Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 “dynamitin” protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Centromeric chromatin – spindle microtubule interactions mediated by kinetochores drive chromosome segregation. We have developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at < 5 nm accuracy — to elucidate the protein architecture of human metaphase kinetochores. Delta analysis, when correlated with tension states of spindle-attached sister kinetochore pairs, provided information on mechanical properties of protein linkages within kinetochores. Treatment with taxol—which suppresses microtubule dynamics, eliminates tension at kinetochores, and activates the spindle checkpoint—resulted in specific large-scale changes in kinetochore architecture. Cumulatively, Delta analysis revealed compliant linkages close to the centromeric chromatin, suggests a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and reveals architectural changes induced by taxol treatment. The methods described here should also be applicable to other intermediate-scale biological machines in cells.