Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy.
In response to chemotactic signals, motile cells develop a single protruding front to persistently migrate in direction of the chemotactic gradient. The highly dynamic reorganization of the actin cytoskeleton is an essential part during this process and requires the precise interplay of various actin filament assembly factors and actin-binding proteins (ABPs). Although many ABPs have been implicated in cell migration, as yet only a few of them have been well characterized concerning their specific functions during actin network assembly and disassembly. In this chapter, we describe a versatile method that allows the direct visualization of the assembly of single actin filaments and higher structures in real time by in vitro total internal reflection fluorescence microscopy (TIRF-M) using purified and fluorescently labeled actin and ABPs.
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Moritz
M
Winterhoff
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Stefan
S
Brühmann
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Christof
C
Franke
NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
Dennis
D
Breitsprecher
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany. faix.jan@mh-hannover.de.
Jan
J
Faix
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011817:Rabbits; D013194:Staining and Labeling
D000199:Actins; D008840:Microfilament Proteins
Actin; Actin-binding proteins; Fluorescence labeling; In vitro TIRF microscopy; In vitro analysis; SNAP tag