RECONSTRUCTION OF THE CENTROSOME CYCLE FROM CRYOELECTRON MICROGRAPHS

The absence of detailed in vitro studies leaves the molecular events i nvolved in the centrosome cycle poorly characterized, Most earlier stu dies have employed electron microscopy of thin or thick sections of ce lls. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron micr oscopy of vitrified specimens. The centrosomes were classified into fi ve categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respe ctively). A detailed analysis of the centriole dimensions in these cat egories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its da ughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified spe cimens can be used to obtain detailed structural information on the ce ntrosome cycle. (C) 1997 Academic Press.