Partial loss of actin nucleator actin‐related protein 2/3 activity triggers blebbing in primary T lymphocytes

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities. Quantification of cytotoxic effector T lymphocytes (CTLs) morphology using standard biomedical assays and developed image analysis algorithms showed that the reduction of actin‐related protein (Arp)3 levels impaired F‐actin content maintenance, leading to defects in CTL functionality. The knockdown CTL motility mode, instead of the actin‐rich lamellipodia‐based migratory strategy, displays blebbing‐like migration at the cost of reduced migration speed. Thus, the role of the Arp2/3 complex is not redundant among the various mechanochemical coordinators involved in the leading‐edge formation in CTLs.