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Figure 1: Relation between FRET, fluorescence lifetime, and force applied to the EcadTSMod biosensor.
Schematic representation of the EcadTSMod biosensor. In absence of tension (top) the TSMod is not elongated, the distance between the two fluorescent proteins is the shortest, meaning that the FRET is maximum. As the relation between the FRET and donor fluorescence lifetime is inverse, in absence of tension, the donor fluorescence lifetime is low. In presence of force (bottom) the TSMod is elongated, the distance between fluorescent proteins increases, the FRET decreases and the donor fluorescence lifetime increases.
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Figure 2: E-cadherin tension sensor displays a FRET decrease in living Xenopus embryo compared to tail less E-cadherin.
(a) Schematic representations of the different constructions used, showing binding regions of p120 catenin, β-catenin and indirect binding of α-catenin and actin, and their respective localization in epithelial cells of Xenopus laevis blastula (fluorescence), each field of view is composed of about 15 to 20 cells at this stage of development. The fluorescence lifetime images are presented for all constructions (fluorescence lifetime). (b) Boxplot representative of the mean fluorescence lifetime per field obtained over all acquisitions for the various constructs. Number of acquisition Nacq = 48, 105 and 44, number of independent experiment Nexp = 10, 14, 6 and number of embryos Nemb = 22, 55, 15, for respectively EcadTI, EcadTSMod and EcadTL. For each comparison, the p value is indicated. In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dots to outliers.
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Figure 3: EcadTSMod biosensor is integrated into the adherens junction and responds to several treatments.
(a) Scatter plot of the fluorescence lifetime vs fluorescence intensity for each pixel of a representative acquisition. Equation of the linear regression is specified in the top right corner. (b) Indirect immunofluorescences of EcadTSMod detected with anti-GFP antibody (green) and α, β and p120 catenins (red) in Xenopus laevis blastula overexpressing the EcadTSMod protein. Linescan were made on the indicated dotted line, and the normalized fluorescence intensity is shown for both channel (green for EcadTSMod and red for catenins) in the corresponding graph. Scale bar, 20 μm. (c) Boxplot of mean forces applied on EcadTSMod in untreated embryos or embryos treated with EGTA, latrunculin A, morpholino against α-catenin (MO α–catenin) and Calyculin A, with respectively 105, 32, 37, 44 and 29 independent fields (respective number of experiments: 14, 2, 2, 2 and 3; respective number of embryos: 55, 16, 19, 22 and 15). For each comparison, the p value is indicated. In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dot to outlier.
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Figure 4: Spatio-temporal analysis of E-cadherin biosensor in the Xenopus embryo.
(a) Temporal acquisition of EcadTSMod, EcadTL and EcadTI in Xenopus blastula. Fluorescence images are presented in the upper row; the lower row corresponds to fluorescence lifetime images. For each construct, 3 time points are presented 1, 7 and 14 minutes. A kymograph of the whole temporal acquisition is represented in the right part, the corresponding XY coordinates are shown by the red dotted line in fluorescence images. (b) Mean fluorescence lifetimes and standard deviations of all XY coordinates over the time series for EcadTI (purple), EcadTSMod (cyan) and EcadTL (green). Values represent the mean ± SD.
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Figure 5: Forces applied to EcadTSMod are stable during epithelial cell division.
(a) Spatio-temporal acquisition of EcadTSMod fluorescence images and the corresponding force images at different time points. On fluorescence images, the black arrowheads point to the division site and the red dotted lines indicate the cell-cell contact presented as a kymograph below. The temporal resolution is 1 image every 20 seconds. The vertical arrowheads on the kymograph indicate the site of division while the horizontal one marks the starting time of contraction. The length of the kymograph varies over time due to the stretching of the membrane during the division. (b) Mean forces and standard deviations of all XY coordinates over the time series for EcadTSMod in dividing Xenopus embryo epithelial cells, the arrowhead indicates the site of division. Values represent the mean ± SD. (c,d) Ratio of the force measured at the dividing site and on the proximal membrane (c) or on a distal membrane on a non-dividing cell (d), during the progress of cell division. The force is measured at the same time point at the dividing site and in a region in the proximal or distal membrane. The corresponding percentage of progress of division is calculated by measuring the distance of the two dividing point. The graph shows the results obtained for 44 division sites (which represents 7 independent experiments, 18 embryos and 27 cells). In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dots to outliers.
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Fig. S1: Localization and fluorescence lifetime of CcadTSMod biosensor. a. Schematic representation of CcadTSMod construct (left) and its localization in living Xenopus blastula (middle) with the corresponding fluorescence lifetime image (right). b. Boxplot of EcadTSMod and CcadTSMod mean fluorescence lifetimes calcuted with respectively 105 and 134 independent acquisitions (representing for CcadTSMod: 15 independent experiments and 41 embryos).
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Fig. S2: CcadTSMod is integrated and responsive to various stimuli. a. Representative scatter plot of the fluorescence lifetime vs fluorescence intensity for each pixel of an acquisition. Equation of the linear regression is specified in the top right corner. The average equation for the 14 independent experiment is with a R2 = 0.0163. b. Boxplot of the mean fluorescence lifetime of CcadTSMod in untreated embryos and after EGTA, latrunculin A, morpholino against α-catenin or Calyculin A treatment, with respective number of acquisitions N= 134, 27, 45, 65 and 41 (respective number of experiments: 15, 3, 4, 4 and 2; respective number of embryos: 41, 9, 16, 21 and 20). For each comparison, the p value is indicated. Dots correspond to outliers.
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Fig. S3: The fluorescence lifetime can be directly converted into a force value. a. graphical representation of the linear relation between fluorescence lifetime and force, obtained as described in supplementary material. Equation of the linear regression presented was used to convert fluorescence lifetime into force. b. EcadTSMod fluorescence image in living Xenopus embryo (left) and the corresponding fluorescence lifetime image (center) and force image (right).
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Fig. S4: Actin cytoskeleton is disrupted by latrunculin A treatment. Xenopus laevis embryos were treated with 5 μM latrunculin A during 20 minutes (latrunculin A) or not (H2O). Embryo were fixed and actin filaments were labelled using Alexa Fluor 568 phalloidin.
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Fig. S5: α-catenin is depleted after injection of morpholino against α-catenin. Embryos were microinjected with either a Morpholino against α-catenin or a control Morpholino together with GFP-GPI mRNA. GFP-GPI which labels plasma membrane, is used as a tracer. When embryos reached developmental stage 10, they were fixed in TCA 2% and processed for indirect immunofluorescence to detect α-catenin (red) and GFP-GPI (green) was detected by indirect immunofluorescence using anti-GFP antibody.
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Fig. S6: Spatio-temporal analysis of CcadTSMod biosensor in the Xenopus embryo. a. Temporal acquisition of CcadTSMod in living Xenopus embryo at blastula. Fluorescence images are presented on the upper row, the lower row correspond to the measured fluorescence lifetime images. A kymograph of the whole temporal acquisition is represented in the right part, the corresponding XY coordinates are shown by the red dotted line in fluorescence images. b. CcadTSMod mean fluorescence lifetimes and standard deviations of al XY coordinates over the time series. Values represent the mean SD.
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Fig. S7: Fluorescence lifetime of EcadTL and EcadTI is stable during cell division. Representative spatio-temporal acquisition of EcadTL (a) and EcadTI (b) fluorescence images and the corresponding fluorescence lifetime images at different time points. On fluorescence images, the black arrowheads point to the division site and the red dotted lines indicate the cell-cell contact presented as a kymograph below. The temporal resolution is 1 image every minute. The vertical arrowheads on the kymograph indicate the site of division while the horizontal one marks the starting time of ingression. The length of the kymograph varies over time due to the stretching of the membrane during the division. The images are representative of 7 and 8 junctions respectively for EcadTL and EcadTI. c. Mean fluorescence lifetime and standard deviations of all XY coordinates over the time series for EcadTL (green) and EcadTI (purple) in dividing Xenopus embryo epithelial cells. The pixel axis is centered on the division site. Values represent the mean SD.
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Fig. S8: Forces applied to CcadTSMod are stable during epithelial cell division. a. Spatio-temporal acquisition of CcadTSMod fluorescence images and the corresponding fluorescence lifetime images at different time points during cell division. On fluorescence images, the black arrowhead points to the division site and the red dotted line indicates the cell-cell contact presented as a kymograph below. The temporal resolution is 1 image every minute. The vertical arrowheads on the kymograph indicate the site of division while the horizontal one marks the starting time of ingression. The length of the kymograph varies over time due to the stretching of the membrane during the division. b. Mean fluorescence lifetime and standard deviations of all XY coordinates over the time series for CcadTSMod in dividing Xenopus embryo epithelial cells. Values represent the mean SD. c and d. Ratio of the fluorescence lifetime measured at the dividing site and on the proximal membrane (c) or on a distal membrane on a non-dividing cell (d), during the progress of cell division. The fluorescence lifetime is measured at the same time point at the dividing site and in a region in the proximal or distal membrane, the corresponding percentage of progress of division is calculated by measuring the distance of the two dividing point. The graph shows the results obtained for 16 division sites (which represents 7 independent experiments, 10 embryos and 11 cells). Dots corresponds to outliers.
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Figure 1. Relation between FRET, fluorescence lifetime, and force applied to the EcadTSMod biosensor.Schematic representation of the EcadTSMod biosensor. In absence of tension (top) the TSMod is not elongated, the distance between the two fluorescent proteins is the shortest, meaning that the FRET is maximum. As the relation between the FRET and donor fluorescence lifetime is inverse, in absence of tension, the donor fluorescence lifetime is low. In presence of force (bottom) the TSMod is elongated, the distance between fluorescent proteins increases, the FRET decreases and the donor fluorescence lifetime increases.
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Figure 2. E-cadherin tension sensor displays a FRET decrease in living Xenopus embryo compared to tail less E-cadherin.(a) Schematic representations of the different constructions used, showing binding regions of p120 catenin, β-catenin and indirect binding of α-catenin and actin, and their respective localization in epithelial cells of Xenopus laevis blastula (fluorescence), each field of view is composed of about 15 to 20 cells at this stage of development. The fluorescence lifetime images are presented for all constructions (fluorescence lifetime). (b) Boxplot representative of the mean fluorescence lifetime per field obtained over all acquisitions for the various constructs. Number of acquisition Nacq = 48, 105 and 44, number of independent experiment Nexp = 10, 14, 6 and number of embryos Nemb = 22, 55, 15, for respectively EcadTI, EcadTSMod and EcadTL. For each comparison, the p value is indicated. In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dots to outliers.
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Figure 3. EcadTSMod biosensor is integrated into the adherens junction and responds to several treatments.(a) Scatter plot of the fluorescence lifetime vs fluorescence intensity for each pixel of a representative acquisition. Equation of the linear regression is specified in the top right corner. (b) Indirect immunofluorescences of EcadTSMod detected with anti-GFP antibody (green) and α, β and p120 catenins (red) in Xenopus laevis blastula overexpressing the EcadTSMod protein. Linescan were made on the indicated dotted line, and the normalized fluorescence intensity is shown for both channel (green for EcadTSMod and red for catenins) in the corresponding graph. Scale bar, 20 μm. (c) Boxplot of mean forces applied on EcadTSMod in untreated embryos or embryos treated with EGTA, latrunculin A, morpholino against α-catenin (MO α–catenin) and Calyculin A, with respectively 105, 32, 37, 44 and 29 independent fields (respective number of experiments: 14, 2, 2, 2 and 3; respective number of embryos: 55, 16, 19, 22 and 15). For each comparison, the p value is indicated. In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dot to outlier.
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Figure 4. Spatio-temporal analysis of E-cadherin biosensor in the Xenopus embryo.(a) Temporal acquisition of EcadTSMod, EcadTL and EcadTI in Xenopus blastula. Fluorescence images are presented in the upper row; the lower row corresponds to fluorescence lifetime images. For each construct, 3 time points are presented 1, 7 and 14 minutes. A kymograph of the whole temporal acquisition is represented in the right part, the corresponding XY coordinates are shown by the red dotted line in fluorescence images. (b) Mean fluorescence lifetimes and standard deviations of all XY coordinates over the time series for EcadTI (purple), EcadTSMod (cyan) and EcadTL (green). Values represent the mean ± SD.
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Figure 5. Forces applied to EcadTSMod are stable during epithelial cell division.(a) Spatio-temporal acquisition of EcadTSMod fluorescence images and the corresponding force images at different time points. On fluorescence images, the black arrowheads point to the division site and the red dotted lines indicate the cell-cell contact presented as a kymograph below. The temporal resolution is 1 image every 20 seconds. The vertical arrowheads on the kymograph indicate the site of division while the horizontal one marks the starting time of contraction. The length of the kymograph varies over time due to the stretching of the membrane during the division. (b) Mean forces and standard deviations of all XY coordinates over the time series for EcadTSMod in dividing Xenopus embryo epithelial cells, the arrowhead indicates the site of division. Values represent the mean ± SD. (c,d) Ratio of the force measured at the dividing site and on the proximal membrane (c) or on a distal membrane on a non-dividing cell (d), during the progress of cell division. The force is measured at the same time point at the dividing site and in a region in the proximal or distal membrane. The corresponding percentage of progress of division is calculated by measuring the distance of the two dividing point. The graph shows the results obtained for 44 division sites (which represents 7 independent experiments, 18 embryos and 27 cells). In the box plot, bold bars correspond to median value, whiskers to the value 1.5x away from the 1st and 3rd quartiles, and the dots to outliers.
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