Biology subjects

King, M. R.

Publications and source records attributed to King, M. R..

4 recordsLinked to original sources

bioRxiv2021

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

cell biology↗

bioRxiv2021

Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via Death Receptor 4 Upregulation and Lipid Raft Localization

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and chemoresistance. We demonstrate that oxaliplatin-resistant CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this had minimal effects on TRAIL sensitivity following CRISPR-Cas9 deletion of caspase-10 in parental cells. Sensitization effects in oxaliplatin-resistant cells were found to be a result of increased DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable CTCs was observed. Increased DR4/lipid raft colocalization in CTCs was found to correspond with increased oxaliplatin resistance and increased efficacy of TRAIL liposomes. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs. Impact StatementOxaliplatin-resistant colorectal cancer cells exhibit unregulated death receptor 4 expression with increased receptor palmitoylation and translocation into lipid rafts, increasing their sensitivity to apoptosis via TRAIL.

cancer biology↗

bioRxiv2020

Branching microtubule nucleation is controlled by importin-mediated inhibition of TPX2 phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-/{beta}, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-/{beta} with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-. Interestingly, importin-{beta} alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin- and importin-{beta} with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions. Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.

biochemistry↗

bioRxiv2019

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. TPX2 is an effector of microtubule nucleation in spindles, and functions with the substrate tubulin by an unknown mechanism. Here, we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 directly demonstrate that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-/{beta} heterodimer, regulates both co-condensation and activity. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.

biochemistry↗