Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules ...(kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.
Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have ...been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than in those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force-generating interface within kinetochores and a separate passive frictional interface located at least 20 nanometers away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.
The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify ...their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (∼14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes). Each CENP-T molecule recruits ∼2 Ndc80 complexes; one as part of a KMN network. In contrast, ∼40% of CENP-C recruits only a KMN network. Replacing the CENP-C domain that binds KMN with the CENP-T domain that recruits both an Ndc80 complex and KMN network yielded functional kinetochores. These results provide a quantitative picture of the linkages between centromeric chromatin and the microtubule-binding Ndc80 complex at the human kinetochore.
The kinetochore is a control module that both powers and regulates chromosome segregation in mitosis and meiosis. The kinetochore-microtubule interface is remarkably fluid, with the microtubules ...growing and shrinking at their point of attachment to the kinetochore. Furthermore, the kinetochore itself is highly dynamic, its makeup changing as cells enter mitosis and as it encounters microtubules. Active kinetochores have yet to be isolated or reconstituted, and so the structure remains enigmatic. Nonetheless, recent advances in genetic, bioinformatic and imaging technology mean we are now beginning to understand how kinetochores assemble, bind to microtubules and release them when the connections made are inappropriate, and also how they influence microtubule behaviour. Recent work has begun to elucidate a pathway of kinetochore assembly in animal cells; the work has revealed that many kinetochore components are highly dynamic and that some cycle between kinetochores and spindle poles along microtubules. Further studies of the kinetochore-microtubule interface are illuminating: (1) the role of the Ndc80 complex and components of the Ran-GTPase system in microtubule attachment, force generation and microtubule-dependent inactivation of kinetochore spindle checkpoint activity; (2) the role of chromosomal passenger proteins in the correction of kinetochore attachment errors; and (3) the function of microtubule plus-end tracking proteins, motor depolymerases and other proteins in kinetochore movement on microtubules and movement coupled to microtubule poleward flux.
Dementia with Lewy bodies (DLB) was considered to be an uncommon cause of dementia until improved neuropathological staining methods for ubiquitin were developed in the late 1980's. Subsequent ...recognition that 10-15% of dementia cases in older people were associated with Lewy body pathology led to the publication in 1996 of Consensus clinical and pathological diagnostic criteria for the disorder. These have greatly raised global awareness of DLB and helped to generate a body of knowledge which informs modern clinical management of this pharmacologically sensitive group of patients. They have also enabled important issues surrounding the relationships of DLB with Alzheimer's disease and Parkinson's disease to be addressed and partially resolved. A recent re-evaluation of the Consensus criteria has confirmed many aspects of the original recommendations, supplementing these with suggestions for improved pathological characterisation, clinical detection and management. Virtu-ally unrecognised 20 years ago, DLB could within this decade be one of the best characterised and potentially treatable neurodegenerative disorders of late life.
The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that ...congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).
Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule ...attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.
Generation of motile force is one of the main functions of the eukaryotic kinetochore during cell division. In recent years, the KMN network of proteins (Ndc80 complex, Mis12 complex, and KNL-1 ...complex) has emerged as a highly conserved core microtubule-binding complex at the kinetochore. It plays a major role in coupling force generation to microtubule plus-end polymerization and depolymerization. In this review, we discuss current theoretical mechanisms of force generation, and then focus on emerging information about mechanistic contributions from the Ndc80 complex in eukaryotes and the microtubule-binding Dam1/DASH complex from fungi. New information has also become available from super-resolution light microscopy on the protein architecture of the kinetochore-microtubule attachment site in both budding yeast and humans, which provides further insight into the mechanism of force generation. We briefly discuss potential contributions of motors, other microtubule-associated proteins, and microtubule depolymerases. Using the above evidence, we present speculative models of force generation at the kinetochore.
Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner ...kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ∼16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20–60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex.
•High-resolution microscopy reveals CCAN protein architecture in human kinetochores•Proper intrakinetochore stretch depends on the CCAN linkers to the Ndc80 complex•Ndc80/Hec1 phosphorylation is inhibited by hyper-intrakinetochore stretch•CCAN DNA binding proteins play a minor role in the compaction of CENP-A chromatin
Using super-resolution microscopy, Suzuki et al. find that constitutive centromere-associated network proteins mechanically constrain the distance between inner and outer kinetochore components under tension. This constraint on intrakinetochore stretch is important to ensure Aurora B access to its substrates and thus to correct errors in kinetochore-microtubule attachment.
Solid organ transplantations (SOT) are performed successfully in selected HIV‐infected patients. However, multiple and reciprocal drug–drug interactions are observed between antiretroviral (ARV) ...drugs and calcineurin inhibitors (CNIs) through CYP450 metabolization. Raltegravir (RAL), a novel HIV‐1 integrase inhibitor, is not a substrate of CYP450 enzymes. We retrospectively reviewed the outcomes of 13 HIV‐infected transplant patients treated by an RAL + two nucleosidic reverse transcriptase inhibitor (NRTI) regimen, in terms of tolerability, ARV efficacy (plasma viral load, CD4 cell count), drug interactions, RAL pharmacokinetics and transplant outcome. Thirteen patients with liver (n = 8) or kidney (n = 5) transplantation were included. RAL was initiated (400 mg BID) either at time of transplantation (n = 6), or after transplantation (n = 7). Median RAL trough concentration was 507 ng/mL (176–890), which is above the in vitro IC95 for wild type HIV‐1 strains (15 ng/mL). Target trough levels of CNIs were promptly obtained with standard dosages of tacrolimus or cyclosporine. RAL tolerability was excellent. There was no episode of acute rejection. HIV infection remained controlled. After a median follow‐up of 9 months (range: 6–14), all patients were alive with satisfactory graft function. The use of an RAL + two NRTI‐based regimen is a good alternative in HIV‐infected patients undergoing SOT.
Raltegravir use in solid organ transplantation represents a new option to reduce drug interactions in HIV‐infected patients.
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