Complementation of fission yeast kinesin-5/Cut7 with human Eg5 provides a versatile platform for screening of anti-cancer compounds

Kinesin-5 family proteins are essential for bipolar spindle assembly to ensure mitotic fidelity. Here we demonstrate evolutionary functional conservation of kinesin-5 between human and fission yeast. Human Eg5 expressed in the nucleus replaces fission yeast counterpart Cut7. Intriguingly, Eg5 overproduction results in cytotoxicity. This phenotype provides a useful platform for the development of novel kinesin-5 inhibitors as anti-cancer drugs.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

A Cytoskeletal Vortex Drives Phage Nucleus Rotation During Jumbo Phage Replication in E. coli

Vortex-like arrays of cytoskeletal filaments that drive cytoplasmic streaming and nucleus rotation have been identified in eukaryotes, but similar structures have not been described in prokaryotes. The only known example of a rotating intracellular body in prokaryotic cells occurs when nucleus-forming jumbo phages infect Pseudomonas. During infection, a bipolar spindle of PhuZ filaments drives intracellular rotation of the phage nucleus, a key aspect of the replication cycle. Here we show the E. coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of mutant PhuZ strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes, this work identifies the first known example of a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

A force-balance model for centrosome positioning and spindle elongation during Interphase and Anaphase B

A computational model in one dimension is proposed to position a single centrosome using astral microtubules (MTs) interacting with the cell cortex. The mechanism exploits mutually antagonistic pulling and pushing forces arising from the astral MTs' binding to cortical dynein motors in the actin-rich cell cortex and their buckling while growing against the cell cortex, respectively. The underlying mechanism of astral MTs is also extended to account for the elongation and positioning of the bipolar spindle during mitotic anaphase B. The model for bipolar spindle involves both IPMTs and astral MTs, can predict its elongation and positioning under various circumstances. The model reveals that the bipolar spindle elongation, weakened by decreasing overlap between the antiparallel interpolar microtubules (IPMTs) in the spindle mid-zone, is recovered by the astral MTs.

Paternal chromosome elimination and X non-disjunction on asymmetric spindles in Sciara male meiosis

Meiosis in male Sciara is unique with a single centrosome. A monopolar spindle forms in meiosis I, but a bipolar spindle forms in meiosis II. The imprinted paternal chromosomes are eliminated in meiosis I; there is non-disjunction of the X in meiosis II. Despite differences in spindle construction and chromosome behavior, both meiotic divisions are asymmetric, producing a cell and a small bud. Observations of live spermatocytes made with the LC-PolScope, differential interference contrast optics and fluorescence revealed maternal and paternal chromosome sets on the monopolar spindle in meiosis I and formation of an asymmetric monastral bipolar spindle in meiosis II where all chromosomes except the X congress to the metaphase plate. The X remains near the centrosome after meiosis I and stays with it as the spindle forms in meiosis II. Electron microscopy revealed amorphous material between the X and the centrosome. Immunofluorescence with an antibody against the checkpoint protein Mad2 stains the centromeres of the maternal X dyad in late meiosis I and in meiosis II where it fails to congress to the metaphase plate. Mad2 is also present throughout the paternal chromosomes destined for elimination in meiosis I, suggesting a possible role in chromosome imprinting. If Mad2 on the X dyad mediates a spindle checkpoint in meiosis II, it may delay metaphase to facilitate formation of the second half spindle through a non-centrosomal mechanism.

Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and the chromatin is crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. We propose that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.

Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.