The endothelium serves as a size-selective barrier and tightly controls the fluid exchange from the circulation to the surrounding tissues. In this study, a multiplexed microscopy characterization is developed to study the spatio-temporal effects of Abl kinases on endothelial cytoskeletal structure using AFM, SEM, and immunofluorescence. Sphingosine 1-phosphate (S1P) produces significant endothelial barrier enhancement by means of peripheral actin rearrangement. However, Abl kinase inhibition by imatinib reduces rapid redistribution of the important cytoskeletal proteins to the periphery and their association with the cortical actin ring. Herein, it moderates the thickness of the cortical actin ring, and diminishes the increase in elastic modulus at the periphery and cytoplasm. These findings demonstrate that imatinib attenuates multiple cytoskeletal changes associated with S1P-mediated endothelial barrier enhancement and suggest a novel role for Abl kinases in mediating these S1P effects. These observations bridge the gap between molecule dynamics, structure complexity and function connectivity across varied length-scales to improve our understanding on human pulmonary endothelial barrier regulation. Moreover, our study suggests a framework for understanding form-function relationships in other biomechanical subsystems, wherein complex hierarchical organization programmed from the molecular scale to the cellular and tissue levels has an intimate relationship to the overall physiological function.