Cutting Edge of Atomic Force Microscopy (AFM) of the Cell: From Live Cell Imaging to High-resolution Structural Analysis of Cytoskeletal Actin Filaments

Abstract

Increasing in scanning speed and mechanical stability improved in AFM facilitated live cell imaging and high-resolution structural analysis of organelles in cells. AFM also is an important tool for observing cells in physiological or native condition at higher resolution than light microscope (LM), and therefore enable to observe endocytosis and movement of cortical actin [1]. Even in the samples fixed chemically, AFM described intracellular fine structures containing water at comparable resolution to electron microscopy (EM) [2][3]. In addition, it should be noted that images formed by scanning needle while touching the sample surface in AFM bring peculiar structural information different from those in LM and EM, in which images are formed by scattering, absorption or interference of electron or photon. Meanwhile, it has been not fully understood how the cantilever detects movement of actin filaments in cytoplasm beneath the cell membrane in living cells. In practice, however, recent high-speed AFM described moving filaments beyond cell membrane, despite such detection mechanism by cantilever remained unknown. Here, we will show various movements of filaments (mostly actin) depending on their spatial assignment in living cells, and also high-resolution structures of actin filaments in unroofed and fixed cells. More recently, AFM detected successfully movement of fine filaments layered in relatively deep inside of living cells through the cell membrane at complete contact mode. When cantilever was approached slowly and contacted onto the living cell, AFM begin to display the surface structure of the cell. We call this state as the surface mode conveniently, in which movement of filo podia, lameli-podia and endocytosis were captured (Fig. 1). When cantilever was pushed down further a little bit, movement of many fine filaments (probably actin filaments) was visualized (Fig.2). We call such measurement as a cytoskeleton mode in live cell imaging. In EM, three types of actin filaments had been recognized beneath the cell membrane, i.e. actin filaments firmly attached to the membrane, actin filaments loosely attached to the membrane and actin filaments forming stress fibers in cytoplasm. Fine filaments extending randomly were disposed over the stress fibers clearly. Therefore, such filaments appeared to be located between cell membrane and stress fibers. Probably, these filaments seem to correspond to the type of actin filaments loosely attached to the membrane seen in EM. However, it is very curious that actin filaments firmly attached to the membrane as well as clathrin coat were not detected from outside of cell membrane at contact mode, despite these organelles were located just beneath the cell membrane in AFM of unroofed cells. Anyway, actin filaments extending randomly showed higher motility than ones forming stress fibers (Fig. 2). In general, stress fibers terminate on the cytoplasmic surface of ventral membrane corresponding to the focal contact between cell membrane and matrix or substrate. Similar focal points were also found in cytoplasm, in which many actin filaments were concentrated in one point. Such bundled actin filaments showed low motility similar to the stress fiber when compared with actin filaments arranged randomly and individually. In order to view actin filaments and organelle more in detail, apical cell membrane was unroofed mechanically with customized low power sonication. All actin filaments showed short periodicity with