Molecular analysis of the INCENPs (inner centromere proteins): separate domains are required for association with microtubules during interphase and with the central spindle during anaphase

It has recently been proposed that mitotic chromosomes transport certain cytoskeletal proteins to the metaphase plate so that these proteins are able to subsequently participate in the assembly of the anaphase spindle and the cleavage furrow. To understand how such proteins accomplish their dual chromosomal: cytoskeletal role, we have begun a molecular and functional analysis of the inner centromere proteins (INCENPs), founder members of the class of "chromosome passenger proteins". cDNA clones encoding the open reading frames of the two chicken INCENPs were recovered. The predicted proteins, class I INCENP (96,357 D) and class II INCENP (100,931 D) are novel, and differ from each other by the inclusion of a 38-codon insert within the class II coding region. Transient expression of the chicken INCENPs in mammalian cells confirms that the signals and structures required for the transfer of these proteins from chromosomes to cytoskeleton are evolutionarily conserved. Furthermore, these studies reveal that INCENP association with the cytoskeleton is complex. The amino-terminal 42- amino acid residues are required for transfer of the INCENPs from the chromosomes to the mitotic spindle at anaphase, but not for binding of INCENPs to cytoplasmic microtubules. In contrast, an internal 200 amino acid coiled-coil domain was required for association with microtubules, but dispensable for spindle association. These experiments suggest that proteins required for assembly of specialized cytoskeletal structures during mitosis from anaphase onwards might be sequestered in the nucleus throughout interphase to keep them from disrupting the interphase cytoskeleton, and to ensure their correct positioning during mitosis.