Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

The functional organization of microtubules in eukaryotic cells requires a combination of their inherent dynamic properties, interactions with motor machineries, and interactions with accessory proteins to affect growth, shrinkage, stability, and architecture. In most organisms, the Kinesin-8 family of motors play an integral role in these organizations, well known for their mitotic activities in microtubule (MT) length control and kinetochore interactions. In Dictyostelium discoideum, the function of Kinesin-8 remains elusive. We present here some biochemical properties and localization data that indicate that this motor (DdKif10) shares some motility properties with other Kinesin-8s but also illustrates differences in microtubule localization and depolymerase action that highlight functional diversity.

Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.

The distance between fluorescent spots formed by various kinetochore proteins (delta) is commonly interpreted as a manifestation of intrakinetochore tension (IKT) caused by microtubule-mediated forces. However, large-scale changes of the kinetochore architecture (such as its shape or dimensions) may also contribute to the value of delta. To assess contributions of these non-elastic changes, we compare behaviour of delta values in human kinetochores with small yet mechanically malleable kinetochores against compound kinetochores in Indian muntjac (IM) cells whose architecture remains constant. Due to the micrometre-scale length of kinetochore plates in IM, their shape and orientation are discernible in conventional light microscopy, which enables precise measurements of IKT independent of contributions from changes in overall architecture of the organelle. We find that delta in IM kinetochores remains relatively constant when microtubule-mediated forces are suppressed by Taxol, but it prominently decreases upon detachment of microtubules. By contrast, large decreases of delta observed in Taxol-treated human cells coincide with prominent changes in length and curvature of the kinetochore plate. These observations, supported by computational modelling, suggest that at least 50% of the decrease in delta in human cells reflects malleable reorganization of kinetochore architecture rather than elastic recoil due to IKT.

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct mechanisms: (1) capture of astral microtubules nucleated at the centrosome by the chromosomes' kinetochores or (2) attachment of kinetochores to noncentrosomal microtubules with subsequent transport of the minus ends of these microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal microtubules appear in the immediate vicinity of the kinetochores. Initially, these microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end-directed kinetochore-associated kinesin CenpE.

We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.

Numerous studies have highlighted the self-centering activities of individual microtubule (MT) arrays in animal cells, but relatively few works address the behavior of multiple arrays that coexist in a common cytoplasm. In multinucleated Dictyostelium discoideum cells, each centrosome organizes a radial MT network, and these networks remain separate from one another. This feature offers an opportunity to reveal the mechanism(s) responsible for the positioning of multiple centrosomes. Using a laser microbeam to eliminate one of the two centrosomes in binucleate cells, we show that the unaltered array is rapidly repositioned at the cell center. This result demonstrates that each MT array is constantly subject to centering forces and infers a mechanism to balance the positions of multiple arrays. Our results address the limited actions of three kinesins and a cross-linking MAP that are known to have effects in maintaining MT organization and suggest a simple means used to keep the arrays separated.

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

The variability in centrosome size, shape, and activity among different organisms provides an opportunity to understand both conserved and specialized actions of this intriguing organelle. Centrosomes in the model organism Dictyostelium sp. share some features with fungal systems and some with vertebrate cell lines and thus provide a particularly useful context to study their dynamics. We discuss two aspects, centrosome positioning in cells and their interactions with nuclei during division as a means to highlight evolutionary modifications to machinery that provide the most basic of cellular services.