title: Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis.
abstract: Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1-containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.
authors.affiliation: Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre and Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 OXZ, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Mahen
authors.forename: Anand D
authors.initials: AD
authors.lastname: Jeyasekharan
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Barry
authors.forename: Ashok R
authors.initials: AR
authors.lastname: Venkitaraman
mesh_terms: D002384:Catalysis; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D018385:Centrosome / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D018613:Microscopy, Confocal / Q000379:methods; D008938:Mitosis; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology; D012984:Software; D013053:Spectrophotometry / Q000379:methods; D000097869:Polo-Like Kinase 1
chemical_list: D018797:Cell Cycle Proteins; D049452:Green Fluorescent Proteins; D046911:Macromolecular Substances; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1