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. 2015 Oct 1:5:14589.
doi: 10.1038/srep14589.

An optogenetic system for interrogating the temporal dynamics of Akt

Yoshihiro Katsura  1 , Hiroyuki Kubota  2   3   4 , Katsuyuki Kunida  2   4 , Akira Kanno  1 , Shinya Kuroda  2   4 , Takeaki Ozawa  1   4
Affiliations

An optogenetic system for interrogating the temporal dynamics of Akt

Yoshihiro Katsura et al. Sci Rep. .

Abstract

The dynamic activity of the serine/threonine kinase Akt is crucial for the regulation of diverse cellular functions, but the precise spatiotemporal control of its activity remains a critical issue. Herein, we present a photo-activatable Akt (PA-Akt) system based on a light-inducible protein interaction module of Arabidopsis thaliana cryptochrome2 (CRY2) and CIB1. Akt fused to CRY2phr, which is a minimal light sensitive domain of CRY2 (CRY2-Akt), is reversibly activated by light illumination in several minutes within a physiological dynamic range and specifically regulates downstream molecules and inducible biological functions. We have generated a computational model of CRY2-Akt activation that allows us to use PA-Akt to control the activity quantitatively. The system provides evidence that the temporal patterns of Akt activity are crucial for generating one of the downstream functions of the Akt-FoxO pathway; the expression of a key gene involved in muscle atrophy (Atrogin-1). The use of an optical module with computational modeling represents a general framework for interrogating the temporal dynamics of biomolecules by predictive manipulation of optogenetic modules.

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Figures

Figure 1
Figure 1. Photo-activatable Akt (PA-Akt) system.
(a) Schematic of a PA-Akt system. Upon light stimulation, inactive CRY2-Akt in cytosol is translocated to the plasma membrane and is subsequently activated by upstream kinases. CRY2–CIBN dimer dissociates under a dark condition, enabling reversible spatiotemporal control of Akt activity. Fusion proteins were labeled with a fluorescent protein (F.P.). (b) Light-induced membrane localization of CRY2-Akt in HEK293 cells. The cell expressing CRY2-Akt and Myr-CIBN was stimulated with 440-nm laser light for 5 s. A kymograph shows the change of fluorescence intensity of CRY2-Akt over 8 min along the green line in the left image. Green, Myr-CIBN; Red, CRY2-Akt; Scale bar, 5 μm. The graph shows the time course of normalized fluorescence intensity of CRY2-Akt at cytoplasm. Bars: Mean ± S.D. (N = 19). (c) Dose-dependent activation of Akt signaling with light. C2C12 cells expressing Myr-CIBN and CRY2-Akt were stimulated with different times of light pulses at 1-min interval. One minute later after the final light pulse, the cells were collected and subjected to Western blot assay. Insulin was added for 15 min. (d) Reversibility of CRY2-Akt activation. CRY2-Akt was activated three times each at an interval of 60 min. In each activation, cells were stimulated 12 times with 1-min interval of light pulses at an intensity of 4 mW/cm2.
Figure 2
Figure 2. Optical control of Akt-FoxO pathway.
(a) Time-lapse images of FoxO1 upon CRY2-Akt activation in C2C12 cells. Myr-CIBN was labeled with ECFP, and CRY2-Akt with Venus. Scale bars, 10 μm. (b) (Left) Representative time courses of Nuclear/Cytoplasm (N/C) fluorescence ratio of FoxO1-mCherry. (Right) Box and whisker plot of the N/C ratio change upon light illumination with outliers. N/C ratio (before light) was obtained by the average of 5 images taken during 15 min prior to light illumination, while N/C ratio (after light) was obtained by the average of 5 images during 15 min, which were taken from 10 min later post light illumination. (N = 30 for control cells expressing Myr-CIBN and CRY2, N = 32 for cells expressing Myr-CIBN and CRY2-Akt, ***p < 0.001 by a two-tailed Student’s t-test). (c) Light-induced down-regulation of FoxO1-regulated gene expression. Cells expressing Myr-CIBN and CRY2-Akt were continuously stimulated with 1-min interval of light pulses at 1 mW/cm2 intensity or 10 nM insulin. Bars: Mean ± s.e.m. (N = 4).
Figure 3
Figure 3. Computational modeling of temporal dynamics of CRY2-Akt activity.
(a) Time courses of CRY2-Akt activity. C2C12 cells expressing Myr-CIBN and CRY2-Akt were stimulated 3, 6, or 12 times with light pulses at 1-min intervals. The relative CRY2-Akt activity was calculated using Western blot from Thr308 phosphorylation level of CRY2-Akt divided by the total amount of CRY2-Akt. The initiation of the first light pulse was set as time 0. Bars: Mean ± s.e.m. (N = 4, each in independent experiment). (b) Simulations of CRY2-Akt activation based on computational models of Non-feedback model (Supplementary Fig. 7) and Feedback model (Supplementary Fig. 8). The graph shows the time courses of CRY2-Akt activity in simulation (lines) and experiments (dots). A lower AIC value stands for a better fit. (c) Schematic of feedback-mediated PIP3 production by PI3K and the hydrolysis by PTEN. (d) Effects of PTEN inhibition on the activation of CRY2-Akt and endogenous Akt. C2C12 cells expressing Myr-CIBN and CRY2-Akt were pretreated with PTEN inhibitor, VO-OHpic for 15 min, and subsequently stimulated with 12 times of light pulses at 1-min interval.
Figure 4
Figure 4. Predictive control of temporal patterns of CRY2-Akt activity with computational modeling.
(a) Prediction of temporal patterns of CRY2-Akt activity in different intervals of light stimulation. C2C12 cells expressing Myr-CIBN and CRY2-Akt were stimulated with different intervals of light. Circles show the relative CRY2-Akt activity evaluated from the Thr308 phosphorylation divided by the total amount of CRY2-Akt. Lines and dotted lines respectively show simulations by the Feedback model and the Non-feedback model. Bars: Mean ± s.e.m. (N = 4, each in independent experiment). (b) Functional analysis of temporal Akt activity. Each of the CRY2-Akt activation pattern was generated by illuminating cells with 1-min interval of light pulses at 1 mW/cm2 intensity. Atrogin-1 expression was measured at a time point of 200 min after the onset of light illumination. Activation interval: 16.0 min (Light-3), 36.5 min (Light-6), 91.0 min (Light-12). Last light pulse was added 6.0 min (Light-3), 12.5 min (Light-6), and 7.0 min (Light-12) before the collection of cells. *p < 0.05, ***p < 0.001 by a two-tailed Student’s t-test. n.s.: not significant. Bars: Mean ± s.e.m. (N = 3).
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