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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Published on: August 13, 2012
Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes
Noriko Yokoyama1, Urszula Golebiewska, Hsien-yu Wang
1Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. noriko@pharm.stonybrook.edu
Journal of Cell Science
|October 14, 2010
Summary
Dishevelled-3 (Dvl3) protein forms large, dynamic complexes crucial for Wnt signaling. Wnt3a stimulation enhances these complexes, impacting developmental pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Dishevelled-3 (Dvl3) is a key scaffold protein in Wnt signaling, essential for embryonic development.
- The physical nature and dynamics of Dvl3-based protein complexes have remained largely unknown, despite visualization via microscopy.
Purpose of the Study:
- To characterize the physical properties and dynamics of supermolecular Dvl3-based complexes in totipotent mouse F9 cells.
- To investigate the impact of Wnt3a stimulation and pathway inhibitors on Dvl3 complex formation and function.
Main Methods:
- Steric-exclusion chromatography to determine molecular mass.
- Affinity pull-downs and proteomics for complex composition analysis.
- Fluorescence correlation microscopy to study complex dynamics in live cells.
Main Results:
- Dvl3 forms stable complexes ranging from homodimers to 0.4-2.0 MDa.
- Wnt3a stimulation rapidly increases the molecular mass of Dvl3 complexes.
- DKK1 and Dvl protein knockdown inhibit the formation of large Dvl3 complexes and Wnt3a-induced signaling.
- Live-cell microscopy revealed supermolecular Dvl3 complexes exceeding 30 MDa, further increasing upon Wnt3a stimulation.
Conclusions:
- Establishes the existence and characterization of very large, dynamic, supermolecular Dvl3-based complexes.
- Demonstrates that Wnt3a signaling modulates the size and formation of these complexes.
- Highlights the critical role of Dvl3 complex assembly in Wnt/canonical pathway activation.
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