科研之友
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摘要
The molecular chaperones alpha A- and alpha B-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alpha A-/- and alpha B-/- lens epithelial cells. However, no report to date has studied the effect of alpha-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alpha A- and alpha B-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wildtype, alpha A-/-, alpha B-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mu m long and similar to 25 nm wide and had a characteristic protofilament structure, but alpha B-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and beta B2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alpha A-/- or alpha B-/- microtubules, extraction of the MAPs gave very long (14 - 20 mu m) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alpha A+ alpha B) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. alpha-Crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.
- 出版日期2006-5
