title: Rad53 homologue forkhead-associated kinase A (FhkA) and Ca2+-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in Dictyostelium.
abstract: BACKGROUND
  During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca2+-binding protein 4a (CBP4a), which is a binding partner of NumA1.
  
  
  METHODS
  Polyclonal antibodies were produced in-house. Cells were fixed and probed with either anti-FhkA or anti-CBP4a in order to determine cellular localization during interphase and throughout the stages of mitosis. Colocalization with DAPI nuclear stain allowed us to determine the location of the nucleus and nucleolus while colocalization with anti-α-tubulin allowed us to determine the cell cycle stage.
  
  
  RESULTS
  Here we verify two novel nucleolar proteins, Rad53 homologue FhkA which localized around the edge of the nucleolus and CBP4a which was detected throughout the entire nucleolus. Treatment with the Ca2+ chelator BAPTA (5 mM) showed that the nucleolar localization of CBP4a is Ca2+-dependent. In response to actinomycin D (0.05 mg/mL) CBP4a disappeared from the nucleolus while FhkA protruded from the nucleus, eventually pinching off as cytoplasmic circles. FhkA and CBP4a redistributed differently during mitosis. FhkA redistributed throughout the entire cell and at the nuclear envelope region from prometaphase through telophase. In contrast, during prometaphase CBP4a relocated to many large, discrete "CBP4a islands" throughout the nucleoplasm. Two larger "CBP4a islands" were also detected specifically at the metaphase plate region.
  
  
  CONCLUSIONS
  FhkA and CBP4a represent the sixth and seventh nucleolar proteins that have been verified to date in Dictyostelium and the third and fourth studied during mitosis. The protein-specific distributions of all of these nucleolar proteins during interphase and mitosis provide unique insight into nucleolar protein dynamics in this model organism setting the stage for future work.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord st,, Toronto, ON M5S 3G5, Canada. acatalano@ccny.cuny.edu.
authors.forename: Andrew
authors.initials: A
authors.lastname: Catalano
authors.forename: Danton H
authors.initials: DH
authors.lastname: O'Day