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RGS14 is a microtubule-associated protein
Luke Martin-McCaffrey1, Francis S Willard, Agnieszka Pajak
1Siebens-Drake Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine, University of Western Ontario, London, Ontario, Canada.
Cell Cycle (Georgetown, Tex.)
|May 27, 2005
Summary
Regulator of G protein signaling 14 (RGS14) directly binds microtubules and promotes tubulin polymerization. This microtubule-associated protein is crucial for mitotic spindle formation and cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Heterotrimeric G-proteins and their regulators are increasingly recognized for their roles in microtubule dynamics and spindle force generation during cell division across various species.
- Previous research established the requirement of RGS14 for the first mitotic division in mouse embryos and its in vivo regulation of microtubule organization.
Purpose of the Study:
- To investigate the role of RGS14 as a microtubule-associated protein (MAP) and its function in mitotic spindle organization and microtubule polymerization.
- To elucidate the direct interaction of RGS14 with tubulin and its influence on microtubule dynamics in vitro and in cell extracts.
Main Methods:
- Co-immunoprecipitation assays to detect RGS14 interaction with Taxol-stabilized tubulin.
- Biochemical purification of RGS14 with tubulin from porcine brain and in vitro binding assays with purified tubulin.
- In vitro tubulin polymerization assays in the presence of RGS14 and Galpha(i1) (GTP/GDP forms).
- Formation of mitotic asters in vitro using HeLa cell extracts and assessment of RGS14 depletion effects.
Main Results:
- RGS14 directly binds to polymerized microtubules in vitro (KD = 1.3 +/- 0.3 microM) and co-immunoprecipitates with stabilized tubulin.
- Both RGS14 and Galpha(i1) promote tubulin polymerization in a GTP-dependent manner, requiring additional MAPs.
- RGS14 is a component of mitotic asters, and its depletion from cell extracts inhibits aster formation.
Conclusions:
- RGS14 functions as a microtubule-associated protein that directly interacts with tubulin.
- RGS14, in conjunction with Galpha(i1), modulates microtubule polymerization dynamics, dependent on GTP/GDP cycling.
- RGS14 plays a critical role in mitotic spindle formation, specifically in the assembly of mitotic asters.