Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.