Biased localization of actin binding proteins by actin filament conformation.

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells.

Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2.

Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks.

Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency.

Novel in situ normal streaming potential device for characterizing electrostatic properties of confluent cells.

The characteristics of transport across confluent cell monolayers may often be attributed to its electrostatic properties. While tangential streaming potential is often used to quantify these electrostatic properties, this method is not effective for transport normal to the apical cell surface where the charge properties along the basolateral sides may be important (i.e.