often only the model for the front/rear of a spindle is need for the purpose of the thermal evaluation simplification and data overhead reduction in engineering. For this, we devote to exploring how the coolant passage affects the heat dissipation of the front/rear halves of spindles by a constructed scaling factor, and accordingly create a simpler thermal evaluation model of spindles. Firstly, the experiments about a coolant unit effect on the thermal performance of spindles were first implemented, and thus, the temperature difference was noticed under various coolant parameter settings. Through the regressive analysis on the test data, the peak temperature area along axial direction was found and then the scaling factor was proposed to describe the effect of a cooling system on the temperature field of the front/rear half spindle. A similar structural coefficient, meanwhile, was also set up to state clearly the role of motor armature heating in the spindle temperature rise. Next, the thermal equivalent convection for a coolant passage was modeled based on the thermal resistance theory. Based on the abovementioned work, we planned a novel thermal network for only the front half of a motored spindle, in which the cooling mechanism was integrated, and the structural constraints were considered by the aid of the proposed scaling factors. Finally, the simplification and solution for this developed thermal grid were done, and the corresponding numerical results were compared with the test values. At the same time, the bearing heating with no or only 50% of a coolant unit, for the sake of better contrast and validation, was also simulated. The comparison results are indicative of a better agreement with real values when the proposed scaling factors are employed to considering the cooling unit impact on the high-speed motorized spindle temperature.

Dynamic changes in chromatin and microtubules at the first cell cycle in SCNT or IVF goat embryos.

We investigated the dynamic changes in chromatin and microtubules at the first cell cycle in goat somatic cell nuclear transfer (SCNT)-derived and in vitro fertilization (IVF)-derived embryos. Stage-dependent and characteristic changes to chromatin and microtubules occurred in SCNT-derived embryos at different times after activation. About half donor nuclei underwent premature chromosome condensation (PCC) at 1 h post activation, and furtherly reached telophase at 2 h after activation. However, we discovered that the separated chromosomes reaggregated, not keeping two independent nuclei; and formed one pronucleus at 2.5 h after activation. One pronucleus was found in all reconstructed oocytes except other no nucleus oocytes from 3 to 22 h after activation. Reconstructed oocytes reached the first mitotic metaphase at 23 h post activation, which was later than that of IVF-derived embryos at 16 h after insemination. SCNT-derived embryos showed significantly higher abnormalities in the first mitotic metaphase spindle, compared with IVF-derived embryos. Abnormal spindles included multi polar and half spindles. SCNT-derived embryos began to cleave at 24 h after activation, which was later than that of IVF-derived embryos at 21 h after insemination. SCNT-derived embryos showed delayed conversion from telophase to interphase than IVF-derived embryos during cleavage. These might lead to poor development in SCNT-derived embryos.

The Process of First Polar Body Formation in Eggs of the Androgenetic ClamCorbicula fluminea

The hermaphorodic clam Corbicula fluminea has the unusual reproduction mode of androgenesis. In fertilized eggs, all maternal chromosomes and centrosomes are simultaneously extruded as two polar bodies. To reveal the process of this unusual polar body formation, the process was observed by confocal microscopy. In particular, actin organization of oocytes labeled with anti-α- and γ-tubulin antibodies and rhodamine phalloidin were observed. The meiotic spindle was organized near the animal pole and was parallel to the oocyte surface at metaphase of the first meiosis. A thin actin layer was uniformly distributed across the oocyte membrane at metaphase. At anaphase of the first meiosis, two circular, actin-poor regions appeared where the meiotic half spindles docked. All chromosomes and centrosomes were located within the two bulges formed at anaphase in the actin-poor regions. No overlapping of microtubules was observed from either centrosome at anaphase. The centrosomes were located at the apical region of the bulges. Microtubules radiated for the bulge base. At late anaphase, around the actin-poor regions, actin accumulated and two actin rings became distinct. The diameter of these rings gradually decreased and cytokinesis occurred at the base of the bulges. Thus, all centrosomes and chromosomes were extruded together with the polar body. The present study suggests that centrosomes at the first meiosis could have the equal ability to approach and attach to the membrane and induce polar body formation.

Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division.

Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. Here we identify Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. We propose that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.

Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

Recent data have uncovered that spindle size asymmetry (SSA) is a key component of asymmetric cell division (ACD) in the mouse cerebral cortex (Delaunay et al., 2014). In the present study we show that SSA is independent of spindle orientation and also occurs during cortical progenitor divisions in the ventricular zone (VZ) of the macaque cerebral cortex, pointing to a conserved mechanism in the mammalian lineage. Because SSA magnitude is smaller in cortical precursors than in invertebrate neuroblasts, the unambiguous demonstration of volume differences between the two half spindles is considered to require 3D reconstruction of the mitotic spindle (Delaunay et al., 2014). Although straightforward, the 3D analysis of SSA is time consuming, which is likely to hinder SSA identification and prevent further explorations of SSA related mechanisms in generating ACD. We therefore set out to develop an alternative method for accurately measuring spindle asymmetry. Based on the mathematically demonstrated linear relationship between 2D and 3D analysis, we show that 2D assessment of spindle size in metaphase cells is as accurate and reliable as 3D reconstruction provided a specific procedure is applied. We have examined the experimental accuracy of the two methods by applying them to different sets of in vivo and in vitro biological data, including mouse and primate cortical precursors. Linear regression analysis demonstrates that the results from 2D and 3D reconstructions are equally powerful. We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle

This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained.

Augmin Plays a Critical Role in Organizing the Spindle and Phragmoplast Microtubule Arrays in Arabidopsis

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.

Dividing without centrioles: innovative plant microtubule organizing centres organize mitotic spindles in bryophytes, the earliest extant lineages of land plants

As remnants of the earliest land plants, the bryophytes (liverworts, mosses and hornworts) are important in understanding microtubule organization in plant cells. Land plants have an anastral mitotic spindle that forms in the absence of centrosomes, and a cytokinetic apparatus comprised of a predictive preprophase band (PPB) before mitosis and a phragmoplast after mitosis. These microtubule arrays have no counterpart in animal cells and the nature of the plant microtubule organizing centre (MTOC) remained an enigma for many years until antibodies to γ-tubulin, an essential component of the MTOC in all eukaryotes, became available for tracing the origin of microtubule arrays. We used immunofluorescence techniques to colocalize γ-tubulin, microtubules and chromosomes in mitotic cells of a representative liverwort, moss and hornwort to study the organization of microtubules during mitotic cell division. THE FUTURE DIVISION SITE IS MARKED BY A PPB IN ALL TAXA BUT THE MTOCS INITIALLY GENERATING THE HALF SPINDLES DIFFER: polar organizers in the liverwort, plastid MTOCs in the hornwort, and nuclear envelope-associated MTOCs in the moss. By mid-prophase, the forming spindles become more similar as γ-tubulin begins to spread around the polar regions of the nuclear envelope. Regardless of origin, mature metaphase spindles are identical and indistinguishable from the typical anastral spindle of higher plants with broad polar regions consisting of numerous subsets of converging microtubules. A curious phenomenon of plant spindles, true of bryophytes as well as higher plants, is the movement of γ-tubulin into the metaphase spindle itself. The bipolar arrays of phragmoplast microtubules are organized by diffuse γ-tubulin located at proximal surfaces of reforming nuclear envelopes. Phragmoplast development appears similar in the three taxa and to vascular plants as well.

Coupling between microtubule sliding, plus‐end growth and spindle length revealed by kinesin‐8 depletion

Mitotic spindle length control requires coordination between microtubule (MT) dynamics and motor-generated forces. To investigate how MT plus-end polymerization contributes to spindle length in Drosophila embryos, we studied the dynamics of the MT plus-end depolymerase, kinesin-8, and the effects of kinesin-8 inhibition using mutants and antibody microinjection. As expected, kinesin-8 was found to contribute to anaphase A. Furthermore, kinesin-8 depletion caused: (i) excessive polymerization of interpolar (ip) MT plus ends, which "overgrow" to penetrate distal half spindles; (ii) an increase in the poleward ipMT sliding rate that is coupled to MT plus-end polymerization; (iii) premature spindle elongation during metaphase/anaphase A; and (iv) an increase in the anaphase B spindle elongation rate which correlates linearly with the MT sliding rate. This is best explained by a revised "ipMT sliding/minus-end depolymerization" model for spindle length control which incorporates a coupling between ipMT plus end dynamics and the outward ipMT sliding that drives poleward flux and spindle elongation.

Influence of centriole number on mitotic spindle length and symmetry

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

Centrosome Size Sets Mitotic Spindle Length in Caenorhabditis elegans Embryos

Just as the size of an organism is carefully controlled, the size of intracellular structures must also be regulated. The mitotic spindle is a supramolecular machine that generates the forces which separate sister chromatids during mitosis. Although spindles show little size variation between cells of the same type, spindle length can vary at least 10-fold between different species. Recent experiments on spindle length showed that in embryonic systems spindle length varied with blastomere size. Furthermore, a comparison between two Xenopus species showed that spindle length was dependent on some cytoplasmic factor. These data point toward mechanisms to scale spindle length with cell size. Centrosomes play an important role in organizing microtubules during spindle assembly. Here we use Caenorhabditis elegans to study the role of centrosomes in setting spindle length. We show that spindle length correlates with centrosome size through development and that a reduction of centrosome size by molecular perturbation reduces spindle length. By systematically analyzing centrosome proteins, we show that spindle length does not depend on microtubule density at centrosomes. Rather, our data suggest that centrosome size sets mitotic spindle length by controlling the length scale of a TPXL-1 gradient along spindle microtubules.

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of gamma-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from gamma-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.

Heterochromatic Threads Connect Oscillating Chromosomes during Prometaphase I in Drosophila Oocytes

In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.

Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable “spindle matrix” to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction–diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.

The Multiple Roles of Mps1 in Drosophila Female Meiosis

The Drosophila gene ald encodes the fly ortholog of mps1, a conserved kinetochore-associated protein kinase required for the meiotic and mitotic spindle assembly checkpoints. Using live imaging, we demonstrate that oocytes lacking Ald/Mps1 (hereafter referred to as Ald) protein enter anaphase I immediately upon completing spindle formation, in a fashion that does not allow sufficient time for nonexchange homologs to complete their normal partitioning to opposite half spindles. This observation can explain the heightened sensitivity of nonexchange chromosomes to the meiotic effects of hypomorphic ald alleles. In one of the first studies of the female meiotic kinetochore, we show that Ald localizes to the outer edge of meiotic kinetochores after germinal vesicle breakdown, where it is often observed to be extended well away from the chromosomes. Ald also localizes to numerous filaments throughout the oocyte. These filaments, which are not observed in mitotic cells, also contain the outer kinetochore protein kinase Polo, but not the inner kinetochore proteins Incenp or Aurora-B. These filaments polymerize during early germinal vesicle breakdown, perhaps as a means of storing excess outer kinetochore kinases during early embryonic development.

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.

Stable Kinetochore-Microtubule Attachment Constrains Centromere Positioning in Metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.

Drosophila dd4mutants reveal that γTuRC is required to maintain juxtaposed half spindles in spermatocytes

The weak spindle integrity checkpoint in Drosophila spermatocytes has revealed a novel function of the gamma-tubulin ring complex (gammaTuRC) in maintaining spindle bipolarity throughout meiosis. Bipolar and bi-astral spindles could form in Drosophila mutants for dd4, the gene encoding the 91 kDa subunit of gammaTuRC. However, these spindles collapsed around metaphase and began to elongate as if attempting anaphase B. The microtubules of the collapsing spindle folded back on themselves, their putative plus ends forming the focused apexes of biconical figures. Cells with such spindles were unable to undergo cytokinesis. A second type of spindle, monopolar hemi-spindles, also formed as a result of either spindle collapse at an earlier stage or failure of centrosome separation. Multiple centrosome-like bodies at the foci of hemi-spindles nucleated robust asters of microtubules in the absence of detectable gamma-tubulin. Time-lapse imaging revealed these to be intermediates that developed into cones, structures that also had putative plus ends of microtubules focused at their tips. Unlike biconical figures, however, cones seemed to contain a central spindle-like structure at their apexes and undergo cytokinesis. We conclude that spermatocytes do not need astral microtubules nucleated by opposite poles to intersect in order to form a central spindle and a cleavage furrow.

PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone.

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres.

The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.