Biology subjects

Carvalho, A. X.

Publications and source records attributed to Carvalho, A. X..

3 recordsLinked to original sources

bioRxiv2018

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

The kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into {micro}m-scale filaments, and we identify the ROD-1 {beta}-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZs capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

cell biology↗

bioRxiv2017

TPXL-1 Activates Aurora A to Clear Contractile Ring Components from the Polar Cortex During Cytokinesis

During cytokinesis, a signal from the bundled microtubules that form between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.\n\nSUMMARYDuring cytokinesis, centrosomal asters inhibit cortical contractility at the cell poles. Mangal et al. provide molecular insight into this phenomenon, showing that TPXL-1, which localizes to astral microtubules, activates Aurora A kinase to clear contractile ring proteins from the polar cortex.

cell biology↗

bioRxiv2017

Dynactin Binding To Tyrosinated Microtubules Promotes Centrosome Centration In C. Elegans By Enhancing Dynein-Mediated Organelle Transport

The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactins microtubule binding activity, located in p150s CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Using precise mutants engineered by genome editing, we show that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating cortical pulling forces. Instead, p150 CAP-Gly mutants inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of the C-terminal tyrosine of -tubulin, and both p150 CAP-Gly and tubulin tyrosination mutants decrease the frequency of organelle transport from the cell periphery towards centrosomes during centration. In light of recent work on dynein-dynactin motility in vitro, our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing embryo, and that this function of dynactin is important for generating robust cytoplasmic pulling forces for centrosome centration.

cell biology↗