ABSTRACT
The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cell Cycle Proteins/metabolism , Centrosome/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Centrosome/diagnostic imaging , Metabolic Networks and Pathways , Phosphorylation , Polymerization , Protein Binding , Protein Structure, Tertiary , Ultrasonography , Polo-Like Kinase 1Subject(s)
Centrosome , Cilia , Ciliary Motility Disorders , Centrosome/diagnostic imaging , Cilia/ultrastructure , Ciliary Motility Disorders/etiology , Ciliary Motility Disorders/genetics , Ciliary Motility Disorders/metabolism , Eye Diseases/genetics , Genotype , Hedgehog Proteins/metabolism , Humans , Kidney Diseases/genetics , Mutation , Phenotype , Signal Transduction , UltrasonographyABSTRACT
Aggregation of proteins in the centrosome is implicated in the pathophysiology of Parkinson's disease. However, the relevance of the centrosome in neurodegeneration is still obscure. Centrosome duplication is initiated by the cyclin E/cyclin-dependent kinase 2 (Cdk2) complex. The present study determined changes in cyclin E or Cdk2 expression and in the centrosomal structure in dopaminergic neuronal CATH.a cells exposed to 50, 100 and 150 micromolar dopamine (DA) for 24 h. DA induced significant increase in Cdk2 protein and cyclin E protein, but not cyclin e mRNA. In DA-treated cells, the intense cyclin E- and Cdk2-immunofluorescence signals were co-localized around large and supernumerary centrosomes, and these two parameters of centrosome amplification were significantly increased compared with the control. Simultaneous co-treatment with DA and a Cdk2 inhibitor blocked centrosome amplification and enhanced cell viability. Our results demonstrated that DA could lead to cyclin E accumulation and Cdk2 up-regulation triggering supernumerary centrosomes and apoptotic cell death.
