Fuller, B.G., Lampson, M.A., Foley, E.A., Rosasco-Nitcher, S., Le, K.V., Tobelman, P., Brautigan, D.L., Stukenberg, P.T. and Kapoor, T.M. (2008) Midzone Activation of Aurora B in Anaphase Produces an Intracellular Phosphorylation Gradient. Nature, 453, 1132-6.
Summary: Error-free cell division depends on spatial and temporal cues regulating micron-scale organization. In current models, substrate phosphorylation plays a central role in generating these regulatory cues. While the kinase and phosphatase localizations during cell division have been extensively analyzed, the dynamics of phosphorylation remain poorly characterized. To fill this gap in our knowledge, we developed FRET-based sensors to examine in dividing cells the substrate phosphorylation dynamics that depend on Aurora kinase, a conserved cell cycle regulator in eukaryotes and an anti-cancer drug target. Quantitative analysis of phosphorylation dynamics, using sensors targeted to chromosomes or centromeres, revealed that as chromosomes segregate substrate phosphorylation levels depend more on intracellular position than on time elapsed after anaphase. These data, along with immunofluorescence analyses using phosphorylation-specific antibodies, revealed that a spatial phosphorylation gradient is established during anaphase. By combining this analysis with chemical inhibition, we show that formation of the gradient depends on proper Aurora B targeting to a subset of microtubules that activate the kinase. Further, our data suggest that Aurora kinase activity organizes the microtubules to which it targets to generate a structure-based feedback loop. These phosphorylation dynamics are likely to be specific for Aurora kinase, as we do not observe similar spatial gradients for Polo-like kinase, another important regulator of the cell cycle whose localization during anaphase is similar to that of Aurora kinase. Together, our findings suggest a model in which an Aurora kinase-dependent spatial phosphorylation gradient dynamically ‘marks’ the middle of the anaphase spindle and regulates the final stages of cell division. This interdisciplinary work has established an important paradigm for how spatial and temporal regulation in cells can be achieved by the reaction and localization dynamics of essential enzymes. Our findings also reveal how nanometer-sized proteins can effectively coordinate the assembly of dynamic micron-sized intracellular assemblies.
Summary: Error-free cell division depends on spatial and temporal cues regulating micron-scale organization. In current models, substrate phosphorylation plays a central role in generating these regulatory cues. While the kinase and phosphatase localizations during cell division have been extensively analyzed, the dynamics of phosphorylation remain poorly characterized. To fill this gap in our knowledge, we developed FRET-based sensors to examine in dividing cells the substrate phosphorylation dynamics that depend on Aurora kinase, a conserved cell cycle regulator in eukaryotes and an anti-cancer drug target. Quantitative analysis of phosphorylation dynamics, using sensors targeted to chromosomes or centromeres, revealed that as chromosomes segregate substrate phosphorylation levels depend more on intracellular position than on time elapsed after anaphase. These data, along with immunofluorescence analyses using phosphorylation-specific antibodies, revealed that a spatial phosphorylation gradient is established during anaphase. By combining this analysis with chemical inhibition, we show that formation of the gradient depends on proper Aurora B targeting to a subset of microtubules that activate the kinase. Further, our data suggest that Aurora kinase activity organizes the microtubules to which it targets to generate a structure-based feedback loop. These phosphorylation dynamics are likely to be specific for Aurora kinase, as we do not observe similar spatial gradients for Polo-like kinase, another important regulator of the cell cycle whose localization during anaphase is similar to that of Aurora kinase. Together, our findings suggest a model in which an Aurora kinase-dependent spatial phosphorylation gradient dynamically ‘marks’ the middle of the anaphase spindle and regulates the final stages of cell division. This interdisciplinary work has established an important paradigm for how spatial and temporal regulation in cells can be achieved by the reaction and localization dynamics of essential enzymes. Our findings also reveal how nanometer-sized proteins can effectively coordinate the assembly of dynamic micron-sized intracellular assemblies.
