Abstract
Purpose: :
The molecular chaperones αA– and αB–crystallins associate with the tubulin cytoskeleton. Previous work suggests that the mitotic spindle is abnormally assembled in a number of αA–/– and αB–/– lens epithelial cells. However, the mechanism by which α–crystallin expression affects microtubule (MT) assembly in lens epithelial cells has not been investigated. In the current work we tested MT assembly in wild type and α–crystallin knockout cells.
Methods: :
MTs were reconstituted from freshly dissected explants of wild type, αA–/–, αB–/– and αA/B–/– (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. MT associated proteins (MAPs) were extracted with 0.35 M NaCl. Tubulin levels were determined by immunoblot analysis. Temperature–induced aggregation of tubulin was measured from 37 to 80°C by SDS–PAGE and densitometric analysis. Sedimentation analysis and 90° light scattering measurements were performed to assess overall tubulin assembly in the presence of α–crystallin. GTPase activity was determined spectrophotometrically.
Results: :
Tubulin levels were ≥ 30% lower in lens epithelial cells of αA–/–, αB–/– and DKO mice as compared to wild type controls. Tubulin was extremely sensitive to denaturation in α–crystallin–/– MT fractions. α–Crystallin prevented heat–induced aggregation of purified tubulin in vitro, suggesting that α–crystallin may affect MT assembly by maintaining the pool of unassembled tubulin. α–Crystallin did not have a strong effect on the GTPase activity of purified tubulin. DKO MTs were smaller than wild type MTs and demonstrated breaks, loosening and unraveling of the protofilaments by electron microscopic analysis. In DKO lens epithelial MTs but not in wild type, αA–/– or αB–/– MTs, extraction of MAPs gave very long (20 µm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous α–crystallin (αA+ αB) was ineffective in preventing polyfilament formation. However, normal MT structure could be restored by including MAPs derived from wild type lens epithelial cells during MT reconstitution. Sedimentation analysis and 90° light scattering measurements showed that α–crystallin suppressed tubulin assembly in vitro.
Conclusions: :
α–Crystallin is important for maintaining the unassembled pool of tubulin in a native state in lens epithelial cells. Lack of α–crystallin expression leads to lower tubulin levels, poor MT nucleation, and destabilized MTs. Intriguingly our data also suggest that α–crystallin may interact with MAPs to inhibit aggregation of MTs in lens epithelial cells.
Keywords: chaperones • cytoskeleton • protein structure/function