contained αB-crystallin. Electron micrographs clearly showed direct interactions of purified αB-crystallin with the surface of microtubule-associated proteins (MAPs) attached to MTs. Purified αB-crystallin bound to MAPMTs in a concentration-dependent manner. However, αB-crystallin did not bind MTs reconstituted from purified tubulin. Finally, we observed that αB-crystallin increased the resistance of MTs to depolymerization in cells and in vitro. Taken together, these results suggest that one of the functions of αB-crystallin is to bind MTs via MAP(s) and to give the MTs resistance to disassembly.

The cytoskeletal component tubulin/microtubule commonly allows the cell to respond mechanically to the environment. The concentration of free tubulin dimer is autoregulated in the balance of free dimer and polymeric forms of microtubule (MT) protein, having an intrinsic property of "dynamic instability", and through cotranslational beta-tubulin mRNA degradation. Recently, we have demonstrated that alphaB-crystallin is a key molecule of muscle atrophy, since alphaB-crystallin has a chaperone-like-activity that suppresses tubulin aggregation and protects the MT disassembly against both Ca2+ and depolymelizing alkaloid in vitro. Most of the small heat-shock proteins (sHsps), including alphaB-crystallin, are expressed in skeletal muscle. However, no report to date has studied the changes of tubulin/MT during muscle adaptation. Here, we examined changes in tubulin content in rat soleus muscles after hindlimb suspension (HS) with/without passive stretch and the recovery. HS induced rapid decreases of soleus muscle mass, most Hsps (alphaB-crystallin, Hsp90, Hsp70, Hsp27, and p20) and tubulin contents in soleus muscle, while heat-shock cognate 70-kDa protein (Hsc70) did not decrease. Soleus muscle mass, most Hsps, and tubulin were maintained with passive stretch. After 5 days' recovery, the levels of tubulin and Hsps, but not Hsc70, were restored to control levels. The interactions of alphaB-crystallin and tubulin/MT were observed with immunoprecipitation with an anti-alpha-tubulin antibody and taxol-dependent MT assembly. Other sHsps were also associated with alphaB-crystallin and MT, whereas Hsp90 and Hsp70 did not co-precipitate with them. These data imply an interaction and close relationship between alphaB-crystallin and tubulin/MTs in muscle tissues. The amount of mRNA of alphaB-crystallin decreased with the muscle atrophy level, whereas the gene expression level of betaI-tubulin was maintained during HS. This means a significant role of post-transcriptional regulation in tubulin/MT system in muscle adaptation, whereas alphaB-crystallin and most sHsps are regulated at the transcriptional level. Additional functional contribution of alphaB-crystallin to tubulin/MTs during myotube formation was examined using C2C12 myoblast cultured cells, the alphaB-crystallin expression of which was decreased or increased. It indicated the necessity of alphaB-crystallin during microtubule reorganization. In conclusion, tubulin/MTs were revealed to be one of the substrates of alphaB-crystallin, and also serial decreases of alphaB-crystallin and tubulin/MT in early soleus muscle atrophy suggest that the chaperone effect of alphaB-crystallin on the cytoskeleton, which may be also dynamically regulated in the muscle cell, is a key mechanism for muscle adaptation and protection of the atrophy and also muscle differentiation.

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. ␣B-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the ''␣-crystallin domain'' common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal ␣-crystallin domain of ␣B-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of ␣B-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the ␣-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that ␣B-crystallin recognizes a variety of substrates and especially that ␣-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.

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