Biology subjects

Wilson, C. J.

Publications and source records attributed to Wilson, C. J..

10 recordsLinked to original sources

bioRxiv2026

An engineered streptavidin condensate platform for chemically inducible control of endogenous proteins in mammalian cells

Inducible control of protein activity with temporal precision is essential for understanding and engineering dynamic cellular behaviors. However, current inducible molecular tools largely rely on overexpression of target proteins, which often disrupts the signaling pathways and cellular functions under investigation. A generalizable method to achieve inducible control of endogenous proteins in mammalian cells remains an unmet need. Here, we present a versatile platform based on engineered streptavidin biomolecular condensates to trap and release endogenously tagged proteins. By tagging endogenous loci with a short streptavidin-binding peptide via CRISPR knock-in, our synthetic streptavidin condensates efficiently partition and functionally inhibit the tagged endogenous proteins. The sequestered cargo protein is rapidly released upon the addition of biotin, restoring protein activity within minutes. We demonstrated the broad applicability of this system by controlling diverse endogenous targets: the anterograde motor KIF5B and retrograde motor DYNC1H1, which regulate intracellular vesicle trafficking, and the Arp2/3 complex subunit ARPC3, which regulates actin dynamics. Furthermore, we developed a dual-inducible system based on rapamycin-dependent condensation of streptavidin, enabling both rapid sequestration and release of endogenous proteins at user-defined time points. Altogether, this engineered streptavidin condensate platform provides a robust, rapid, and scalable approach for manipulating endogenous protein function under physiologically relevant conditions in both basic and translational research.

synthetic biology↗

bioRxiv2025

Improving conformational ensembles of folded proteins in GoMartini

The Martini coarse-grained (CG) force field enables efficient simulations of biomolecular systems but cannot reliably maintain folded protein structures. To stabilize proteins during simulation, Martini is typically combined with structure-based force fields such as elastic network models (ENMs) or G[o] models. While these approaches preserve global folds and capture protein flexibility, their ability to reproduce conformational dynamics remains unclear. Here, we benchmark Martini combined with ENMs or G[o] models on three folded proteins and show that both approaches struggle to sample the conformational space observed in atomistic simulations, even when uniform interaction strengths or equilibrium bond distances are adjusted. This limitation arises from the assumption of a uniform interaction network, in which all bond energies are equal. To overcome this, we present a fully automated, perturbation-based optimization approach for G[o] networks, PoG[o], that iteratively refines a non-uniform G[o] network against a pre-computed atomistic free energy landscape in essential conformational space. Our approach converges rapidly, yielding CG ensembles in close agreement with reference atomistic simulations. As a cross-validation, the optimization also improves the root-mean-square fluctuation profile.

biophysics↗

bioRxiv2025

STIP1/HOP Promotes the Formation of Cytotoxic α-Synuclein Oligomers

The accumulation of alpha-synuclein (a-Syn) as toxic oligomers, and subsequently in Lewy bodies, is a pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Molecular chaperones and co-chaperones are expected to act in concert to maintain physiological activities of proteins, including a-Syn, but in neurodegeneration this process can become mal-adaptive. Transcript levels of Stress inducible phosphoprotein 1 (STIP1), a co-chaperone of Hsp90/Hsp70, are elevated in brain samples from PD patients. In synucleinopathy mouse models, STIP1 has unexpected bidirectional effects on a-Syn, with overexpression of STIP1 aggravating a-Syn toxicity, whereas knockdown of STIP1 improves toxicity and behavioural phenotypes. However, it is unclear how STIP1 enhances the toxicity of a-Syn. Here we unravel the mechanisms by which the direct interaction between STIP1/HOP and a-Syn regulates the neurotoxicity of a-Syn. Specifically, two binding motifs in the C-terminus of a-Syn directly interact with the TPR2A domain of STIP1/HOP in a dynamic manner, competing for a shared interface on TPR2A. Binding of STIP1/HOP to a-Syn attenuates the formation of a-Syn fibrils while promoting the accumulation of high molecular weight amorphous a-Syn species. Samples of a-Syn aggregated in the presence of STIP1/HOP contain significantly more A11-positive oligomeric species and cause a greater reduction in cell viability than a-Syn aggregated in the absence of STIP1/HOP in neuronal cells. Our results provide a mechanism by which the direct interaction between STIP1/HOP and the C-terminus of a-Syn promotes the formation of cytotoxic, non-amyloidogenic, high molecular weight a-Syn species. Our model offers an explanation for the unexpected pathological link between STIP1 and a-Syn toxicity, thus opening new therapeutic avenues for the treatment of synucleinopathies.

biochemistry↗

bioRxiv2024

Phase delays between mouse globus pallidus neurons entrained by common oscillatory drive arise from their intrinsic properties, not their coupling

A hallmark of Parkinsons disease is the appearance of correlated oscillatory discharge throughout the cortico-basal ganglia (BG) circuits. In the primate globus pallidus (GP), where the discharge of GP neurons is normally uncorrelated, pairs of GP neurons exhibit oscillatory spike correlations with a broad distribution of pairwise phase delays in experimental parkinsonism. The transition to oscillatory correlations is thought to indicate the collapse of the normally segregated information channels traversing the BG. The large phase delays are thought to reflect pathological changes in synaptic connectivity in the BG. Here we study the structure and phase delays of spike correlations measured from neurons in the mouse external GP (GPe) subjected to identical 1-100 Hz sinusoidal drive but recorded in separate experiments. First, we find that spectral modes of a GPe neurons empirical instantaneous phase response curve (iPRC), elucidate at what phases of the oscillatory drive the GPe neuron locks when it is entrained, and the distribution of phases at which it spikes when it is not. Then, we show that in this case the pairwise spike cross-correlation equals the cross-correlation function of these spike phase distributions. Finally, we show that the distribution of GPe phase delays arises from the diversity of iPRCs, and is broadened when the neurons become entrained. Modeling GPe networks with realistic intranuclear connectivity demonstrates that the connectivity decorrelates GPe neurons without affecting phase delays. Thus, common oscillatory input gives rise to GPe correlations whose structure and pairwise phase delays reflect their intrinsic properties captured by their iPRCs. Significance StatementThe external globus pallidus (GPe) is a hub in the basal ganglia, whose neurons impose a barrage of inhibitory synaptic currents on neurons of the subthalamic nucleus, substantia nigra and internal globus pallidus. GPe neurons normally fire independently, but in experimental parkinsonism, they become correlated in the frequency range associated with the pathological rhythms seen in human Parkinsons disease, raising the possibility that they may be generators of the pathological oscillation. We drove individual pallidal neurons with an oscillatory input over a wide range of frequencies. Cross-correlations of these neurons reproduced many of the features seen in parkinsonism, suggesting that their correlated oscillations might derive from a shared input rather than internal interconnections.

neuroscience↗

bioRxiv2023

Age-dependent extinction and the neutral theory of biodiversity

Red Queen (RQ) theory states that adaptation does not protect species from extinction because their competitors are continually adapting alongside them. RQ was founded on the apparent independence of extinction risk and fossil taxon age, but analytical developments have since demonstrated that age-dependent extinction is widespread, usually most intense among young species. Here we develop ecological neutral theory as a general framework for modeling fossil species survivorship under incomplete sampling. We show that it provides excellent fit to a high-resolution dataset of species durations for Paleozoic zooplankton, and more broadly can account for age-dependent extinction seen throughout the fossil record. Unlike widely used alternative models, the neutral model has parameters with biological meaning, thereby generating testable hypotheses on changes in ancient ecosystems. The success of this approach suggests novel interpretations of mass extinctions and of scaling in eco-evolutionary systems. Intense extinction among young species does not necessarily refute RQ or require a special explanation, but can instead be parsimoniously explained by neutral dynamics operating across species regardless of age. Significance StatementRed Queen theory predicts that competition among species should cause extinction risk to be independent of species age, but recent analyses have refuted this central prediction. To fill the resulting theoretical vacuum, we used ecological neutral theory to build a model of the lifespans of incompletely sampled species evolving under zero-sum competition. This model predicts survivorship among fossil zooplankton with surprising accuracy and accounts for empirical deviations from the predictions of Red Queen more generally. A neutral model of background extinction allows for interpreting survivorship curves in terms of biological process, suggests a novel understanding of mass extinctions, and supports a role for competition in extinction.

paleontology↗

bioRxiv2023

A synthetic biology approach to assemble and reboot clinically-relevant Pseudomonas aeruginosa tailed phages

The rise in frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically-relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination (TAR) cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step towards the construction of clinically-relevant, engineered P. aeruginosa phages. ImportancePseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically-relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

synthetic biology↗

bioRxiv2021

AlphaFold2: A role for disordered protein prediction?

The development of AlphaFold2 was a paradigm-shift in the structural biology community; herein we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that a naaive use of Dictionary of Secondary Structure of Proteins (DSSP) to separate ordered from disordered regions leads to a dramatic overestimation in disorder content, and that the predicted Local Distance Difference Test (pLDDT) provides a much more rigorous metric. In addition, we show that even when used for disorder prediction, conventional predictors can outperform the pLDDT in disorder identification, and note an interesting relationship between the pLDDT and secondary structure, that may explain our observations, and hints at a broader application of the pLDDT to IDP dynamics.

bioinformatics↗

bioRxiv2019

Constitutive Expression of the Progesterone Receptor Isoforms Promotes Hormone-Dependent Development of Ovarian Neoplasms

Abnormal expression of the progesterone receptor (PGR) isoforms, PGRA and PGRB, is often observed in women with reproductive tract cancer. To assess the importance of the PGR isoform ratio in the maintenance of the healthy reproductive tract, mice with Cre recombinase- activated PGRA and PGRB transgenes were bred with the PGRCre mouse model to generate strains expressing either PGRA or PGRB in PGR positive tissues. The PGRB mice developed ovarian neoplasms at 23 weeks of age derived from ovarian luteal cells, while the PGRA expressing mice displayed a reduced frequency of tumor development. Transcriptomic analyses of the ovarian tumors revealed an enhanced AKT pathway signature, which is in agreement with expression changes found in human ovarian adenocarcinoma. Effective treatment with the PGR antagonist RU486 reduced tumor growth and the expression of cell cycle genes. We concluded that tumor growth and proliferation is hormone and PGR isoform dependent. Further analysis of the PGRB cistrome identified binding events of critical mitotic phase entry genes. This work suggests an intriguing mechanism whereby the expression of the PGR isoforms determines in vivo neoplasia through high-jacking of the cell cycle pathway.

cancer biology↗

bioRxiv2018

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

We report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporated randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

bioengineering↗

bioRxiv2017

The experimental design and data interpretation in “Unexpected mutations after CRISPR–Cas9 editing in vivo” by Schaefer et al. are insufficient to support the conclusions drawn by the authors

To the Editor To the Editor Conflict of Interest Statement References References The recent correspondence to the Editor of Nature Methods by Schaefer et al.1 has garnered significant attention since its publication as a result of its strong conclusions contradicting numerous publications in the field using similar analytical approaches and methods2-4. The authors suggest that the CRISPR-Cas9 system is highly mutagenic in genomic regions not expected to be targeted by the gRNA. We believe that the conclusions drawn from this study are unsubstantiated by the disclosed experiments as they were designed and carried out. Further, it is impossible to ascribe the observed differences in the subject mice to the effects of CRISPR per se. The genetic differences seen in ...

genetics↗