Biology subjects

Hawkins, T. L.

Publications and source records attributed to Hawkins, T. L..

3 recordsLinked to original sources

bioRxiv2020

The GINS complex is required for the survival of rapidly proliferating retinal and tectal progenitor cells during zebrafish development

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the GINS complex as neither Gins1 nor Gins2 seem to be present during these stages.

genetics↗

bioRxiv2019

C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

Microtubules are biopolymers that perform diverse cellular functions. The regulation of microtubule behavior occurs in part through post-translational modification of both the - and {beta}- subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and glutamate residues to the disordered C-terminal tails of tubulin. Due to their prevalence in stable, high stress cellular structures such as cilia, we sought to determine if these modifications alter the intrinsic stiffness of microtubules. Here we describe the purification and characterization of differentially-modified pools of tubulin from Tetrahymena thermophila. We found that glycylation on the -C-terminal tail is a key determinant of microtubule stiffness, but does not affect the number of protofilaments incorporated into microtubules. We measured the dynamics of the tail peptide backbone using nuclear magnetic resonance spectroscopy. We found that the spin-spin relaxation rate (R2) showed a pronounced decreased as a function of distance from the tubulin surface for the -tubulin tail, indicating that the -tubulin tail interacts with the dimer surface. This suggests that the interactions of the -C-terminal tail with the tubulin body contributes to the stiffness of the assembled microtubule, providing insight into the mechanism by which glycylation and glutamylation can alter microtubule mechanical properties.\n\nSIGNIFICANCEMicrotubules are regulated in part by post-translational modifications including the heterogeneous addition of glycine and glutamate residues to the C-terminal tails. By producing and characterizing differentially-modified tubulin, this work provides insight into the molecular mechanisms of how these modifications alter intrinsic microtubule properties such as flexibility. These results have broader implications for mechanisms of how ciliary structures are able to function under high stress.

biophysics↗

bioRxiv2017

Sorting of molecular shuttles by designing electrical and mechanical properties of microtubules

Kinesin-driven microtubules have been a focus to serve as molecular shuttles to replace multiple on-chip functions in micro total analysis systems ({micro}TAS). Although transport, concentration, and detection of target molecules have been demonstrated, controllability of transport directions is still a major challenge. To define multiple moving directions for selective molecular transport, we integrated the bottom-up molecular design of microtubules and the top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively-charged soft microtubules with approximately 80% efficiency under an electric field. Our method is the first to sort multiple microtubules by integrating molecular control and microfluidic device design, and is applicable to multiplexed molecular sorters.

bioengineering↗