STAT5A–α-actinin axis links actin integrity to mitochondrial homeostasis and innate immune activation

ABSTRACT Coordination between actin cytoskeleton networking and mitochondrial organization and health underpins cellular homeostasis. Here, we found STAT5A to be a pivotal transcription factor that sustains the expression of key actin regulators, including ACTN1. STAT5A, but not STAT5B deficiency dismantles F-actin architecture leading to amorphous cell shape, reduced cellular motility, and corrals mitochondria around the nucleus. This actin networking disruption impairs mitochondrial DRP1 recruitment and dynamic equilibrium, leading to ROS-associated DNA damage and cGAS–STING mediated type I IFN production. Consequently, this establishes a chronically IFN-stimulated state in neighboring cells. Conversely, overexpression of STAT5A increases actin cytoskeleton networking and promotes faster cell motility. Moreover, ectopic expression of ACTN1 in the background of STAT5A-knockout cells is sufficient to restore the actin cytoskeleton organization and mitochondrial network morphology, eliminating DNA damage and IFN-signaling. Giving the importance of actin in cellular homeostasis, our findings place actin abundance, as regulated by the STAT5A–ACTN1 axis as essential for linking cytoskeletal integrity with mitochondrial health, restraining aberrant innate immune activation. Graphic Abstract Key points STAT5A deficiency disrupts cytoskeletal integrity via ACTN1 loss. The disrupted actin-cytoskeleton impairs mitochondria organization, causing ROS production and DNA damage. STAT5A knockout activates cGAS–STING signaling and IFN-β production. Restoring ACTN1 rescues cytoskeleton, mitochondrial function, and immune balance.