Microinjection of antibodies to centromere protein CENP-A arrests cells ininterphase but does not prevent mitosis

Centromere protein CENP-A is a histone H3-like protein associated specifica lly with the centromere and represents one of the human autoantigens identi fied by sera taken from patients with the CREST variant of progressive syst emic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events, It has been assumed th at this effect is due to CENP-E and CENP-C autoantigens, because of the eff ects of injecting monospecific sera to those proteins into culture cells. H ere we have used an antibody raised against an N-terminal peptide of the hu man autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused ce lls to arrest in interphase before mitosis. These cells showed highly conde nsed small nuclei, a granular cytoplasm and loss of their division capabili ty. On the other hand, microinjection of nocodazole-blocked HeLa cells in m itosis resulted in the typical punctate staining pattern of CENP-A for cent romeres during different stages of mitosis and apparently normal cell divis ion. This was corroborated by time-lapse imaging microscopy analysis of mid -interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitoti c stages was observed. These results suggest that CENP-A is involved predom inantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere.