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Centrosomal control of microtubule dynamics
V Rodionov1, E Nadezhdina, G Borisy
1Laboratory of Molecular Biology, R. M. Bock Laboratories, University of Wisconsin, Madison, WI 53706, USA.
Abstract:
In many animal cells, minus ends of microtubules (MTs) are thought to be capped by the centrosome whereas plus ends are free and display dynamic instability. We tested the role of the centrosome by examining MT behavior in cytoplasts from which the centrosome was removed. Cells were injected with Cy3-tubulin to fluorescently label MTs and were enucleated by using a centrifugation procedure. Enucleation resulted in a mixture of cytoplasts containing or lacking the centrosome. Fibroblast (CHO-K1) and epithelial (BSC-1) cells were investigated. In fibroblast cytoplasts containing the centrosome, MTs showed dynamic instability indistinguishable from that in intact cells. In contrast, in cytoplasts lacking the centrosome, MTs treadmilled-shortened at the minus end at about 12 micrometers/min while growing at the plus end at the same rate. The change in behavior of the plus end from dynamic instability to persistent growth correlated with an elevated level of free tubulin subunits (78% in centrosome-free cytoplasts vs. 44% in intact cells) generated by minus-end depolymerization. In contrast to fibroblast cells, in centrosome-free cytoplasts prepared from epithelial cells, MTs displayed dynamic instability at plus ends and relative stability at minus ends presumably because of specific minus-end stability factors distributed throughout the cytoplasm. We suggest that, in fibroblast cells, a minus-end depolymerization mechanism functions to eliminate errors in MT organization and that dynamic instability of MT plus ends is a result of capping of minus ends by the centrosome.
Insights
The centrosome caps microtubule minus ends, preventing depolymerization and promoting dynamic instability at plus ends in fibroblast cells. Removing the centrosome causes minus-end shortening and plus-end growth.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
- Microtubule Organization
Background:
- Microtubule (MT) minus ends are typically centrosome-capped, while plus ends exhibit dynamic instability.
- The precise role of the centrosome in regulating MT dynamics remains incompletely understood.
Purpose of the Study:
- To investigate the function of the centrosome in MT organization and dynamics.
- To determine how MT behavior changes in the absence of the centrosome.
Main Methods:
- Utilized enucleation via centrifugation to create cytoplasts with or without centrosomes.
- Employed fluorescently labeled tubulin (Cy3-tubulin) to visualize MTs in real-time.
- Examined MT behavior in both fibroblast (CHO-K1) and epithelial (BSC-1) cell types.
Main Results:
- In fibroblast cytoplasts lacking centrosomes, MTs exhibited treadmilling: minus-end shortening (12 µm/min) and simultaneous plus-end growth.
- This treadmilling correlated with increased free tubulin subunits (78% vs. 44% in intact cells) due to minus-end depolymerization.
- Epithelial cell cytoplasts lacking centrosomes maintained MT dynamic instability, suggesting cytoplasmic minus-end stabilizing factors.
Conclusions:
- In fibroblasts, centrosome capping of MT minus ends is crucial for preventing depolymerization and maintaining plus-end dynamic instability.
- Minus-end depolymerization in centrosome-free cells may serve as an error-correction mechanism for MT organization.
- Specific cytoplasmic factors likely contribute to minus-end stability in epithelial cells, independent of the centrosome.