Abstract
Purpose:
Previous studies show that caspase-3 signaling has a requisite role in lens differentiation initiation, but the target of caspase-3 in the developing lens remains unknown. In muscle differentiation similar low level activation of caspase-3 signals differentiation by cleaving ICAD, releasing CAD, which executes a limited DNA cleavage and genetic reprograming of the cells. Such processes are regulated by chaperone proteins. The concept of crystallins as molecular chaperone proteins is widely acknowledged yet their full potential as chaperones in lens differentiation and specific chaperone targets remain to be explored. These studies examined whether α-crystallin is a molecular chaperone of ICAD/CAD in signaling lens differentiation.
Methods:
E10 lenses were microdissected into central epithelium, equatorial epithelium, cortical fiber and central fiber zones. Fractions were analyzed by direct immunoblot for expression of αA-crystallin, αB-crystallin, CAD and ICAD. Association between these proteins was examined by co-immunoprecipitation. E10 chick embryo lenses were also fixed, sectioned and labeled for CAD, αA-crystallin, nuclei and/or actin and imaged by confocal microscopy.
Results:
CAD is most highly expressed in the equatorial epithelium, consistent with a role in signaling lens differentiation initiation. Both ICAD and α-crystallin are expressed in all zones of the E10 lens. Co-immunoprecipitation demonstrated that α-crystallin specifically interacted with both CAD and ICAD. This association was found to be specific for αA-crystallin; no interaction could be detected with αB-crystallin. Linkage of αA-crystallin to ICAD/CAD was strongest in the cortical fiber zone, the site of morphogenesis following differentiation initiation. Immunostaining confirmed the co-localization of CAD and αA-crystallin. These results suggest the possibility that αA-crystallin acts as a co-chaperone with ICAD to turn off CAD activity after differentiation initiation is complete.
Conclusions:
The αA-crystallin chaperone is associated with the ICAD/CAD complex in the developing lens, likely to regulate the action of CAD in lens differentiation.
Keywords: 450 chaperones •
500 differentiation •
497 development