Curated Optogenetic Publication Database

Abstract: Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular “clutch” at tricellular vertices which stabilizes vertex motion upon tensional load.

Abstract: Spatiotemporal localization of protein function is essential for physiological processes from subcellular to tissue scales. Genetic and pharmacological approaches have played instrumental roles in isolating molecular components necessary for subcellular machinery. However, these approaches have limited capabilities to reveal the nature of the spatiotemporal regulation of subcellular machineries like those of cytoskeletal organelles. With the recent advancement of optogenetic probes, the field now has a powerful tool to localize cytoskeletal stimuli in both space and time. Here, we detail the use of tunable light-controlled interacting protein tags (TULIPs) to manipulate RhoA signaling in vivo. This is an optogenetic dimerization system that rapidly, reversibly, and efficiently directs a cytoplasmic RhoGEF to the plasma membrane for activation of RhoA using light. We first compare this probe to other available optogenetic systems and outline the engineering logic for the chosen recruitable RhoGEFs. We also describe how to generate the cell line, spatially control illumination, confirm optogenetic control of RhoA, and mechanically induce cell-cell junction deformation in cultured tissues. Together, these protocols detail how to probe the mechanochemical circuitry downstream of RhoA signaling. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a stable cell line expressing TULIP constructs Basic Protocol 2: Preparation of collagen substrate for imaging Basic Protocol 3: Transient transfection for visualization of downstream effectors Basic Protocol 4: Calibration of spatial illumination Basic Protocol 5: Optogenetic activation of a region of interest.

Abstract: Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.

Abstract: Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodelling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodelling and continuous strain relaxation. First, a critical strain threshold for tension remodelling triggers irreversible junction length changes for sufficiently strong contractions, making the system robust to small fluctuations in contractile activity. Second, continuous strain relaxation allows for mechanical memory removal, enabling frequency-dependent modulation of cell shape changes via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodelling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.

Abstract: Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.