Real-Time Scanning Ion Conductance Microscopy Reveals MARCKS-Driven Morphological Remodeling and Nanomechanical Softening during LPS-Induced Macrophage Polarization.

Macrophages initiate acute immune responses to danger signals through highly plastic polarization and dynamic remodeling of cell mechanics and morphology. While physical cues are increasingly recognized as coregulators of macrophage activation alongside biochemical signals, the role of pathogen-induced biophysical remodeling in shaping immune function remains poorly understood. In this study, we employed noncontact scanning ion conductance microscopy (SICM) to monitor real-time nanomechanical and morphological changes in immortalized bone marrow-derived macrophages (iBMDMs) at single-cell resolution during lipopolysaccharide (LPS)-induced M1 polarization. We found that M1 activation significantly increased the surface area-to-volume (SA/V) ratio and decreased cellular stiffness─biophysical adaptations that may support enhanced pro-inflammatory responses. Mechanistically, these changes were driven by NF-κB-dependent activation of the cytoskeletal regulator MARCKS. The inhibition of MARCKS disrupted both morphological remodeling and inflammatory activation, highlighting its essential role in coordinating immune signaling with nanomechanical plasticity. This study provides the first real-time, quantitative evidence linking inflammatory signaling to macrophage morphological and nanomechanical remodeling, and shows that MARCKS inhibition suppresses, while PMA-mediated activation mimics, LPS-induced responses.