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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

Published: April 14th, 2023

DOI:

10.3791/65314

1Department of Biological Sciences, Dartmouth College, 2School of Life Sciences, Westlake University, 3Department of Molecular Biology, Princeton University

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

Actomyosin contractility, the contractile force exerted by non-muscle myosin II (hereafter 'myosin') on the F-actin network, is one of the most important forces in changing cell shape and driving tissue-level morphogenesis1,2. For example, the activation of actomyosin contractility at the apical domain of the epithelial cells results in apical constriction, which facilitates a variety of morphogenetic processes, including epithelial folding, cell extrusion, delamination, and wound healing3,4,5,

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1. Setting up the genetic cross and preparing the egg collection cup

  1. Select female flies (virgin) from the optogenetic line UASp-CIBNpm (I); UASp-CRY2-Rho1DN-mCherry (III) on a CO2 pad under a stereomicroscope and set up a cross with male flies from the maternal GAL4 driver line 67 Sqh-mCherry; 15 E-cadherin-GFP.
    NOTE: The 67 and 15 stand for maternal-Tubulin-GAL4 inserted into the second (II) and third (III) chromosomes, respectively52. The GAL4 line.......

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In the unstimulated embryos undergoing apical constriction, Sqh-mCherry became enriched at the medioapical region of the ventral mesodermal cells, whereas CRY2-Rho1DN-mCherry was cytosolic (Figure 1A). Laser ablation within the constriction domain led to a rapid tissue recoil along the A-P axis (Figure 1B,C). In the stimulated embryos, the CRY2-Rho1DN-mCherry signal became plasma membrane localized, whereas the medioapical signal of Sqh-mCherry .......

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This protocol described the combined use of optogenetics and laser ablation to probe changes in tissue tension immediately after the inactivation of actomyosin contractility. The optogenetic tool described here takes advantage of the dominant negative form of Rho1 (Rho1DN) to acutely inhibit endogenous Rho1 and Rho1-dependent actomyosin contractility. Previous characterization of Opto-Rho1DN in the context of Drosophila ventral furrow formation demonstrated that the tool is highly effective in mediating the rapi.......

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The authors thank Ann Lavanway for imaging support. The authors thank the Wieschaus lab and the De Renzis lab for sharing reagents and the Bloomington Drosophila Stock Center for fly stocks. This study is supported by NIGMS ESI-MIRA R35GM128745 and American Cancer Society Institutional Research Grant #IRG-82-003-33 to BH.

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NameCompanyCatalog NumberComments
35 mm glass-bottom dishMatTekP35G-1.5-10-CUsed for sample preparation
60 mm × 15 mm Petri dish with lidFalcon351007Used for sample preparation
Black cloth for covering the microscopeOnlineNAUsed to avoid unwanted light stimulation
Clorox Ultra Germicadal Bleach (8.25% sodium hypochlorite)VWR10028-048Used for embryo dechorination
CO2 padGenesee Scientific59-114Used for cross set-up
ddH2ONANAUsed for sample preparation
Dumont Style 5 tweezersVWR102091-654Used for sample preparation
Eyelash tool (made from pure red sable round brush #2)VWR22940-834Used for sample preparation
FluoView (Software)OlympusNAUsed for image acquisition and optogenetic stimulation
Halocarbon oil 27Sigma AldrichH8773-100MLUsed for embryo stage visualization
ImageJ/FIJINIHNAUsed for image analysis
MATLABMathWorksNAUsed for image analysis
Nikon SMZ-745 stereoscopeNikonNAUsed for sample preparation
Olympus FVMPE-RS multiphoton microscope with InSight DS Dual-line Ultrafast Lasers for simultaneous dual-wavelength multiphoton imaging, , a 25x/NA1.05 water immersion objective (XLPLN25XWMP2), and an IR/VIS stimulation unit for photo-activation/stimulation. This system is also equipped with a TRITC filter (39005-BX3; AT-TRICT-REDSHFT 540/25x, 565BS, 620/60M), and a fluorescence illumination unit that emits white light.OlympusNAUsed for image acquisition and optogenetic stimulation
SP Bel-Art 100-place polypropylene freezer storage box (Black, light-proof box for sample transfer)VWR30621-392Used to avoid unwanted light stimulation
UV Filter Shield for FM1403 Fluores (Orange-red plastic shield)BoliopticsFM14036151Used to avoid unwanted light stimulation
VITCHELO V800 Headlamp with White and Red LED LightsAmazonNAUsed to avoid unwanted light stimulation

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