Abstract
Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.
Highlights
Remodeling of the cortical cytoskeleton plays a central role in cell locomotion, cytokinesis, endocytosis, phagocytosis and tissue organization
The development of live-cell fluorescence single-molecule imaging has opened a window for the direct viewing of assembly and remodeling processes of the actin filament network
Prior to the development of this approach, two methods were mainly employed to monitor the dynamics of actin filament turnover in live cells
Summary
Remodeling of the cortical cytoskeleton plays a central role in cell locomotion, cytokinesis, endocytosis, phagocytosis and tissue organization. The development of live-cell fluorescence single-molecule imaging has opened a window for the direct viewing of assembly and remodeling processes of the actin filament network. Many of these processes have turned out to occur much faster than predicted by in vitro biochemical data [1]. As discussed in detail in previous studies [1,17], FRAP, PAF and FLAP experiments may exhibit potential problems, such as low spatial resolution, insufficiency in monitoring subpopulation, local reincorporation of disassembled probes and photodamage during the photobleaching procedure. Single-molecule imaging directly visualizes assembly, dwell time and movement of individual fluorescently labeled molecules. Diffusing EGFP-actin in the monomeric form are blurred on the image
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