Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Determination of Rod and Cone Influence to the Early and Late Dynamic of the Pupillary Light Response.

PURPOSE: This study aims to identify which aspects of the pupil light reflex are most influenced by rods and cones independently by analyzing pupil recordings from different mouse models of photoreceptor deficiency. METHODS: One-month-old wild type (WT), rodless (Rho-/-), coneless (Cnga3-/-), or photoreceptor less (Cnga3-/-; Rho-/- or Gnat1-/-) mice were subjected to brief red and blue light stimuli of increasing intensity. To describe the initial dynamic response to light, the maximal pupillary constriction amplitudes and the derivative curve of the first 3 seconds were determined. To estimate the postillumination phase, the constriction amplitude at 9.5 seconds after light termination was related to the maximal constriction amplitude. RESULTS: Rho-/- mice showed decreased constriction amplitude but more prolonged pupilloconstriction to all blue and red light stimuli compared to wild type mice. Cnga3-/- mice had constriction amplitudes similar to WT however following maximal constriction, the early and rapid dilation to low intensity blue light was decreased. To high intensity blue light, the Cnga3-/- mice demonstrated marked prolongation of the pupillary constriction. Cnga3-/-; Rho-/- mice had no pupil response to red light of low and medium intensity. CONCLUSIONS: From specific gene defective mouse models which selectively voided the rod or cone function, we determined that mouse rod photoreceptors are highly contributing to the pupil response to blue light stimuli but also to low and medium red stimuli. We also observed that cone cells mainly drive the partial rapid dilation of the initial response to low blue light stimuli. Thus photoreceptor dysfunction can be derived from chromatic pupillometry in mouse models.