In an attempt to discover novel proteins functioning in both interphase nucleus and mitotic spindle as NuMA does, we carried out cDNA library screening with pooled autoimmune antibodies. Among positive clones we found a recently identified transcription regulatory protein (CDCA4) with the distinctive nuclear-mitotic apparatus distribution. CDCA4 localizes at metaphase spindle poles and the midzone in later stages. Additionally, an intensive CDCA4 accumulation parallel to spindle was observed in half of metaphase cells but not in later stages, implying a transient form of CDCA4 binding to midzone from anaphase. Mitotic arrest dissolved CDCA4 from centrosomes but during the spindle recovery, CDCA4 invariably colocalized with the microtubule nucleation foci as a component of microtubule organization center. RNA interference of CDCA4 resulted in significant increase of multinuclei and multipolar spindles, suggesting impaired function in chromosome segregation or cytokinesis. However, the spindle checkpoint and the centrosome cycle appeared not to be affected by such interference. Furthermore, CDCA4 depletion resulted in accelerated cell proliferation, perhaps due to the disruption of CDCA4 nuclear function as a transcription suppressor. Interphase CDCA4 is localized in nucleoli by immunofluorescence, although GFP-CDCA4 expressed in the nucleoplasm. An N-terminal KRKC domain appears to be the nuclear localization signal as identified by sequence alignment and the expression of truncated mutants. Taken together, our results suggested that as a novel nuclearmitotic apparatus protein, CDCA4 is involved in spindle organization from prometaphase. When anaphase begins, CDCA4 may play a different role as a midzone factor involved in chromosome segregation or cytokinesis.
(c) 2008 Wiley-Liss, Inc.