Probing in vivo dynamics of mitochondria and cortical actin networks using high‐speed atomic force/fluorescence microscopy

Abstract

The dynamics of the cell membrane and submembrane structures are closely linked, facilitating various cellular activities. Although cell surface research and cortical actin studies have shown independent mechanisms for the cell membrane and the actin network, it has been difficult to obtain a comprehensive understanding of the dynamics of these structures in live cells. Here, we used a combined atomic force/optical microscope system to analyze membrane-based cellular events at nanometer-scale resolution in live cells. Imaging the COS-7 cell surface showed detailed structural properties of membrane invagination events corresponding to endocytosis and exocytosis. In addition, the movement of mitochondria and the spatiotemporal dynamics of the cortical F-actin network were directly visualized in vivo. Cortical actin microdomains with sizes ranging from 1.7 3 10 4 to 1.4 3 10 5 nm 2 were dynamically rearranged by newly appearing actin filaments, which sometimes accompanied membrane invaginations, suggesting that these events are integrated with the dynamic regulation of submembrane organizations maintained by actin turnovers. These results provide novel insights into the structural aspects of the entire cell membrane machinery which can be visualized with high temporal and spatial resolution.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

“…To reveal protein-induced membrane deformation during CME in a living cell, we first established a hybrid imaging system with HS-AFM and a confocal laser scanning microscope. We previously reported the development of a tip-scanning AFM unit and its combination with an inverted optical microscope with a fluorescence illumination unit 21,22 . In this study, we combined the HS-AFM unit with an inverted optical microscope equipped with a confocal laser scanning unit to increase the optical resolution.…”

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

“…Current high spatiotemporal resolution imaging is commonly obtained on microbial cells which have a stiff and well-defined cell surface 43,47 and animal adherent cells which can tightly adhere and spread on a substrate. 52,58,60 The dynamics of fine structures or even single molecules 194 can be captured on these cell surfaces. For animal suspended cells which do not spread on a substrate and are very soft, the imaging resolution is limited.…”

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

“…The cantilever precisely approached the target region of the retinal tissue as observations were made with CLSM. The images were recorded in the phase-modulation mode in which the amplitude domain was approximately 1–5 nm 39 . This AFM technique is characterized by a small and soft cantilever (2 μm wide, 9 μm long, and 0.1 μm thick in size) with a spring constant of 0.1 N/m (USC-F0.8-k0.1-T12-10: NanoWorld AG, Neuchatel, Switzerland).…”