Rad53 homologue forkhead-associated kinase A (FhkA) and Ca2+-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in Dictyostelium

Abstract

BackgroundDuring 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.MethodsPolyclonal 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.ResultsHere 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.ConclusionsFhkA 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.

Evidence for nucleolar subcompartments in Dictyostelium

“…This diversity in localization patterns suggests diversity in function. Accordingly, most of these eight proteins are each thought to function differently; Hsp32 is involved in heat shock response, TRAP-1 in the formation of the spore coat, NumA1 and CBP4a have ties to cell cycle regulation, and Snf12 is involved in stress response [13,[15][16][17]22,28,29]. As more nucleolar proteins are identified it will be interesting to determine if each nucleolar subcompartment houses proteins with similar functions and if additional subcompartments exist.…”

“…4A). Moreover, during mitosis these proteins each exhibit a unique pattern of redistribution [15][16][17]. Dictyostelium undergoes a semi-open mitosis and the nucleocytoplasmic translocation of all known nuclear proteins, including those in the nucleolus, has recently been reviewed [27].…”

“…CBP4a is a nucleolar binding partner of NumA1 [15,23]. Colocalization of either GFP-eIF6 or GFP-Bud31 with anti-CBP4a revealed that CBP4a occupies only a portion of the nucleolar area occupied by GFP-eIF6 and GFP-Bud31 (Fig.…”

“…Anti-NumA1, anti-CBP4a, and anti-Snf12 were produced and verified as previously described [15][16][17]19]. Cell lines expressing either C-GFP-Snf12 or GFP-NLS-3 (GFP-KRKR) from Snf12 were made and verified as previously described [17].…”

“…Calcium-binding protein 4a (CBP4a) and forkhead-associated kinase A (fhkA) are two recently identified proteins in Dictyostelium that localize to different nucleolar locales; FhkA localizes around the nucleolar periphery while CBP4a resides within the nucleoplasm [15]. BAF60a/SMARCD1 homologue Snf12 and NumA1, two nucleolar proteins linked to cell cycle in Dictyostelium, localize differently since Snf12 localizes to a smaller area of the nucleolus than NumA1 [16,17].…”