Dhanya K. Cheerambathur

Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

A microtubule-binding site in the extreme N terminus of KNL-1 is dispensable for load-bearing attachments but participates in checkpoint silencing at the kinetochore.

A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint

A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint.

Kinesin-5–dependent Poleward Flux and Spindle Length Control in Drosophila Embryo Mitosis

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motors concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole–pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation.

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.

Crosstalk Between Microtubule Attachment Complexes Ensures Accurate Chromosome Segregation

Dissecting Chromosome Segregation During cell division, the centromere regions of chromosomes assemble multiprotein organelles called kinetochores that form attachments to spindle microtubules. Working in Caenorhabditis elegans, Cheerambathur et al. (p. 1239, published online 14 November) describe a mechanism controlling the formation of kinetochore-spindle microtubule attachments that is essential for accurate chromosome segregation. The findings suggest the existence of crosstalk between the two major protein complexes involved in forming spindle microtubule attachments: the kinetochore dynein module, which initially captures spindle microtubules, and the Ndc80 complex, which ultimately forms the dynamic end-coupled attachments that segregate chromosomes. Chromosome partitioning involves regulatory crosstalk between two major microtubule-binding complexes at the kinetochore. The microtubule-based mitotic spindle segregates chromosomes during cell division. During chromosome segregation, the centromeric regions of chromosomes build kinetochores that establish end-coupled attachments to spindle microtubules. Here, we used the Caenorhabditis elegans embryo as a model system to examine the crosstalk between two kinetochore protein complexes implicated in temporally distinct stages of attachment formation. The kinetochore dynein module, which mediates initial lateral microtubule capture, inhibited microtubule binding by the Ndc80 complex, which ultimately forms the end-coupled attachments that segregate chromosomes. The kinetochore dynein module directly regulated Ndc80, independently of phosphorylation by Aurora B kinase, and this regulation was required for accurate segregation. Thus, the conversion from initial dynein-mediated, lateral attachments to correctly oriented, Ndc80-mediated end-coupled attachments is actively controlled.

NOCA-1 functions with γ-tubulin and in parallel to Patronin to assemble non-centrosomal microtubule arrays in C. elegans

Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify Caenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families. DOI: http://dx.doi.org/10.7554/eLife.08649.001

Linked in: formation and regulation of microtubule attachments during chromosome segregation.

Accurate segregation of the replicated genome during cell division depends on dynamic attachments formed between chromosomes and the microtubule polymers of the spindle. Here we review recent advances in mechanistic analysis of microtubule attachment formation and regulation.

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.

A Nucleoporin Docks Protein Phosphatase 1 to Direct Meiotic Chromosome Segregation and Nuclear Assembly.

During M-phase entry in metazoans with open mitosis, the concerted action of mitotic kinases disassembles nuclei and promotes assembly of kinetochores-the primary microtubule attachment sites on chromosomes. At M-phase exit, these major changes in cellular architecture must be reversed. Here, we show that the conserved kinetochore-localized nucleoporin MEL-28/ELYS docks the catalytic subunit of protein phosphatase 1 (PP1c) to direct kinetochore disassembly-dependent chromosome segregation during oocyte meiosis I and nuclear assembly during the transition from M phase to interphase. During oocyte meiosis I, MEL-28-PP1c disassembles kinetochores in a timely manner to promote elongation of the acentrosomal spindles that segregate homologous chromosomes. During nuclear assembly, MEL-28 recruits PP1c to the periphery of decondensed chromatin, where it directs formation of a functional nuclear compartment. Thus, a pool of phosphatase activity associated with a kinetochore-localized nucleoporin contributes to two key events that occur during M-phase exit in metazoans: kinetochore disassembly and nuclear reassembly.