Protein Architecture of the Human Kinetochore Microtubule Attachment Site

Abstract

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.

A super-resolution map of the vertebrate kinetochore

“…Our data could be explained by a modification of the "amphipathic" superhelix model if helical segments were oriented radially with their long axes perpendicular to the chromosome axis. Such an orientation would expose some CENP-A and H3 on the outer surface of the chromosome, but is difficult to reconcile with immunoelectron microscopy and super-resolution colocalization of other kinetochore proteins with CENP-A by fluorescence microscopy, all of which indicate that CENP-A seems to provide a basal layer to the kinetochore that does not penetrate significantly into the chromosome interior (13,46,47).…”

“…In both models, CENP-A and H3 nucleosomes face the external surface, enabling the binding of all CCAN proteins. CENP-C could bind to the more internal CENP-A blocks, crosslinking several layers and explaining the similar oscillations undergone by CENP-A and CENP-C when kinetochores are under tension exerted by microtubules (47). The KMN network assembles in mitosis on top of the CCAN and binds microtubules.…”

“…Detailed maps of kinetochore proteins in fixed chromosomes derived from measurements using two-color fluorescence light microscopy place CENP-C external, but very close to, CENP-A, with CENP-T slightly external to CENP-C (46,47). Current kinetochore chromatin folding models based on data available from localization of canonical histones and CENP-A on unfolded chromatin fibers propose that CENP-A and H3 coexist in the same fiber and are sorted on different faces of an "amphipathic" superhelix, in which CENP-A faces the outer kinetochore and the H3-containing blocks are embedded in the centromere (7,13).…”

Phosphorylation of DYNLT1 at Serine 82 Regulates Microtubule Stability and Mitochondrial Permeabilization in Hypoxia

“…Since the above mentioned reports indicated a key role of DYNLT1 in modulating mPT and MT network stability, we hypothesized that phosphorylation of DYNLT1, especially on S82, might occur in hypoxia, based on our previous work in which we showed that hypoxia-activated p38/MAPK signaling pathway initiates MT disruption and alters cell viability by phosphorylating the downstream effector (Hu et al, 2010). In order to generalize these observations, we chose H9c2 and HeLa cells (Brito and Rieder, 2009;Wan et al, 2009) to study hypoxiareduced MTs disruption and mitochondrial permeabilization related to S82. We used site-directed mutagenesis of the phosphorylation site to obtain phosphomimic and dephosphomimic mutants.…”

Stepwise unfolding supports a subunit model for vertebrate kinetochores

“…Although numerous immuno-electron microscopy (16)(17)(18) and superresolution microscopy (19)(20)(21) studies have mapped the locations of CCAN components relative to one another, the packing of the chromatin fiber in centrochromatin remains unknown. Early studies of stretched chromosomes suggested a repeating "subunit" structure for the kinetochore (1,22).…”