Biology subjects

Welburn, J.

Publications and source records attributed to Welburn, J..

5 recordsLinked to original sources

bioRxiv2023

A BubR1-independent pathway for CENP-E targeting to the outer corona of kinetochores

For chromosome segregation to take place, unattached kinetochores expand in early mitosis, forming a fibrous structure called the fibrous corona that is captured by microtubules. The corona is assembled from the RZZ complex, Spindly, CENP-E and the Mad1/Mad2 spindle assembly checkpoint proteins. CENP-E aligns chromosomes along the mitotic spindle by moving them to the plus ends of microtubules. Here, we show that CENP-E is recruited to the outer corona independently of BubR1 in a dynein-dependent fashion. We determine the structure of this domain and show that a conserved loop is essential for CENP-E targeting to the outer corona. We show that both domains are essential for CENP-E recruitment to unattached kinetochores. We also report that the kinetochore-targeting domain of CENP-E contributes to the recruitment of the RZZ complex, Mad1 and Spindly, providing a feedback loop to assemble the outer corona. In this study, we propose that CENP-E uses 2 pathways to target to the kinetochore, which allows it to optimize kinetochore capture by microtubules for chromosome alignment and mitotic progression.

cell biology↗

bioRxiv2022

Understanding molecular mechanisms and predicting phenotypic effects of pathogenic tubulin mutations

Cells rely heavily on microtubules for several processes, including cell division and molecular trafficking. Mutations in the different tubulin- and -{beta} proteins that comprise microtubules have been associated with various diseases and are often dominant, sporadic and congenital. While the earliest reported tubulin mutations affect neurodevelopment, mutations are also associated with other disorders such as bleeding disorders and infertility. We performed a systematic survey of tubulin mutations across all isotypes in order to improve our understanding of how they cause disease, and increase our ability to predict their phenotypic effects. Both protein structural analyses and computational variant effect predictors were very limited in their utility for differentiating between pathogenic and benign mutations. This was even worse for those genes associated with non-neurodevelopmental disorders. We selected tubulin- and -{beta} disease mutations that were most poorly predicted for experimental characterisation. These mutants co-localise to the mitotic spindle in HeLa cells, suggesting they may exert dominant-negative effects by altering microtubule properties. Our results show that tubulin mutations represent a blind spot for current computational approaches, being much more poorly predicted than mutations in most human disease genes. We suggest that this is likely due to their strong association with dominant-negative and gain-of-function mechanisms.

genetics↗

bioRxiv2022

Reconstitution of an active human CENP-E motor

CENP-E is a large kinesin motor protein which plays pivotal roles in mitosis by facilitating chromosome capture, alignment and promoting microtubule flux in the spindle. So far, it has not been possible to obtain active human CENP-E to study its molecular properties. Xenopus CENP-E motor has been characterised in vitro and is used as a model motor, however its protein sequence differs significantly from human CENP-E. Here, we characterise human CENP-E motility in vitro. Full-length CENP-E exhibits an increase in run length and longer residency times on microtubules when compared to CENP-E motor truncations, indicating that the C-terminal microtubule binding site enhances the processivity when the full-length motor is active. In contrast to constitutively active human CENP-E truncations, full-length human CENP-E has a reduced microtubule landing rate in vitro, suggesting that the non-motor coiled coil regions self-regulate motor activity. Together, we demonstrate that human CENP-E is a processive motor, providing a useful tool to study the mechanistic basis for how human CENP-E drives chromosome congression and spindle organisation during human cell division.

biochemistry↗

bioRxiv2020

Emergent properties of a mitotic Kif18b-MCAK-EB network

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. Prior work has identified key molecular players in this process, including the kinesin-18 Kif18b and the kinesin-13 Kif2C/MCAK, which both promote microtubule depolymerization. MCAK acts as a potent microtubule depolymerase diffusing short distances on microtubules, while Kif18b is a mitotic processive motor with weak depolymerase activity. However the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 act in an integrated manner to potently promote microtubule depolymerization. We find Kif18b acts as a microtubule plus end delivery factor for its cargo MCAK, and that Kif18b also promotes EB accumulation to plus ends independently of lattice nucleotide state. Together, our work defines the mechanistic basis for a cooperative Kif18b-EB-MCAK network with emergent properties, that acts to efficiently shorten microtubules in mitosis.

biochemistry↗

bioRxiv2018

Microtubule end tethering of a processive Kinesin-8 motor Kif18b is required for spindle positioning

Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning is therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. Here we show using gene knockout that the Kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using an integrated approach, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.

cell biology↗