TY - CHAP
T1 - Fluorescence single-molecule imaging of actin turnover and regulatory mechanisms
AU - Watanabe, Naoki
N1 - Funding Information:
This work was supported in part by the Cabinet Office, Government of Japan through its Funding Program for Next Generation World-Leading Researchers (LS013), and the grants from the Human Frontier Science Program and the Takeda Science Foundation.
PY - 2012
Y1 - 2012
N2 - Cells must rapidly remodel the actin filament network to achieve various cellular functions. Actin filament turnover is a dynamic process that plays crucial roles in cell adhesion, locomotion, cytokinesis, endocytosis, phagocytosis, tissue remodeling, etc., and is regulated by cell signaling cascades. Success in elucidating dynamic biological processes such as actin-based motility relies on the means enabling real time monitoring of the process. The invention of live-cell fluorescence single-molecule imaging has opened a window for direct viewing of various actin remodeling processes. In general, assembly and dissociation of actin and its regulators turned out to occur at the faster rates than previously estimated by biochemical and structural analyses. Cells undergo such fast continuous exchange of the components perhaps not only to drive actin remodeling but also to facilitate rapid response in many other cell mechanics and signaling cascades. This chapter describes how epifluorescence single-molecule imaging which visualizes deeper area than the TIRF microscopy is achieved in XTC cells, the currently best platform for this approach.
AB - Cells must rapidly remodel the actin filament network to achieve various cellular functions. Actin filament turnover is a dynamic process that plays crucial roles in cell adhesion, locomotion, cytokinesis, endocytosis, phagocytosis, tissue remodeling, etc., and is regulated by cell signaling cascades. Success in elucidating dynamic biological processes such as actin-based motility relies on the means enabling real time monitoring of the process. The invention of live-cell fluorescence single-molecule imaging has opened a window for direct viewing of various actin remodeling processes. In general, assembly and dissociation of actin and its regulators turned out to occur at the faster rates than previously estimated by biochemical and structural analyses. Cells undergo such fast continuous exchange of the components perhaps not only to drive actin remodeling but also to facilitate rapid response in many other cell mechanics and signaling cascades. This chapter describes how epifluorescence single-molecule imaging which visualizes deeper area than the TIRF microscopy is achieved in XTC cells, the currently best platform for this approach.
KW - XTC cell
KW - abelson kinase
KW - actin turnover
KW - defective CMV promoter
KW - drug perfusion
KW - epi-fluorescence
KW - flow cell
KW - formin homology protein
KW - photobleaching normalization
KW - single-molecule imaging
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U2 - 10.1016/B978-0-12-388448-0.00020-6
DO - 10.1016/B978-0-12-388448-0.00020-6
M3 - Chapter
AN - SCOPUS:84856425806
T3 - Methods in Enzymology
SP - 219
EP - 232
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -