Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium.
Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.
Institute of Physiology II, University of Münster, Münster, Germany.
Cornelius
C
Kronlage
Institute of Cell Dynamics and Imaging, University of Münster, Münster, Germany.
Marco
M
Schäfer-Herte
Institute of Medical Physics and Biophysics, University of Münster, Münster, Germany.
Daniel
D
Böning
Institute of Physiology II, University of Münster, Münster, Germany.
Hans
H
Oberleithner
Institute of Physiology II, University of Münster, Münster, Germany; Institute of Cell Dynamics and Imaging, University of Münster, Münster, Germany. Electronic address: felsj@uni-muenster.de.
Johannes
J
Fels
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001011:Aorta / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D047630:Depsipeptides / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology
D000199:Actins; D000970:Antineoplastic Agents; D047630:Depsipeptides; D019384:Nucleic Acid Synthesis Inhibitors; C057531:jasplakinolide; D015638:Cytochalasin D; D002118:Calcium