Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain
Riasat Zaman1, Andrew Lombardo1, Cécile Sauvanet1
1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY.
The Journal of Cell Biology
|April 9, 2021
Summary
New research reveals that activated ezrin-radixin-moesin (ERM) proteins are crucial for cell structure. The study identifies LOK/SLK kinases as key activators of ERM proteins, uncovering a novel feedback loop essential for epithelial cell apical morphology.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Ezrin-radixin-moesin (ERM) proteins connect the plasma membrane to the actin cytoskeleton, forming apical structures like microvilli.
- Kinases, including LOK and SLK, are implicated in ERM protein activation.
- Proper regulation of ERM proteins is vital for maintaining cell shape and function.
Purpose of the Study:
- To investigate the role of LOK and SLK kinases in ERM protein activation.
- To elucidate the relationship between ERM proteins, LOK/SLK, and RhoA signaling.
- To understand the molecular mechanisms governing apical structure formation in epithelial cells.
Main Methods:
- CRISPR/Cas9 gene editing was employed to generate knockout cell lines for ERM proteins and LOK/SLK.
- Immunoblotting and phospho-specific antibodies were used to assess ERM phosphorylation levels.
- RhoA activity assays and myosin light chain phosphorylation analysis were performed.
- Microscopy was utilized to examine cellular morphology, including microvilli and actin organization.
Main Results:
- LOK/SLK knockout abolished ERM-activating phosphorylation, demonstrating LOK/SLK's essential role in ERM activation.
- Cells lacking LOK/SLK or ERMs exhibited similar apical defects, including loss of microvilli and altered junctional actin.
- Constitutively active ezrin rescued the phenotypes in both knockout models, confirming LOK/SLK's function in activating ERMs.
- Both knockout lines showed elevated active RhoA and myosin light chain phosphorylation, indicating active ERMs negatively regulate RhoA.
Conclusions:
- LOK/SLK kinases are critical upstream activators of ERM proteins.
- Active ERM proteins act as negative regulators of RhoA, establishing a novel feedback loop.
- This ERM-RhoA feedback loop is essential for maintaining proper apical morphology in epithelial cells.
- The findings provide new insights into the regulation of cytoskeleton dynamics and cell polarity.
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