ABSTRACT
The centromere is the chromosomal region in which the kinetochore is assembled to orchestrate chromosome segregation. It is defined by the presence of a histone H3 variant called Centromere Protein A (CENP-A) or CenH3. Propagation of centromere identity entails deposition of new CENP-A upon exit from mitosis in vertebrate cells. A group of 16 proteins that co-immunoprecipitate with CENP-A, the Constitutive Centromere Associated Network or CCAN, contribute to kinetochore assembly and function. For most of them it is still unclear how and when they are recruited to centromeres and whether they have a role in CENP-A deposition. Taking advantage of the Xenopus egg cell-free system, we have addressed these issues for CCAN proteins CENP-C, CENP-T and CENP-W. CENP-C recruitment occurs as soon as sperm DNA, containing CENP-A, is added to the egg extract, and continues after de novo incorporation of CENP-A in early interphase. In contrast, centromeric recruitment of CENP-T occurs in late interphase and precedes that of CENP-W, which occurs in mitosis. Unlike CENP-C, CENP-T and CENP-W do not participate in CENP-A deposition. However, like CENP-C, they play a major role in kinetochore assembly. Depletion of CENP-C results in reduced amount of CENP-T at centromeres, an effect more prominent in mitosis than in interphase. In spite of this, kinetochores can still be assembled under this condition although the recruitment of Ndc80 and Mis12 is decreased. Our results support the existence of 2 pathways for kinetochore assembly directed by CENP-C and CENP-T/W, which can be reconstituted in Xenopus egg extracts.
