Curated Optogenetic Publication Database

Abstract: The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. One possibility is that the autonomous local action of a leader cell is sufficient to drive migration of the group. Yet another possibility is that a global mechanical organization is required for the group to move cohesively. Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. We find that the induced leader can robustly drag one follower but is generally unable to direct larger groups. By measuring traction forces and tension propagation in groups of increasing size, we establish a quantitative relationship between group velocity and the asymmetry of the traction and tension profiles. We propose a model of the motile cluster as an active polar fluid that explains this force-velocity relationship in terms of asymmetries in the distribution of active tractions. Our results challenge the notion of autonomous leader cells by showing that collective cell migration requires a global mechanical organization within the cluster.

Abstract: Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Abstract: What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

Abstract: Optogenetics uses light to manipulate protein localization or activity from subcellular to supra-cellular level with unprecedented spatiotemporal resolution. We used it to control the activity of the Cdc42 Rho GTPase, a major regulator of actin polymerization and cell polarity. In this chapter, we describe how to trigger and guide cell migration using optogenetics as a way to mimic EMT in an artificial yet highly controllable fashion.

Abstract: Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.

Abstract: Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.