Structural insight into the Phosphoinositide-Regulated Cellular Dynamics of Alpha-Actinin

Abstract

Alpha-actinin2 is a fundamental component of striated muscle sarcomeres. It localizes to the Z-disk, the macromolecular assembly that connects two adjacent sarcomeres where it provides mechanical stability during contraction but also plays a pivotal role during Z-disk assembly. A regulatory role by phosphatidylinositol-bis-phosphate (PiP2) has been proposed for all 4 alpha-actinin isoforms but not validated at structural level. The X-ray structure of full-length alpha-actinin2 suggests the existence of two different conformational states. In the ground state, alpha-actinin exists in an intramolecular autoinhibited conformation, where the C-terminal EF-hands bind a titin-like pseudoligand at the connection between rod and actin-binding domains. PiP2 acts as a potential trigger for the transition to an open state capable of titin-binding. We hypothesize this mechanism plays a key role during Z-disk maturation, with the open-state allowing interaction with key binding partners. Super-resolution and correlative electron microscopy of mutant-transfected Neonatal Rat Cardiomyocytes (NRC) shed new light on the internal organization of the Z-disk and the regulatory mechanism of alpha-actinin ligand binding. Combining fluorescence confocal microscopy and live-cell imaging in NRC, we identified a crucial role for PiP2, which regulates the intracellular dynamics of alpha-actinin as seen in Fluorescence Recovery After Photobleaching experiments with a PiP2-binding deficient mutant. A second alpha-actinin mutant, in constitutively open-conformation, impaired overall sarcomere assembly by reduced intracellular dynamics, suggesting that PiP2 might not be limited to a positive regulation, but is also essential for the dynamic equilibrium of the protein.