An official website of the United States government
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.
more »
« less
Small organic osmolytes accelerate actin filament assembly and stiffen filaments
Abstract Actin filament assembly and mechanics are crucial for maintenance of cell structure, motility, and division. Actin filament assembly occurs in a crowded intracellular environment consisting of various types of molecules, including small organic molecules known as osmolytes. Ample evidence highlights the protective functions of osmolytes such as trimethylamine‐N‐oxide (TMAO), including their effects on protein stability and their ability to counteract cellular osmotic stress. Yet, how TMAO affects individual actin filament assembly dynamics and mechanics is not well understood. We hypothesize that, owing to its protective nature, TMAO will enhance filament dynamics and stiffen actin filaments due to increased stability. In this study, we investigate osmolyte‐dependent actin filament assembly and bending mechanics by measuring filament elongation rates, steady‐state filament lengths, and bending persistence lengths in the presence of TMAO using total internal reflection fluorescence microscopy and pyrene assays. Our results demonstrate that TMAO increases filament elongation rates as well as steady‐state average filament lengths, and enhances filament bending stiffness. Together, these results will help us understand how small organic osmolytes modulate cytoskeletal protein assembly and mechanics in living cells.
more »
« less
- Award ID(s):
- 1943266
- PAR ID:
- 10589016
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytoskeleton
- Volume:
- 82
- Issue:
- 5
- ISSN:
- 1949-3584
- Format(s):
- Medium: X Size: p. 281-290
- Size(s):
- p. 281-290
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Gelsolin is a calcium (Ca2+) dependent, pH sensitive actin-binding protein that regulates actin filament dynamics to remodel the actin cytoskeleton. It is known that gelsolin binding induces conformational changes of actin filaments, leading to filament severing. However, the influence of physiological conditions, such as pH variations, on gelsolin-mediated filament severing activities, mechanics and conformations remains unclear despite their role in actin-actin interactions. Using Total Internal Reflection Fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays, we demonstrate that filament severing efficiencies by gelsolin are enhanced in acidic conditions. In addition, analysis of filament thermal fluctuations using TIRF reveals that gelsolin binding stiffens actin filaments. Furthermore, we show that gelsolin binding induces conformational changes in filaments by measuring the filament half-pitch using high resolution Atomic Force Microscopy imaging. Together, our results suggest that pH modulation plays a key role in gelsolin-mediated filament severing activities, bending mechanics, and conformational changes, which have implications in many cellular processes including cell motility and morphogenesis.more » « less
-
Abstract Growth and turnover of actin filaments play a crucial role in the construction and maintenance of actin networks within cells. Actin filament growth occurs within limited space and finite subunit resources in the actin cortex. To understand how filament growth shapes the emergent architecture of actin networks, we developed a minimal agent‐based model coupling filament mechanics and growth in a limiting subunit pool. We find that rapid filament growth induces kinetic trapping of highly bent actin filaments. Such collective bending patterns are long‐lived, organized around nematic defects, and arise from competition between filament polymerization and bending elasticity. The stability of nematic defects and the extent of kinetic trapping are amplified by an increase in the abundance of the actin pool and by crosslinking the network. These findings suggest that kinetic trapping is a robust consequence of growth in crowded environments, providing a route to program shape memory in actin networks.more » « less
-
The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.more » « less
-
Abstract Precise control over how and where actin filaments are created leads to the construction of unique cytoskeletal arrays within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved actin-related protein 2/3 (Arp2/3) complex as well as a large family of formins. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this mechanism is conserved, we combined the ability to image single filament dynamics in the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contribute to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both classes of nucleator increased overall actin filament abundance and enhanced the frequency of de novo nucleation events by an unknown mechanism. Collectively, our findings suggest that multiple actin nucleation mechanisms cooperate to generate and maintain the homeostatic cortical array of plant epidermal cells.more » « less
