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Alpha-crystallin expression affects microtubule assembly and prevents their aggregation

Jing-hua Xi^*, Fang Bai^*, Rebecca McGaha^* and Usha P. Andley^*,,1

^* Department of Ophthalmology and Visual Sciences and of

Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA

¹Correspondence: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110, USA. E-mail: andley{at}vision.wustl.edu

The molecular chaperones A- and 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 A–/– and B–/– lens epithelial cells. However, no report to date has studied the effect of -crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of A- and B-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, A–/–, B–/–, and (A/B) –/– (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 µm long and 25 nm wide and had a characteristic protofilament structure, but B–/– microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, -enolase, and ßB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, A–/– or B–/– microtubules, extraction of the MAPs gave very long (14–20 µm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous -crystallin (A+ 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 -crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90° light scattering measurements showed that -crystallin suppressed tubulin assembly in vitro. -Crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that -crystallin prevented heat-induced aggregation of tubulin, suggesting that -crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin. Xi, J.-h, Bai, F., McGaha, R., Andley, U. P. Alpha-crystallin expression affects microtubule assembly and prevents their aggregation.

Key Words: tubulin • lens • cytoskeleton • chaperone

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