Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy.
In this chapter, we describe how to shield a patch of a cell membrane against extracellularly applied chemoattractant stimuli. Classical patch clamp methodology is applied to allow for controlled shielding of a membrane patch by measuring the seal resistivity. In Dictyostelium cells, a seal resistivity of 50 MΩ proved to be tight enough to exclude molecules from diffusing into the shielded membrane region. This allowed for separating a shielded and a non-shielded region of a cell membrane to study the spatiotemporal dynamics of intracellular chemotactic signaling events at the interface between shielded and non-shielded areas. The spatiotemporal dynamics of signaling events in the membrane was read out by means of appropriate fluorescent markers using laser scanning confocal microscopy.
Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476, Potsdam, Germany.
Matthias
M
Gerhardt
Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476, Potsdam, Germany.
Michael
M
Walz
Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476, Potsdam, Germany. beta@uni-potsdam.de.
Carsten
C
Beta
D000199:Actins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002633:Chemotaxis; D004023:Dictyostelium / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods; D018408:Patch-Clamp Techniques
D000199:Actins
Actin dynamics; Chemotaxis; Confocal microscopy; Dictyostelium discoideum; Excitable dynamics; Patch clamp