Project Summary Actin, which exists in filamentous (F-actin) and monomeric (G-actin) forms, is the most abundant protein in the cytoplasm of eukaryotic cells and a key player in countless processes in health and disease, including cell motility, muscle contraction, cancer metastasis, and cardiomyopathies. F-actin is a polar filament, with fast polymerizing barbed (+) and depolymerizing pointed (-) ends. The control of monomer addition/dissociation at the ends of the actin filament is a key event that affects all cellular functions of actin in health and disease. This process is tightly regulated in cells by actin's ATPase activity and many proteins that control subunit exchange at the ends of the filament. Among those are proteins that accelerate subunit addition at the barbed end (formins), accelerate subunit dissociation from the pointed end (cyclase-associated protein and cofilin), or “cap” the barbed (CapZ, gelsolin) and pointed (Tmod-Tpm) ends of F-actin. Barbed end capping by CapZ is in turn regulated by several proteins, including CARMIL. The structural-functional mechanisms that control the activities of these proteins and filament end dynamics are poorly understood. To address this lack of understanding, this grant will tackle the following Aims: 1) Control of actin dynamics at the barbed end, 2) Mechanism of barbed end uncapping by CARMIL, 3) Control of actin dynamics at the pointed end. The rationale and likelihood of success of the Aims is substantiated by extensive preliminary work, including: a) all the relevant proteins have been obtained, including a majority in human cells, b) determination of preliminary cryo-electron microscopy (cryo-EM) structures of the free and CapZ-capped barbed end, c) determination of preliminary cryo-EM structures of the free and Tmod-Tpm-capped pointed end, d) determination of preliminary cryo-EM structures of CARMIL alone and in complex with CapZ, e) a CARMIL knockout cell line obtained by CRISPR/Cas9 (collaboration with the Lappalainen lab, Helsinki), and f) proteomics data revealing key post- translational modifications in CARMIL. The proposed studies should substantially advance our understanding of F-actin barbed and pointed end dynamics, a process that impacts cellular activities of actin in health and disease.