Drexler, T. | Search bioRxiv

bioRxiv2026

Layered helical order reveals assembly states in podosome actin networks

How stochastic molecular events are converted into ordered cellular machines that perform mechanical work remains a central question in biology. Cryo-electron tomography enables visualization of macromolecular assemblies inside cells at molecular resolution, but static tomograms are generally interpreted as structural snapshots, with limited access to assembly state and temporal progression. Here, we show that cellular tomograms retain interpretable information about the assembly of a force-generating actin network. Using human macrophage podosomes as a physiologically relevant system, we combine spatial mapping of actin filaments (F-actin) and Arp2/3-mediated branch junctions, orientation analysis of deep-learning-based filament segmentations and Markov-chain modeling to infer actin-network assembly in situ. We identify favored membrane-directed actin polymerization and Arp2/3-mediated branching, establishing a local growth axis for podosome network organization. At the network scale, podosomes display layered helical order: filament-orientation classes recur at intervals of approximately 33 nm along the membrane-normal axis, forming a three-class cycle that returns to an approximately equivalent non-polar orientation. Markov modeling identifies preferred mother-to-daughter filament-orientation transitions and yields a stationary composition of branch-orientation states that closely matches that observed in structurally more advanced network regions. Together, these results link local actin nucleation, network-scale architecture and structural assembly state in a native cellular machine. Our study advances cryo-electron tomography from structural description toward temporal inference and 4D in situ structural biology.

cell biology↗

bioRxiv2021

A nanoscale reciprocating rotary mechanism with coordinated mobility control

Biological molecular motors transform chemical energy into mechanical work by coupling a cycle of catalytic reactions to large scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the celebrated example of F1Fo ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in the structure of the surrounding stator orchestrated by mechanical deformation. We designed the mechanism using DNA origami, characterized the structure of the components and the entire mechanism using cryo-electron microscopy, and examined the mechanisms dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. The data indicates that, while the camshaft can rotate inside the stator by diffusion, mechanical deformation of the stator makes the camshaft pause at a set of preferred orientations. By changing the mechanical stiffness of the stator, we could accelerate or suppress the Brownian rotation within the mechanism, thereby demonstrating an allosteric coupling between the movement of the camshaft and of the stator, and the ability to tailor the free energy landscape that governs the rotary motion. Our mechanism provides a framework for the manufacture of artificial nanomachines that, just like the man-made machines in the macroscopic world, function because of coordinated movements of their components.

biophysics↗