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Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level.
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This content will become publicly available on December 23, 2026
Structural reorganization underlying stress-induced cytoplasmic solidification in yeast
SUMMARY Cells employ diverse strategies to rapidly adapt to sudden environmental changes. In yeast, cytoprotective solidification in response to starvation and energy depletion (ED) has been reported and associated with extensive mesoscale macromolecular assembly. Yet, the structural and molecular basis underlying such whole-cell level liquid-to-solid phase transitions remain unknown. Here, we use cryo-electron tomography to characterize the subcellular organization of intact yeast cells exposed to ED and other stressors, and to untangle the effects of molecular crowding versus cytoplasmic acidification previously suggested to underpin solidification. We visualize self-assembly of macromolecules and complexes into ordered assemblies and condensates under ED, and quantify ribosome and polysomes concentrations to probe changes in cytoplasmic crowding. Combined with live-cell microscopy, we pinpoint supramolecular assembly induced by acidification, rather than a uniform increase in intracellular crowding, as the structural basis of cytoplasmic solidification that supports yeast cells’ adaptation in response to environmental stresses.
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- Award ID(s):
- 2243257
- PAR ID:
- 10686248
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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