Abstract
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.
Highlights
Ezrin can establish a dynamic linkage between plasma membrane and cytoskeleton
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 filamentous actin (F-actin) network
Colloidal probe microscopy enabled us to describe this interaction on a molecular level, dissecting the individual contributions of ezrin phosphorylation and PIP2 binding
Summary
Bruckner§1,2, Stefan Nehls§, Anna Pietuch§, Volker Gerke¶, Ingo Mey‡, Andreas Janshoff§, and Claudia Steinem‡3 From the ‡Institute of Organic and Biomolecular Chemistry, University of Gottingen, Tammannstrasse 2, 37077 Gottingen, Germany, the §Institute of Physical Chemistry, University of Gottingen, Tammannstrasse 6, 37077 Gottingen, Germany, and the ¶Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Munster, Von-Esmarch-Strasse 56, 48149 Munster, Germany
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