Dynamic insights into cellular mechanics and membrane undulations in vascular smooth muscle cells

Dynamic oscillations in cell mechanics are fundamental yet poorly understood features of living cells. In vascular smooth muscle cells (VSMCs), such oscillations may play important roles in regulating contractility, mechanosensitivity, and vascular function. Here, real-time atomic force microscopy (AFM), advanced signal processing, biochemical analysis, and machine learning-based image quantification were combined to investigate the spatiotemporal coupling between cellular mechanics, membrane undulation, cytoskeletal organization, and actomyosin signaling in VSMCs. Continuous AFM force and height mapping revealed intrinsic, low-frequency oscillations in both elastic modulus and membrane roughness, with dominant modes at ∼0.55, ∼1.6, and ∼3.5 mHz, that were absent in passive material controls. Pharmacological modulation of the actin cytoskeleton demonstrated frequency-dependent regulation of these oscillations: stabilization of F-actin with jasplakinolide increased cellular stiffness and selectively enhanced low-frequency mechanical oscillations while suppressing membrane roughness fluctuations, whereas actin depolymerization with latrunculin A reduced stiffness and mechanical oscillations but markedly amplified membrane undulations. Confocal imaging and deep learning-based analysis confirmed corresponding changes in actin fiber density and organization. Moreover, inhibition of myosin light chain kinase (MLCK) signaling reduced cell stiffness and preferentially attenuated higher-frequency oscillatory modes, whereas biochemical analysis revealed differential regulation of MLCK phosphorylation following actin perturbation. Together, our findings suggest that changes in actin organization and MLCK-driven contractility control different patterns of mechanical oscillation and membrane behavior in VSMCs. This helps us better understand how smooth muscle mechanics are regulated across different scales and why disruptions in these processes could influence vascular function and disease.NEW & NOTEWORTHY In this study, we observed previously unrecognized mechanical oscillations in vascular smooth muscle cells that occur at specific frequencies and remain synchronized across the cell, alongside coordinated membrane movements. By combining real-time atomic force microscopy with targeted perturbations of the cytoskeleton and myosin light chain kinase activity, we found that different oscillatory behaviors depend on actin organization and contractile signaling. These results outline a multiscale framework for understanding how smooth muscle mechanics are dynamically controlled.