Structure of the native γ-tubulin ring complex capping spindle microtubules.

Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive.

Centriolar satellites are acentriolar assemblies of centrosomal proteins.

Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)-based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions.

Lessons from yeast: the spindle pole body and the centrosome.

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted.

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles.

CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response.

The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells.

Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication.

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat.

Interactions between centromere complexes in Saccharomyces cerevisiae.

We have purified two new complexes from Saccharomyces cerevisiae, one containing the centromere component Mtw1p together with Nnf1p, Nsl1p, and Dsn1p, which we call the Mtw1p complex, and the other containing Spc105p and Ydr532p, which we call the Spc105p complex. Further purifications using Dsn1p tagged with protein A show, in addition to the other components of the Mtw1p complex, the two components of the Spc105p complex and the four components of the previously described Ndc80p complex, suggesting that all three complexes are closely associated. Fluorescence microscopy and immunoelectron microscopy show that Nnf1p, Nsl1p, Dsn1p, Spc105p, and Ydr532p all localize to the nuclear side of the spindle pole body and along short spindles.

Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication.

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized.

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells.

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint.