Chapter 17: Application of laser tweezers to studies of membrane-cytoskeleton adhesion.
Interaction between the plasma membrane and cytoskeleton is important in regulating cell functions such as lamellipodial extension, cell motility, and endocytosis. Here, we describe a method to determine plasma membrane-cytoskeleton adhesion by measuring tether force using laser optical tweezers. We show the basic components and instrumental setup of a laser optical tweezers system which is used to pull thin membrane tethers from the cell surface using one micron polystyrene beads coated with membrane binding ligands. We also show an application of tether force measurements in elucidating regulation of membrane-cytoskeleton adhesion. That phosphatidylinositol-4,5-biphosphate (PIP2) may play an important role in regulating membrane-cytoskeleton adhesion is suggested by correlating the plasma membrane concentration of PIP2 with membrane-cytoskeleton adhesion using tether force measurements. The assay described here enables us to investigate mechanochemical components of many important cellular processes, such as lamellipodial extension, cell motility, membrane resealing after cell wounding, and endocytosis. Furthermore, this method could be used to establish the link between biochemical function and physical properties of the plasma membrane which drives these dynamic cellular processes.
Raucher
Drazen
D
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
D016475:3T3 Cells; D000818:Animals; D001696:Biomechanical Phenomena / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D052898:Optical Tweezers