Phosphatidylinositol 4,5-bisphosphate alters the number of attachment sites between ezrin and actin filaments: a colloidal probe study.
Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.
From the Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany.
Julia A
JA
Braunger
Bastian R
BR
Brückner
Stefan
S
Nehls
Anna
A
Pietuch
Volker
V
Gerke
Ingo
I
Mey
Andreas
A
Janshoff
Claudia
C
Steinem
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D000099149:Ezrin
Actin; Atomic Force Microscopy; Ezrin; Phosphatidylinositol; Phosphorylation