Krishnamurthy VV et al. (2016), Reversible optogenetic control of kinase activi...

XB-ART-52569

Development 2016 Nov 01;14321:4085-4094. doi: 10.1242/dev.140889.

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Reversible optogenetic control of kinase activity during differentiation and embryonic development.

Krishnamurthy VV , Khamo JS , Mei W , Turgeon AJ , Ashraf HM , Mondal P , Patel DB , Risner N , Cho EE , Yang J , Zhang K .

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A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

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Species referenced: Xenopus
Genes referenced: cry2 mapk1 odc1 raf1 tbxt

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	Fig. 1. Correlation between the differentiation ratio and the fluorescence intensity ratio (GFP/mCherry) in PC12 cells co-transfected with CIBN-GFP-CaaX and CRY2-mCherry-Raf1. (A) Light-induced binding between CIBN and CRY2 leads to membrane recruitment of CRY2-mCherry-Raf1, which activates the Raf/MEK/ ERK signaling pathway and induces PC12 cell differentiation. (B) Representative fluorescence images of PC12 cells co-transfected with CIBN-GFP-CaaX (green) and CRY2-mCherry-Raf1 (red). A differentiated cell is marked with a dashed circle; an undifferentiated cell is marked with a dashed square. Differentiated cells are defined as those with at least one neurite of length equal to or longer than the diameter of their cell bodies. (C) A representative image of cells transfected with Raf1- GFP-CaaX, a constitutively active Raf1 (CA-Raf1). A differentiated cell is marked with a dashed circle; an undifferentiated cell is marked with a dashed square. (D) Differentiation ratios of PC12 cells transfected with CA-Raf1, co-transfected with CRY2-mCherry-Raf1 and CIBN-GFP-CaaX, singly transfected with CRY2- mCherry-Raf1, or singly transfected with CIBN-GFPCaaX. Twenty four hours after transfection, cells were either exposed to light (0.2 mW/cm2) or incubated in the dark for another 24 h. Values represent mean±s.d. from four independent data sets. (E) Histograms of fluorescence intensity ratio (GFP/mCherry) for differentiated and undifferentiated cells in co-transfected cultures. Number of cells analyzed: differentiated cells (n=430), undifferentiated cells (n=430). (F) Quantification of GFP/mCherry ratio for the data shown in E. Values represent mean±s.d. Differentiated cells show a significantly higher GFP/mCherry ratio than undifferentiated cells. P-value was determined by a two-tailed, unpaired t-test. (G) Fluorescence intensities of GFP and mCherry in differentiated and undifferentiated cells. Values represent mean±s.d. P-values (D,F) were determined by a two-tailed, unpaired t-test.
	Fig. 2. Characterization of the bicistronic system based on the P2A peptide. (A) Design of CRY2-2A-CIBN, a bicistronic optogenetic system. Upon ribosomal skipping, one mRNA transcript generates two proteins: CRY2-mCherry-Raf1-N2A (ending with amino acids NPG) and proline-CIBN-GFP-CaaX. (B) Western blot analysis of the CRY2-2A-CIBN system in BHK-21 cells. The blot was probed sequentially with anti-GFP (bands in the green rectangle) and anti-mCherry (bands in the pink rectangle). A composite image is shown. Lane 1: protein ladder; lane 2: non-transfection; lane 3: CRY2-mCherry-Raf1 single transfection; lane 4: CIBN-GFP-CaaX single transfection; lane 5: co-transfection of CRY2-mCherry-Raf1 and CIBN-GFP-CaaX; lane 6: CRY2-2A-CIBN single transfection. Arrows show that sizes of cleaved products from CRY2-2A-CIBN match with that of singly transfected cells. The asterisk indicates residual full-length CRY2-2A-CIBN. (C) Quantification of the intensity ratio of CIBN-GFP-CaaX to CRY2-mCherry-Raf1 on western blot. Values represent mean±s.d. (n=4). (D) Confocal fluorescence images of BHK-21 cells that were co-transfected with CRY2-mCherry-Raf1 and CIBN-GFP-CaaX. A cell expressing CIBN-GFP-CaaX, but not CRY2-mCherry- Raf1, is marked (arrow). (E) In cell cultures that were transfected with CRY2-2A-CIBN, all cells show fluorescence in both GFP and mCherry channels. (F,G) Correlation analyses between CRY2-mCherry-Raf1 and CIBN-GFP-CaaX in co-transfected and CRY2-2A-CIBN transfected cells. Fluorescence intensities between mCherry and GFP arewell correlated (R2=0.836) in CRY2-2A-CIBN transfected cells (F) but are poorly correlated (R2=0.336) in co-transfected cells (G). Number of cells analyzed: CRY2-2A-CIBN (n=100), co-transfected (n=100).
	Fig. 3. Characterization of bicistronic constructs with varying number of CIBNs. (A) Schematic of the CRY2-2A-(n)CIBN construct. (B) An anti-GFP western blot analysis showing the expression of (n)CIBN-GFP-CaaX in BHK-21 cells transfected with bicistronic systems. The expected sizes of (n)CIBN-GFP-CaaX proteins are 48 kDa (single-CIBN), 68 kDa (double-CIBN), and 87 kDa (triple-CIBN). The cell culture co-transfected with CRY2-mCherry-Raf1/CIBN-GFP-CaaX (lane 1) shows a band at the position of single CIBN (lane 3). (C) The same blot consequently probed with anti-mCherry without stripping. All cell cultures show a primary band that corresponds to CRY2-mCherry-Raf1 (arrow, expected size 161 kDa) in the pink rectangle. (D-F) Confocal fluorescence imaging of cells transfected with CRY2-2A-(n)CIBN. The number of CIBN is 1 (D), 2 (E) and 3 (F). In each panel, the first panel shows cleaved (n)CIBN-GFP-CaaX localized on the plasma membrane; the second panel shows a snapshot of CRY2-mCherry-Raf1 before blue-light stimulation; the third panel shows a snapshot of CRY2-mCherry- Raf1 after ten pulses of blue-light stimulation. The bottom panel shows normalized intensity profiles across the cell (marked by yellow dashed line) before and after light stimulation. Blue light-induced membrane recruitment of CRY2-mCherry-Raf1 can be observed under all three conditions but to different extents. Scale bars: 10 μm. (G) Quantification of CRY2-mCherry-Raf1 membrane translocation. The membrane/cytoplasm mCherry intensity ratios were calculated before and after light stimulation. The fold increase was then calculated by dividing the after-stimulation ratio by the before-stimulation ratio. The single-CIBN construct is less effective at inducing membrane recruitment of CRY2-mCherry-Raf1 compared with the multiple-CIBN constructs. Number of cells analyzed: 1-CIBN (n=6), 2-CIBN (n=5), 3-CIBN (n=7). Values represent mean±s.d. (H) Supplementing 2A-CIBN-GFP-CaaX to CRY2-2A-CIBN improves the membrane translocation of cytosolic CRY2-mCherry-Raf1 after blue-light stimulation. The three images are GFP fluorescence (left), and mCherry fluorescence before (middle) and after (right) ten pulses of blue-light stimulation. (I) Fold increase of the membrane/cytoplasm ratio of CRY2-mCherry-Raf1 fluorescence intensity. Supplementing additional 2A-CIBN-GFP-CaaX improves the capacity for membrane recruitment of CRY2-mCherry-Raf1. Number of cells analyzed: CRY2-2A-CIBN+2A-CIBN-GFP-CaaX(n=6), CRY2-2A-CIBN (n=7). Values represent mean±s.d. (J) Supplementing additional 2A-CIBN-GFP-CaaX increases the differentiation ratio. Values represent mean±s.d. from two independent data sets for each condition. (K) Quantification of differentiation ratios for various transfection conditions. The double-CIBN construct shows the highest differentiation ratio, double that of co-transfected constructs and reaching the limit of CA-Raf1. The single- and triple-CIBN constructs show low differentiation ratios. Number of data sets analyzed: CA-Raf1 and all light conditions (n=4), all dark conditions (n=3). Note that data for CA-Raf1 and co-transfection are the same as those in Fig. 1D. They are presented here for easier comparison between differentiation ratios from all bicistronic constructs. Values represent mean±s.d. ***P<0.001; ns, not statistically significant. P-values were determined by two-tailed, unpaired t-tests.
	Fig. 4. Application of the bicistronic optogenetic Raf1 activation system in Xenopus embryos. (A) Schematic for light-controlled Raf1 activation in Xenopus embryos or animal caps. (B) RT-PCR results showing that exposure to blue light induced the expression of xbra, a pan-mesodermal marker, in CRY2-2A- 2CIBN-injected animal caps at the early gastrula stage. odc was used as loading control. (C) Expression of xbra in whole embryos at the early gastrula stage assessed by whole-mount in situ hybridization. Samples include controls (uninjected, no light treatment); embryos that were uninjected, but treated with blue light; embryos that were injected with CRY2-2A-2CIBN and cultured under normal conditions; and embryos that were injected with CRY2-2A-2CIBN and treated with blue light. Red arrows indicate the cell lineage tracer. Blue arrow indicates ectopic xbra mRNA expression. (D)Western blot analysis showing that in an animal cap assay, CRY2-2A-2CIBN induced blue light-dependent reversible phosphorylation of ERK. (E) Activation of Raf1 by treating CRY2-2A-2CIBN-injected embryos with blue light after the completion of germ layer specification is sufficient for inducing ectopic tail-like structures in the head region (arrowheads). Images show morphology of control embryos; uninjected embryos that were treated with blue light; embryos that were injected with CRY2-2A-2CIBN and cultured under normal conditions; CRY2-2A-2CIBN-injected embryos treated with blue light from stage 8 to stage 25, and CRY2-2A-2CIBN-injected embryos treated with blue light from stage 14 to stage 30. (F) Percentage of embryos with ectopic tail-like structure. (G,H) Histological analysis of the tail-like structures induced by activation of Raf1. (G) Tail-like structure induced by Raf1 in an embryo exposed to blue light from stage 8 to stage 25. (H) Tail-like structure induced by Raf1 in an embryo exposed to blue light from stage 14 to stage 30. Small inserts at the lower left corner are low-power views, which show the location of the Raf1-induced taillike structure (arrowheads). Scale bars: 100 μm.
	Figure S5. The 2-CIBN construct elicited higher percentage of ectopic tail-like structure than the 1- and 3-CIBN constructs. (A) Representative images of embryos at the late tailbud stage. Embryos were either uninjected (upper left), or injected with CRY2-2A-1CIBN (upper right), CRY2-2A- 2CIBN (lower left), and CRY2-2A-3CIBN (lower right) at the 4-cell stage. Light illumination was applied during stage 8-30. (B) Bar graph shows the percentage of embryos with ectopic tail-like structures. The 2-CIBN construct induced ectopic structure in more than 60% of all embryos examined, whereas the 1- and 3-CIBN only elicited 0% and 3.6%, respectively.

References [+] :

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