Live Cell STED-AFM Analysis Correlates Cytoskeletal Structure Remodelling and Membrane Physical Properties during Polarized Migration in Astrocytes

Abstract

The establishment of cell polarity, migration and adhesion is a complex interplay between cytoskeletal organisation and cell physical properties. Measurement of cell physical properties, such as mechanical stiffness, is conventionally performed by scanning probe microscopy – such as atomic force microscopy (AFM). AFM does not allow the specific imaging of subcellular cytoskeletal elements. To overcome this live cell mechanical property maps (AFM) are correlated with STED super-resolution images of the cytoskeleton by mounting the AFM on the optical microscope. Live cell organic dyes (SiR-actin/tubulin) label cytoskeletal elements allowing STED imaging. STED images are correlated with AFM topography and force maps. This technique is used to investigate polarised migration [1].A scratch assay is used to induce polarised migration in astrocytes and it is found that both the actin and tubulin cytoskeleton rearrange during migration. In the leading edge of migration an increase in actin stress fibers is observed. This corresponds to an increase in astrocyte stiffness compared to the basis. Expression of the gap junction protein connexin30, which is known to colocalise with actin and inhibit migration, is found to reduce stress fiber formation and astrocyte stiffness in the leading edge. Overall combining STED and AFM allows investigations into the relation of changes in cell topography to functional mechanical properties with subcellular resolution and will be important when investigating cell migration, adhesion and division in physiological and pathological conditions. [1] Curry N, et al., Frontiers in Cellular Neurscience, 11:104 (2017)