Student Theses and Dissertations


Lin Mei

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Alushin Laboratory


Eukaryotic cells employ the actin cytoskeleton to maintain cell shape, support motility, and sense and respond to external mechanical stimuli. An intricate network of actin filaments constitutes the foundation of tissue architecture by forming key cellular structures including the muscle fiber, the filopodium, the lamellipodium, and the cell cortex. Cellular and tissue level studies have shown that more than 150 actin-binding proteins regulate almost every single aspect of actin physiology, such as actin polymerization, actin severing, and actin crosslinking, but at the molecular level, the structural mechanisms by which different actin binding proteins bind, assemble, and regulate different actin networks remain largely elusive. It is also known that the actin cytoskeleton mediates mechanical coupling between eukaryotic cells and their tissue microenvironments. Although cellular and tissue level studies suggest the architecture and composition of actin networks are modulated by force, it is still unclear how interactions between actin filaments and associated proteins are mechanically regulated. Force-sensitive actin binding interactions are fundamental for cells to sense and respond to mechanical stimuli by converting mechanical cues into biochemical signals, a key process known as cellular mechanotransduction. In this thesis, single molecule biophysical techniques including simultaneous optical trapping and confocal microscopy assays and in vitro reconstituted myosin motor-based total internal reflection fluorescence microscopy assays were employed to study how mechanical forces applied on actin filaments can regulate actin binding. A case study on a homologous pair of essential adhesion actin-binding proteins, α-catenin and vinculin, reveals α-catenin directly senses force on actin, while vinculin does not. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to Factin, structure-guided protein engineering, and ongoing nuclear magnetic resonance and force reconstitution cryo-electron microscopy studies demonstrate that α-catenin’s C-terminus is a modular detector of F-actin tension and suggest a force-sensing mechanism. Cryo-electron microscopy was also employed to study how adhesion actinbinding proteins and the calponin-homology domain actin-binding proteins assemble cellular actin networks by binding and crosslinking actin filaments. Actin binding by another essential adhesion actin-binding protein talin, and actin bundling by both α- catenin and vinculin were additionally structurally characterized. Using a unique calponin-homology domain actin-binding protein, T-plastin, as an example, the thesis established the structural mechanism by which T-plastin crosslinks actin filaments. In summary, with biochemical, biophysical, and structural methods, this thesis systematically studied the molecular mechanisms for the multi-modal regulation of actin networks by actin binding proteins.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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Life Sciences Commons