{'fraction_non_alphanumeric': 0.03916768665850673, 'fraction_numerical': 0.024567232033572303, 'mean_word_length': 4.713786213786213, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
An Optogenetic Method to Modulate Cell Contractility during Tissue Morphogenesis

Summary Morphogenesis of multicellular organisms is driven by localized cell shape changes. How, and to what extent, changes in behavior in single cells or groups of cells influence neighboring cells and large-scale tissue remodeling remains an open question. Indeed, our understanding of multicellular dynamics is limited by the lack of methods allowing the modulation of cell behavior with high spatiotemporal precision. Here, we developed an optogenetic approach to achieve local modulation of cell contractility and used it to control morphogenetic movements during Drosophila embryogenesis. We show that local inhibition of apical constriction is sufficient to cause a global arrest of mesoderm invagination. By varying the spatial pattern of inhibition during invagination, we further demonstrate that coordinated contractile behavior responds to local tissue geometrical constraints. Together, these results show the efficacy of this optogenetic approach to dissect the interplay between cell-cell interaction, force transmission, and tissue geometry during complex morphogenetic processes.

Mean levels of plasma membrane-associated mCherry::CRY2-OCRL in the dark, following photoactivation (black arrow). Photo-activation was achieved at a scanning speed of 1.27x10 -6 s/pixel for a total time of 1s for the entire embryo at 30 s intervals for 4 min using a continuous wave laser (488 nm). x axis displays time (in min). y axis displays the log 2 ratio of mCherry::CRY2-OCRL plasma membrane to cytosol fluorescence intensities. Pooled data are represented as mean ± SD (n=3 embryos). (B) Mean levels of cortical Moesin::mCherry levels in the dark, following photo-activation (black arrow). Photo-activation was achieved as in (A). x axis displays time (in min). y axis displays the log 2 ratio of Moesin::mCherry plasma membrane to cytosol fluorescence intensities. Pooled data are represented as mean ± SD (n=3 embryos).
(C-E) Confocal images of a representative embryo co-expressing a non-tagged version of mCherry::CRY2-OCRL, the actin-binding protein Moesin::mCherry and CIBN::pmGFP before photoactivation (C), 4 min after the beginning of photo-activation (D), and after 20 min in the absence of blue light (E). Scale bars, 10 µm.     CRY2-OCRL plasma membrane recruitment was activated with 488 nm laser illumination in the whole embryo, before ventral mesodermal cells started to constrict. Of note, the punctate fluorescence corresponds to clusters of CRY2-OCRL and CIBN-GFP at the plasma membrane, presumably caused by the tendency of CRY2 to oligomerize (Bugaj et al., 2013).

Movie S9. Ventral furrow formation depends on the number of cells that can constrict, Related to Figure 7.
Confocal movie of an embryo co-expressing CIBN::pmGFP (not shown) and mCherry::CRY2-OCRL (white). CRY2-OCRL plasma membrane recruitment was activated with two-photon laser illumination (λ = 950 nm) in two groups of ventral mesodermal cells, one at the posterior and one at the anterior region of the furrow primordium (red boxes). Scale bar, 10 µm.

Supplemental Experimental Procedures
Cloning Plasmids encoding CIBN::pmGFP and CRY2 PHR were purchased from Addgene (plasmid no. 26867 and 26866, respectively). CIBN::pmGFP and CRY2 PHR were PCR-amplified using gene-specific primers. To generate a non-tagged version of CIBN localizing to the plasma membrane, the sequence encoding CIBN was PCR-amplified from Addgene plasmid no. 26867 and fused C-terminally to the CaaX sequence (KKKKKKSKTKCVIM) of human KRas4B through overlap extension PCR. The catalytic domain (residues 99-519) of OCRL was PCR-amplified from Drosophila melanogaster cDNA using gene-specific primers and fused downstream of CRY2 PHR . To generate a fluorescently tagged version of CRY2 PHR -OCRL, mCherry fused C-terminally to a GAGA linker was cloned upstream of CRY2-OCRL. All constructs were cloned into the pPW vector (Drosophila Genomics Resource Center, Bloomington, IN) using the Gateway cloning system (Life Technologies) according to standard procedures.

Fly genetics
Transgenic flies were obtained by standard methods and all stocks were maintained at 22°C. Standard genetic crosses were used to generate flies having the following genotypes: To test for the effect of CRY2-OCRL plasma membrane recruitment on morphogenesis, All crosses were kept in the dark at 22°C. Flies of the desired genotype were maintained in cages with apple juice plates covered with yeast paste and kept in the dark at 18°C for embryo collection.

Live imaging and Photo-activation Protocol
To avoid unwanted photo-activation, embryos were staged and mounted using a standard upright microscope equipped with a 10x objective (Carl Zeiss) in a dark room. The microscope light source was replaced with a lamp (Tween Light) equipped with a yellow 11W incandescence bulb (Décor Color Yellow 11, Osram). Embryos were visualized using a thin layer of Halocarbon oil 27 (Sigma-Aldrich) and those at the right developmental stage were selected and dechorionated with 100% sodium hypochlorite solution for 2 minutes, washed with water, and mounted on a 35 mm glass bottom dish having a 14 mm microwell (coverslip thickness: 0.16-0.19 mm) (MatTek corporation). Embryos were covered with PBS and imaged at 20°C with a Zeiss LSM 780 NLO confocal microscope (Carl Zeiss) equipped with a 561 diode laser, an argon laser and a two-photon laser (Chameleon; Coherent, Inc.). Since bright field illumination could result in unwanted photo-activation, the microscope light source was covered with a Deep Amber lighting filter (Cabledelight, Ltd). mCherry was excited with λ = 561 nm at a laser intensity of 50.8 µW, measured 1 cm from the objective. EGFP was excited with λ = 488 nm at a laser intensity of 6.9 µW, measured 1 cm from the objective. Photo-activation was tested with λ = 458 nm, 488 nm, 514 nm (one-photon illumination) and with λ = 900 nm, 950 nm and 1000 nm (two-photon illumination) by illuminating a single z-stack 5 µm from the apical surface for a total scan time of 1 s. Between two consecutive photo-activations there was an interval of 30 s and the acquisition of the mCherry channel at 561 nm. The laser intensities used were: 6.1 µW (458 nm), 6.9 µW (488 nm), 10.6 µW (514 nm), 6.5 mW (900 nm), 3.0 mW (950 nm), 1.03 mW (1000 nm). The laser intensities were measured 1 cm from the objective and corresponded to the ones normally used for optical imaging of fluorescent proteins.
Once established the most effective wavelength to trigger CRY2-OCRL recruitment to the plasma membrane, photo-activation with one-photon illumination was achieved with λ = 488 nm by illuminating a single z-stack 5 µm from the apical surface for a total scan time of 1 s. Between two consecutive excitations there was an interval of 30 s and the acquisition of the mCherry channel at 561 nm. The laser power used was equivalent to the one used for EGFP imaging (6.9 µW, measured 1 cm from the objective).
Photo-activation with two-photon illumination was achieved with λ = 950 nm by illuminating five z-stacks (each plane separated by 1 µm) for a total scan time of 2.5 s (~500 ms per plane). Laser powers used were 3.0 mW, 1.5 mW and 0.7 mW, measured 1 cm from the objective. Unless otherwise stated, local photo-activation experiments were achieved with a laser intensity of 3.0 mW. The first frame was acquired at 561 nm to image CRY2-OCRL prior to activation. Between two consecutive two-photon excitations there was an interval of 30 s and the acquisition of the mCherry channel at 561 nm.
To test for photo-activation at different depths, we illuminated for a total scan time of 2.5 s five consecutive z-stacks (each plane separated by 0.5 µm), with the central z-stack being taken at 5 µm, 10 µm, 20 µm and 80 µm from the apical surface. Two-photon activation was achieved as described before with a laser intensity of 3.0 mW (measured 1 cm from the objective).
Videos and images were acquired using a C-Apochromat 63x/NA 1.2 water immersion objective (Carl Zeiss), with the exception of images shown in Figure S1F-L, which were acquired with a 40x/NA 1.1 water immersion objective (Carl Zeiss). Confocal images are single z-stacks taken 5 µm below the apical cortex. Movies and corresponding still frames are sum projections of five z-stacks taken 5 µm below the apical cortex. In Figure S1F-L, confocal images are single z-stacks taken at the embryo cross-section (~80 µm from the apical cortex). Images were acquired using Zen 2011 software (Carl Zeiss). Images were processed with FIJI (Schindelin et al., 2012) and custom-written R software (see "Image Analysis and Quantification of Cell Features" section).

Image Analysis and Quantification of Cell Features
Images were opened and metadata extracted using the RBioFormats software package (https://github.com/aoles/RBioFormats). Images were processed in R using custom-written software that utilized the image processing toolbox EBImage (Pau et al., 2010). Each z-stack was first smoothed with a Gaussian filter of diameter ~1 µm. Membrane was identified using an adaptive thresholding algorithm that used a moving box of diameter ~2.5 µm. Pixel noise on the membrane mask was reduced by performing a closing morphological operation with a filter diameter of ~1 µm. To reduce the number of false positive (non-membrane) pixels, objects with an area smaller than 4 µm 2 were removed at this stage. The membrane mask was inverted to obtain masks for cytoplasmic areas, which were used as seeds in a voronoi-based segmentation algorithm to identify cell borders more precisely. Finally, we omitted objects that were below an area of 4 µm 2 or above 400 µm 2 to reduce the number of false positive cells.
Cell area, major axis, eccentricity, and angle with respect to the a-p axis were calculated for each z-stack separately using the computeFeatures function from the EBImage toolbox. To calculate cell anisotropy we first weighted the unit vector along the major axis of the cell by the cell eccentricity. We defined the first and second components of the resulting vector as the a-p and d-v anisotropy, respectively. This resulted in quantities between zero and one that were invariant with respect to isotropic rescaling of cell size. Since we were primarily interested in quantifying elongation along the a-p axis, we focused on the a-p anisotropy in the analysis.
The level of optogenetic activation of a cell was calculated by manually defining the membrane mask with the EGFP channel. Nuclei, which tended to be darker than true cytoplasmic regions, were also manually removed. We then quantified the level of optogenetic activation of each cell by calculating the log 2 ratio between the average membrane and average cytosol intensities.
A similar procedure was used to quantify the plasma membrane levels of PH PLCδ ::GFP and Moesin::mCherry. Here, for each embryo we calculated the plasma membrane fluorescence as the ratio of the average membrane to average cytosol intensities at the beginning of photo-activation (T 0 ), and after 1 min (for PH PLCδ ::GFP) or 4 min (for Moesin::mCherry) of photo-activation. We then calculated the log 2 ratio of the plasma membrane fluorescence intensity after photo-activation to the plasma membrane fluorescence intensity at T 0 . Plots were generated using R package ggplot2 (http://ggplot2.org).