Biology subjects

Putnam, A. A.

Publications and source records attributed to Putnam, A. A..

4 recordsLinked to original sources

bioRxiv2026

Molecular and cellular characterization of the interaction between the Rho GAP RGA-3/4 and F-actin

During cytokinesis in amphibian and echinoderm embryos, propagating waves of Rho activity and F-actin assembly - cortical excitability - are amplified and focused at the equatorial cortex by the mitotic spindle. Such cortical waves are proposed to result from fast positive feedback based on Rho and the Rho GEF, Ect2, and delayed negative feedback involving F-actin and the Rho GAP, RGA-3/4. Currently, this model is tenuous, in part because the relationship between F-actin and RGA-3/4 is not well understood. Therefore, we investigated how RGA-3/4 regulates F-actin structure both in vivo and in vitro. Full-length RGA-3/4, when expressed alone in Xenopus oocytes, localizes to and bundles cortical F-actin. In vitro, purified full-length RGA-3/4 protein directly binds to and bundles F-actin. TIRF microscopy revealed that RGA-3/4 binds to F-actin cooperatively and accumulates on the filaments before filament crosslinking events. Structure-function analyses demonstrated both the amino-terminal region and the intrinsically disordered region at the carboxyl-terminal portion of RGA-3/4 are essential for normal F-actin interaction. These results provide the first demonstration that RGA-3/4 is a direct, cooperative F-actin binding and bundling protein. We propose that its special features make RGA-3/4 uniquely suited to provide F-actin-dependent negative feedback during cortical excitability and cytokinesis.

cell biology↗

bioRxiv2026

RNA-dependent aggregation of a common TEV protease variant alters in vitro biomolecular condensate reconstitution

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are commonly studied in vitro using protease-mediated removal of solubilizing tags to induce condensation under controlled conditions. Tobacco Etch Virus (TEV) protease is widely used for this purpose and is generally assumed to remain soluble and inert during condensate reconstitution. Here, we show that in RNA-containing systems, TEV protease variants can interact with RNA, leading to aggregation and changes in the phase behavior of the target protein. Using confocal microscopy, turbidity measurements, and mass photometry, we demonstrate that commonly used TEV protease variants differ in their propensity to undergo RNA-dependent aggregation. The widely used pRK793 TEV protease forms large RNA-associated aggregates. We further show that RNA-TEV aggregation alters the morphology and organization of protein-RNA condensates formed by well-characterized phase-separating proteins, including PGL-3 and FUS. Together, our findings show that TEV protease can directly impact in vitro LLPS assays through RNA binding and aggregation. These results underscore the importance of validating protease-based induction strategies and incorporating appropriate controls when reconstituting biomolecular condensates, particularly in RNA-rich systems.

biochemistry↗

bioRxiv2024

High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p

DEAD-box RNA helicases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box helicases unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened. Using high-resolution optical tweezers and fluorescence, we reveal a highly dynamic and stochastic process of multiple Ded1p protomers assembling on and unwinding an RNA duplex. One Ded1p protomer binds to a duplex-adjacent ssRNA tail and promotes binding and subsequent unwinding of the duplex by additional Ded1p protomers in 4-6 bp steps. The data also reveal rapid duplex unwinding and rezipping linked with binding and dissociation of individual protomers and coordinated with the ATP hydrolysis cycle.

biophysics↗

bioRxiv2021

Pickering stabilization of a dynamic intracellular emulsion

Biomolecular condensates are cellular compartments that form by phase separation in the absence of limiting membranes. Studying the P granules of C. elegans, we find that condensate dynamics are regulated by protein clusters that adsorb to the condensate interface. Using in vitro reconstitution, live observations and theory, we demonstrate that localized assembly of P granules is controlled by MEG-3, an intrinsically disordered protein that forms low dynamic assemblies on P granules. Following classic Pickering emulsion theory, MEG-3 clusters lower surface tension and slow down coarsening. During zygote polarization, MEG-3 recruits DYRK/MBK-2 kinase to accelerate localized growth of the P granule emulsion. By tuning condensate-cytoplasm exchange, interfacial clusters regulate the structural integrity of biomolecular condensates, reminiscent of the role of lipid bilayers in membrane-bound organelles. One Sentence SummaryBiomolecular condensates are stabilized by interfacial nanoscale protein clusters.

molecular biology↗