Biology subjects

King, M. R.

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

12 recordsLinked to original sources

bioRxiv2025

Pipette, mix, repeat: A reduced-order fluid model and protocol survey to evaluate 96-well mixing protocols

Mixing in 96-well plates is a fundamental operation in biology and bioengineering, yet the most common strategy, pipetting up and down to "mix well", is rarely quantified or justified. Rules of thumb such as "three mixing cycles" coexist with protocols that recommend 5, 10, or 20 pipette strokes per step, and there is limited fluid-mechanical guidance on when these choices are adequate. Here, a reduced-order compartment model of advection-diffusion in a single 96-well plate well is developed, in which the fluid is discretized into hundreds of macrocells with finite-volume-style neighbor exchange. A pipette jet is represented as a localized enhancement of exchange along a pipette-aligned "jet footprint," parameterized by an effective jet strength, effective jet footprint size as a proxy for cycled volume fraction, pipette angle, and centered versus off-center tip placement. Repeated aspirate-dispense cycles are simulated and mixing quantified by the decay of normalized concentration variance. The reduced model qualitatively reproduces the mixing hierarchy reported in high-fidelity COMSOL simulations of repetitive pipetting, with higher jet strength and jet footprint substantially accelerating homogenization, while gentle, small-footprint jets often require more than three cycles to reach near-uniform thresholds. Systematic variation of pipette angle and tip position shows that moderately off-center, moderately tilted pipetting modestly improves mixing relative to strictly vertical, centered configurations, with angle and tip position effects remaining small compared to jet strength and footprint size. In parallel, a survey of 96-well pipette mixing instructions in online research forums, commercial assay protocols, and published papers was carried out, extracting explicit "pipetted up and down N times" stroke counts. The resulting distribution varies from 2 to 20 strokes per mixing step, with no clear consensus. This combined modeling and survey analysis indicates that three cycles are not sufficient for many instances of 96-well pipette mixing, and provides a fast, interpretable tool for rationalizing and redesigning pipette-mixing protocols in high-throughput experiments.

bioengineering↗

bioRxiv2025

Molecular grammars of intrinsically disordered regions that span the human proteome

Intrinsically disordered regions (IDRs) of proteins are defined by functionally relevant molecular grammars. This refers to IDR-specific non-random amino acid compositions and non-random patterning of distinct pairs of amino acid types. Here, we introduce GIN (Grammars Inferred using NARDINI+) as a resource, which we have used to extract the molecular grammars of all human IDRs and classified them into thirty distinct clusters. Unbiased analyses of IDRome-spanning grammars reveals that specialized IDR grammar features direct biological processes, cellular localization preferences, and molecular functions. IDRs with exceptional grammars, defined as sequences with high-scoring non-random features, are harbored in proteins and complexes that enable spatial and temporal sorting of biochemical activities. Protein complexes within the nucleus recruit specific factors through top-scoring IDRs. These IDRs are frequently disrupted via cancer-associated mutations and fusion oncoproteins. Overall, GIN enables the decoding of sequence-function relationships of IDRs and can be deployed in IDR-specific and IDRome-wide analyses.

bioinformatics↗

bioRxiv2023

Single fluorogen imaging reveals spatial inhomogeneities within biomolecular condensates

Biomolecular condensates are viscoelastic materials. Simulations predict that fluid-like condensations are defined by spatially inhomogeneous organization of the underlying molecules. Here, we test these predictions using single-fluorogen tracking and super-resolution imaging. Specifically, we leverage the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to condensate microenvironments. We deployed three different fluorogens to probe the microenvironments and molecular organization of different protein-based condensates. The spatiotemporal resolution and environmental sensitivity afforded by single-fluorogen imaging shows that the internal environments of condensates are more hydrophobic than coexisting dilute phases. Molecules within condensates are organized in a spatially inhomogeneous manner, and this gives rise to slow-moving nanoscale molecular clusters that coexist with fast-moving molecules. Fluorogens that localize preferentially to the interface help us map their distinct features. Our findings provide a structural and dynamical basis for the viscoelasticity of condensates.

biophysics↗

bioRxiv2023

Dynamical control enables the formation of demixed biomolecular condensates

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

biophysics↗

bioRxiv2022

RanGTP regulates the augmin complex

Spindles are composed of microtubules that must nucleate at the right place and time during mitosis. Spindle microtubule nucleation is regulated by the GTPase Ran, which, through importin-{beta}, releases a gradient of spindle assembly factors (SAFs) centered at chromosomes. Branching MT nucleation generates most spindle MTs and requires the augmin complex. In Xenopus laevis, Ran can control branching through the SAF TPX2, TPX2 is non-essential in other organisms. Thus, how Ran regulates branching MT nucleation in the absence of TPX2 is unknown. Here, we use in vitro pulldowns and TIRF microscopy to show that augmin is itself a SAF. Augmin directly interacts with both importins through two nuclear localization sequences on the Haus8 subunit, which overlap the MT binding site. Moreover, Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. By uncovering that RanGTP directly regulates augmin, we demonstrate how Ran controls branching MT nucleation and, thereby, spindle assembly and cell division.

biochemistry↗

bioRxiv2022

Uncovering molecular grammars of intrinsically disordered regions that organize nucleolar fibrillar centers

The nucleolus is a multilayered structure. Each layer is thought to be a compositionally distinct phase, although how these phases form and interface with one another remains unclear. Using computational, proteomics, in vitro, and in vivo studies, we uncover distinct molecular grammars within intrinsically disordered regions (IDRs) of nucleolar proteins that localize to fibrillar centers (FCs) and dense fibrillar components (DFCs). FC and DFC proteins feature two distinct types of IDRs namely those with long tracts of acidic residues and those with blocks of lysines interspersed by acid-rich-regions. We find that phase separation driven by complex coacervation in mixtures of nucleolar proteins, with their distinctive IDRs, and ribosomal DNA and RNA molecules is sufficient to drive the formation of structural facsimiles of FCs and DFCs. One-Sentence SummaryFacsimiles of core nucleolar substructures were reconstituted via phase separation of key protein and nucleic acid mixtures.

biophysics↗

bioRxiv2022

The soluble N-termini of mechanosensitive ion channels MSL8, MSL9, and MSL10 are environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations. In the past several decades, IDRs have emerged as a core component of many proteomes, comprising [~]30% of all eukaryotic protein sequences. IDRs are ubiquitous throughout different biological pathways, with a notable enrichment in responses to environmental stimuli such as abiotic stress. However, the diversity of IDR-based systems that biology has evolved to respond to different stimuli is expansive, warranting the exploration of IDRs present in unique molecular contexts. Here, we identify and characterize intrinsic disorder in the soluble, cytoplasmic N-terminal domains of three members of the MscS-Like (MSL) family of mechanosensitive ion channels, MSL8, MSL9 and MSL10. In plants, MSL channels are proposed to mediate the reactions to cell swelling, pathogenic invasion, and touch. A series of bioinformatic tools unanimously predicted that the cytosolic N-termini of MSLs are intrinsically disordered. We confirmed this prediction for the N-terminus of MSL10 (MSL10N) via circular dichroism spectroscopy. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE) and underwent structural changes and alterations to homotypic interaction favorability in the presence of molecular crowding agents. Lastly, in vitro imaging of condensates indicated that MSL8N, MSL9N and MSL10N have sharply differing propensities for condensate formation both inherently and in response to salt, temperature, and molecular crowding. Altogether, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond disparately to changes in their physiochemical environment.

biochemistry↗

bioRxiv2022

Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

biophysics↗

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↗