Poleward force at the kinetochore in metaphase depends on the number of kinetochore microtubules

To examine the dependence of poleward force at a kinetochore on the number of kinetochore microtubules (kMTs), we altered the normal balance in the number of microtubules at opposing homologous kinetochores in meiosis I grasshopper spermatocytes at metaphase with a focused laser microbeam. Observations were made with light and electron microscopy. Irradiations that partially damaged one homologous kinetochore caused the bivalent chromosome to shift to a new equilibrium position closer to the pole to which the unirradiated kinetochore was tethered; the greater the dose of irradiation, the farther the chromosome moved. The number of kMTs on the irradiated kinetochore decreased with severity of irradiation, while the number of kMTs on the unirradiated kinetochore remained constant and independent of chromosome-to-pole distance. Assuming a balance of forces on the chromosome at congression equilibrium, our results demonstrate that the net poleward force on a chromosome depends on the number of kMTs and the distance from the pole. In contrast, the velocity of chromosome movement showed little dependence on the number of kMTs. Possible mechanisms which explain the relationship between the poleward force at a kinetochore, the number of kinetochore microtubules, and the lengths of the kinetochore fibers at congression equilibrium include a "traction fiber model" in which poleward force producers are distributed along the length of the kinetochore fibers, or a "kinetochore motor-polar ejection model" in which force producers located at or near the kinetochore pull the chromosomes poleward along the kMTs and against an ejection force that is produced by the polar microtubule array and increases in strength toward the pole.     

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4–6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users.     

Insights into the kinetochore

The Ndc80 complex is a core component of the kinetochore, which links chromosomes to microtubules. Recently, Ciferri et al. (2008) published an atomic-level structure of the complex with implications for kinetochore architecture and for the generation and control of chromosome movements during mitosis.     

Balancing the kinetochore ledger

Reduction of polo-like kinase-1 (Plk1) at kinetochores as cells progress from prometaphase to metaphase is surprising given that the kinase is thought to stabilize kinetochore-microtubule (kt-MT) attachments. In this issue, Liu et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205090) demonstrate that kinetochore-associated Plk1 is a potent suppressor of microtubule plus-end dynamics. The authors propose that Plk1 activity facilitates the establishment of kt-MT attachments in prometaphase by stabilizing microtubules and that reduction of the kinase in metaphase promotes force generation by dynamic microtubules.     

Functional redundancy in the maize meiotic kinetochore

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, approximately 6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.     

Structure of the mammalian kinetochore

The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D₂O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer. We dedicate this paper to Wolfgang Beermann on the occasion of his 60th birthday in appreciation of many years of friendship and his pioneering contributions in the field of chromosome biology     

Organization within the mammalian kinetochore

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25–30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.     

The plant kinetochore

Kinetochores are large protein complexes that bind to centromeres. By interacting with microtubules and their associated motor proteins, kinetochores both generate and regulate chromosome movement. Kinetochores also function in the spindle checkpoint; a surveillance mechanism that ensures that metaphase is complete before anaphase begins. Although the ultrastructure of plant kinetochores has been known for many years, only recently have specific kinetochore proteins been identified. The recent data indicate that plant kinetochores contain homologs of many of the proteins implicated in animal and fungal kinetochore function, and that the plant kinetochore is a redundant structure with distinct biochemical subdomains.     

Visualizing kinetochore architecture

Kinetochores are large macromolecular assemblies that link chromosomes to spindle microtubules (MTs) during mitosis. Here we review recent advances in the study of core MT-binding kinetochore complexes using electron microcopy methods in vitro and nanometer-accuracy fluorescence microscopy in vivo. We synthesize these findings in novel three-dimensional models of both the budding yeast and vertebrate kinetochore in different stages of mitosis. There is a growing consensus that kinetochores are highly dynamic, supra-molecular machines that undergo dramatic structural rearrangements in response to MT capture and spindle forces during mitosis.     

Chromatin assembly: the kinetochore connection

An unexpected new role for the chromatin assembly factor CAF-1 and the histone-regulating Hir proteins has been discovered in budding yeast. Both protein complexes are required together for building functional kinetochores.