Biology subjects

Nachum, S.

Publications and source records attributed to Nachum, S..

3 recordsLinked to original sources

bioRxiv2025

3D-printed toolkit for regulating epithelial unjamming

During wound healing and cancer invasion, epithelial monolayers transition from a solid-like, jammed state to a fluid-like unjammed state. However, affordable and accessible tools for modeling and precise tuning of this response remain limited. Here, we present a simple, lithography-free 3D-printed toolkit of customizable inserts and scratchers for fabricating micro-gaps with defined geometries and widths down to [~]50 {micro}m, without requiring cleanroom facilities or specialized expertise. Using epithelial monolayers, we demonstrate that gap width fundamentally regulates unjamming dynamics. Narrow gaps induced rapid, coordinated migration with high initial velocities, whereas wide gaps suppressed early unjamming and produced slower, spatially heterogeneous closure. Cell shape dynamics confirmed conserved elongation, cell rounding, and closure behaviors across conditions. The presented toolkit provides new opportunities for investigating the mechanical and molecular mechanisms that regulate epithelial plasticity, enabling fine control of unjamming transitions and allowing the modeling of both healthy and pathological wound-response regimes.

biophysics↗

bioRxiv2025

Mutational scan of self-cleavage by HIV-1 protease provides new views of a conformationally dynamic mechanism

The mechanism of conformationally dynamic proteins remains understudied because they are difficult to analyze structurally. For HIV-1 protease the mechanism of cleavage by mature protease is well understood in large part because it forms a stable structure that is amenable to x-ray crystallography. However, self-cleavage or autoproteolysis of protease from the viral polyprotein involves transiently populated structures and is poorly understood. We probed autoproteolysis in HIV-1 using a yeast reporter and mutational scanning. We compared our results with mutational scanning of protease on viral fitness, which integrates both autocleavage and cutting by mature enzyme. We identified 220 mutations that were well tolerated for self-cleavage but not fitness. We analyzed three of these mutations (D30E, W42M and P44L) using independent approaches. All three were capable of efficient self-cleavage in a bacterial assay of autoproteolysis, but had strong defects in mature form for cleavage of a peptide substrate. These separation of function mutations from the mutational scan clustered at hot-spot locations that do not impact autoproteolysis likely because they are conformationally dynamic during self-cleavage. We used the mutational scanning results and molecular simulations to provide models of autoproteolysis conformations. These models provide new views of a structurally dynamic mechanism.

biochemistry↗

bioRxiv2019

Cell cycle oscillators underlying orderly proteolysis of E2F8

E2F8 is a transcriptional repressor that antagonizes the canonical cell cycle transcription factor E2F1. Despite the importance of this atypical E2F family member in cell cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control its dynamics. To address this question, we developed a complementary set of static and dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, and a full transition from pro-metaphase to G1. This revealed an interlocking molecular switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 sites in E2F8 and the activation of APC/CCdh1, but not APC/CCdc20. Further, we revealed a differential stability of E2F8, accounting for its accumulation in late G1 while APC/CCdh1 is still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in promoting orderly cell cycle progression.

cell biology↗