Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/156224

Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin

McCall, P. M.·MacKintosh, F. C.·Kovar, D. R.·Gardel, M. L.
View original source ↗Export metadata ↓

Abstract

The shape of most animal cells is controlled by the actin cortex, a thin, isotropic 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 turnover: Myosin-II molecular motors drive deformations required for cell division, migration, and tissue morphogenesis, while turnover of the molecular components of the actin cortex relax stress and facilitate network reorganization. While many aspects of F-actin network viscoelasticity are well-characterized in the presence and absence of motor activity, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system wherein the steady-state length and turnover rate of F-actin in entangled solutions are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and nucleate filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. Microrheology measurements 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 in spite of sustained severing.\n\nSignificance StatementWhen an animal cell moves or divides, a disordered network of actin filaments (F-actin) plays a central role in controlling the resulting changes in cell shape. While it is known that continual turnover of F-actin by cofilin-mediated severing aids in reorganization of the cellular cytoskeleton, it is unclear how the turnover of structural elements alters the mechanical properties of the network. Here we show that severing of F-actin by cofilin results in a stress relaxation mechanism in entangled solutions characterized by a single-timescale set by the severing rate. Additionally, we identify ATP hydrolysis and nucleotide-dependent cofilin binding as sufficient ingredients to generate a non-equilibrium steady-state in which asymmetric F-actin disassembly preserves filament number in spite of sustained severing.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

McCall, P. M., MacKintosh, F. C., Kovar, D. R., Gardel, M. L.. 2017-06-26. Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin. https://doi.org/10.1101/156224

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Single Molecule Diffusion Studies Reveal Rapid Translations Across Target Membrane Surfaces for both p40Phox PX domain and Class III PI3K VPS34 Complex 1, the Autophagy Lipid Kinase

Single molecule analyses are providing new insights into the molecular mechanisms by which peripheral membrane proteins approach and translate across a target bilayer surface before stable docking. Here we compare and contrast the translational movements of four diffusers on a representative supported lipid bilayer: (i) the fluorescent lipid LRB-(DO)PE, (ii) the G-protein Rab1A anchored to (DO)PE; (iii) the PI(3)P sensor p40Phox PX domain, and (iv) the autophagy lipid kinase VPS34 Complex 1. The latter enzyme is a Class III PI-3-Kinase and its unusual architecture exhibits two distinct membrane-docking "feet" unique to Class III PI3Ks. Notably, the membrane contact surfaces of the PX domain and autophagy lipid kinase each possess multiple positive charges suitable for electrostatic interactions with their anionic target membrane. The present single molecule tracking studies reveal that both the fluorescent lipid and the lipid-anchored G protein Rab1A exhibit 2-dimensional translation typical of simple lipid diffusion in the bilayer. Single molecule density measurements indicate both PX domain and autophagy lipid kinase exhibit significantly reduced membrane docking when the bilayer negative charge is decreased, confirming the importance of their protein-bilayer electrostatic interactions. Single molecule tracking studies reveal that both PX domain and autophagy lipid kinase exhibit hopping movements across the bilayer surface during their initial bilayer contacts, yielding a significantly higher frequency of long range displacements than allowed by lipid 2-D diffusion. After transitioning from hopping to a stable bilayer-docked state, both PX domain and autophagy lipid kinase exhibit 2-D diffusion slower than lipid diffusion indicating the appearance of increased frictional drag against the bilayer due to new protein-bilayer contacts. PX domain requires its target lipid for the transition from electrostatic hopping to stable bilayer docking, while autophagy lipid kinase can dock spontaneously to anionic lipid bilayers even in the absence of its substrate lipid. In principle, the slowly diffusing, bilayer-docked states of PX domain and autophagy lipid kinase could be interrupted by unbinding-hopping-rebinding events. However, such events are not detected for PX domain in the presence of target lipid (DO)PI(3)P nor for autophagy lipid kinase in the presence of substrate lipid (DO)PI. Instead, unbinding-hopping-rebinding events are only observed for autophagy lipid kinase in the absence of substrate lipid, indicating that the presence of a specific binding lipid reduces the frequency or spatial amplitude of such events. Overall, the findings provide evidence that p40Phox PX domain possesses a search mechanism involving electrostatic hopping that speeds its acquisition of target lipid, as previously observed for PH domains. The evidence indicates that the autophagy lipid kinase also exhibits electrostatic hopping/skating behavior that is proposed to play a functional role by speeding regulatory encounters with autophagy lipid kinase activators and effectors. Moreover, the ability of the lipid kinase to spontaneously dock to anionic membranes without a target lipid could facilitate its distribution across broad regions of the growing autophagosome. Finally, the autophagy lipid kinase exhibits multiple translational modes that we hypothesize are enabled by its two membrane docking feet.

biophysics↗
bioRxiv2026

Inferring scaling laws of branching networks from their geometry by global optimization

Optimality principles are widely used to understand branching networks, yet they are tested on individual branchings that cannot reliably distinguish competing scaling laws. We formulate the missing link between the laws and measurements by mapping each law to its predicted branch points through global optimization. Vascular branch points closely match weighted-length optima when each vessel is allowed its own fitted weight. Inferred scaling laws reveal type-specific geometric signatures hidden from local analysis. They beat all tested laws, including Murray's law in 1,679 of 1,744 held-out hepatic and pulmonary trees. For coronary arteries on the heart surface, an exact discrete optimization model extends the framework beyond convex geometry and supports area-preserving scaling. The framework also identifies distinct laws for airways and plant shoots while rejecting weighted-length optimality for neuronal axons. Global optimization turns network geometry into a quantitative test of the principles that govern branching networks.

biophysics↗
bioRxiv2026

Ultra-precision 3D Printed Sample Tubes of Complex Cross Section for Enhanced Electron Paramagnetic Resonance Sensitivity of AqueousSamples

This paper discusses the applications of ultra-precision 3D printing in developing EPR sample tubes for X-band measurements of aqueous samples. The seed idea of this work started from the flat cell idea discussed in Mett and Hyde et al.(2003), Sidabras et al. (2005), and Sidabras et al. (2017), where placing multiple aqueous flat cells perpendicular to the electric field in the microwave cavity reduce RF losses and improve EPR signal sensitivity. In this work, we present the 3D printed aquastar sample holders for dielectric resonators and the aquasun sample holders for cavity resonators. Even though the ultra-precision 3D printing enables the fabrication of complex cross-sections at 25um resolution, the photopolymer material has comparatively higher dielectric losses compared to the conventional materials like glass and PTFE. To reduce the RF losses due to the inherent dielectric properties, we introduced the latticing approach in reducing the amount of plastic without compromising the structural stability. In addition to this, we present the slicing and dielectric approximation approach in reducing the computation cost for these complex cross-sections. The novel sample tubes discussed here present signal improvements through both simulation and experiments, which were validated through power saturation experiments. The sample tube development methodology presented here provide a general framework for designing and implementing 3D printed EPR sample holders, based on resonator electromagnetic field distribution and dielectric properties of the photopolymer material.

biophysics↗