The Borealin dimerization domain interacts with Sgo1 to drive Aurora B-mediated spindle assembly.

The chromosomal passenger complex (CPC), which includes the kinase Aurora B, is a master regulator of meiotic and mitotic processes that ensure the equal segregation of chromosomes. Sgo1 is thought to play a major role in the recruitment of the CPC to chromosomes, but the molecular mechanism and contribution of Sgo1-dependent CPC recruitment is currently unclear. Using Xenopus egg extracts and biochemical reconstitution, we found that Sgo1 interacts directly with the dimerization domain of the CPC subunit Borealin. Borealin and the PP2A phosphatase complex can bind simultaneously to the coiled-coil domain of Sgo1, suggesting that Sgo1 can integrate Aurora B and PP2A activities to modulate Aurora B substrate phosphorylation. A Borealin mutant that specifically disrupts the Sgo1-Borealin interaction results in defects in CPC chromosomal recruitment and Aurora B-dependent spindle assembly, but not in spindle assembly checkpoint signaling at unattached kinetochores. These findings establish a direct molecular connection between Sgo1 and the CPC and have major implications for the different functions of Aurora B, which promote the proper interaction between spindle microtubules and chromosomes.

SAS-6 Association with γ-Tubulin Ring Complex Is Required for Centriole Duplication in Human Cells.

Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cells required for robust spindle bipolarity [1, 2]. They are duplicated once during the cell cycle [3], and the duplication involves assembly of a cartwheel on the pre-existing centriole followed by assembly of triplet microtubules around the cartwheel [4, 5]. Although the molecular details of cartwheel formation are understood [6-13], the mechanisms initiating the formation of centriolar microtubules are not known. Here, we show that the central component of cartwheel, HsSAS-6 plays a crucial role in the formation of centriolar microtubules by interacting with the microtubule nucleation machinery, γ-tubulin ring complex (γ-TuRC) in human cells. The globular N terminus and the central coiled-coil domain of SAS-6 are required for formation of the cartwheel [7, 14], whereas the function of its C-terminal outer cartwheel region in centriole duplication remains unclear. We find that deletion of HsSAS-6 C terminus disrupts microtubule formation in daughter centriole, and as a result, cells fail to form the new centriole. Consequently, this results in mitotic cells having only two centrioles localized at a single site. Detailed molecular analyses showed that HsSAS-6 interacts with the γ-TuRC proteins and associates with the γ-TuRC at the centrosome, and furthermore, the C terminus is essential for this association. High-resolution microscopy revealed localization of the γ-TuRC protein, γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub in the centriole. Together, the results indicate that HsSAS-6 regulates centriolar microtubule assembly by anchoring γ-TuRCs to the pro-centriole at the onset of daughter centriole formation.

EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice.

Kinetochore couples chromosome movement to dynamic microtubules, a process that is fundamental to mitosis in all eukaryotes but poorly understood. In vertebrates, spindle-kinetochore-associated (Ska1-3) protein complex plays an important role in this process. However, the proteins that stabilize Ska-mediated kinetochore-microtubule attachment remain unknown. Here we show that microtubule plus-end tracking protein EB1 facilitates Ska localization on microtubules in vertebrate cells. EB1 depletion results in a significant reduction of Ska1 recruitment onto microtubules and defects in mitotic chromosome alignment, which is also reflected in computational modelling. Biochemical experiments reveal that EB1 interacts with Ska1, facilitates Ska1-microtubule attachment and together stabilizes microtubules. Structural studies reveal that EB1 either with Ska1 or Ska complex forms extended structures on microtubule lattice. Results indicate that EB1 promotes Ska association with K-fibres and facilitates kinetochore-microtubule attachment. They also implicate that in vertebrates, chromosome coupling to dynamic microtubules could be mediated through EB1-Ska extended structures.

Human SAS-6 C-Terminus Nucleates and Promotes Microtubule Assembly in Vitro by Binding to Microtubules.

Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αß-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.

+TIP EB1 downregulates paclitaxel­induced proliferation inhibition and apoptosis in breast cancer cells through inhibition of paclitaxel binding on microtubules.

Microtubule plus­end­binding protein (+TIP) EB1 has been shown to be upregulated in breast cancer cells and promote breast tumor growth in vivo. However, its effect on the cellular actions of microtubule­targeted drugs in breast cancer cells has remained poorly understood. By using cellular and biochemical assays, we demonstrate that EB1 plays a critical role in regulating the sensitivity of breast cancer cells to anti­microtubule drug, paclitaxel (PTX). Cell viability assays revealed that EB1 expression in the breast cancer cell lines correlated with the reduction of their sensitivity to PTX. Knockdown of EB1 by enzymatically­prepared siRNA pools (esiRNAs) increased PTX­induced cytotoxicity and sensitized cells to PTX­induced apoptosis in three breast cancer cell lines, MCF­7, MDA MB­231 and T47D. Apoptosis was associated with activation of caspase­9 and an increase in the cleavage of poly(ADP­ribose) polymerase (PARP). p53 and Bax were upregulated and Bcl2 was downregulated in the EB1­depleted PTX­treated MCF­7 cells, indicating that the apoptosis occurs via a p53­dependent pathway. Following its upregulation, the nuclear accumulation of p53 and its association with cellular microtubules were increased. EB1 depletion increased PTX­induced microtubule bundling in the interphase cells and induced formation of multiple spindle foci with abnormally elongated spindles in the mitotic MCF­7 cells, indicating that loss of EB1 promotes PTX­induced stabilization of microtubules. EB1 inhibited PTX­induced microtubule polymerization and diminished PTX binding to microtubules in vitro, suggesting that it modulates the binding sites of PTX at the growing microtubule ends. Results demonstrate that EB1 downregulates inhibition of PTX­induced proliferation and apoptosis in breast cancer cells through a mechanism in which it impairs PTX­mediated stabilization of microtubule polymerization and inhibits PTX binding on microtubules.

Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomer on binding GTP.

The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 µM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells.