EFhd2 regulates microtubule-dependent mitochondrial fitness at the B cell immune synapse

Abstract B cells can recognize soluble and membrane bound antigens, enabling them to initiate and execute versatile immune responses. This study examines Swiprosin-1/EFhd2 (EFhd2) in regulating mitochondrial function and organization in B cells during B cell receptor (BCR) activation and immune synapse formation. Using EFhd2 knockout (KO) and wild-type (WT) murine B cells, we assessed mitochondrial abundance, membrane potential, and respiratory capacity using soluble anti-IgM and anti-CD40/IL-4 stimulation. EFhd2KO B cells exhibit more functional mitochondria and mitochondrial spare capacity selectively in activated but not in resting cells. This phenotype changed absolutely upon BCR synapse formation: While activated WT B cells enhance basal mitochondrial respiration, ATP production and maximal respiration, with large increments of spare capacity, EFhd2KO B cells fail completely to do so. Actin depolymerization and microtubule destabilization impair functional mitochondrial upregulation in WT but not in EFhd2KO B cells, while microtubule stabilization restores full spare capacity in EFhd2KO B cells at the BCR synapse. Live-cell imaging reveals that EFhd2KO B cells fail to organize mitochondria, microtubules, and BCRs symmetrically. Super-resolution 3D imaging shows that WT B cells condense mitochondria at the synapse, whereas EFhd2KO B cells display dispersed, unorganized mitochondria and BCR clusters. EFhd2 re-expression restores mitochondrial polarization in EFhd2KO B cells, confirming its role in coordinating mitochondrial positioning. These findings highlight EFhd2 as a key integrator of cytoskeletal and mitochondrial functions for optimal B cell responses to membrane bound antigens. Graphical Abstract