Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics

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.

Optogenetic dissection of Rac1 and Cdc42 gradient shaping

“…Cells were confined on round micropatterns to prevent cell shape polarity 48 and gradients of light with slopes ranging from 1x to 4x were applied (Fig 2a). As we could predict in a previous work 49 , recruitment of the cytoplasmic optogenetic partner CRY2 to the basal plasma membrane followed the stimulation signal with the addition of an exponential decaying tail of 5 µm characteristic length due to the diffusion of CIBN-CRY2 dimers at the membrane (Fig 2b). This allowed us to tune precisely the spatial distribution of desired GEFs and test the relationship between the activation input and the output in terms of GTPase activity.…”

“…Plasmids and molecular constructs ITSN-CRY2-mCherry was constructed as detailed previously 49 Stable cell lines were obtained using lentiviral infections: all lentiviruses were produced by transfecting pHR-or pLVX-based plasmids along with the vectors encoding packaging proteins (pMD2.G and psPax2) using HEK-293T cells. Viral supernatants were collected 2 days after transfection and HeLa cells were transduced at an MOI of 2.…”

Molecular flux control encodes distinct cytoskeletal responses by specifying SRC signaling pathway usage

“…Accordingly, with the same photostimulation frequency, lamellipodia were formed immediately after OS activation (1 min +/-0,5 min ), while invadosomes appeared later (15,6 min +/-10 min ). To test the relationship between the intensity of each OS flux on the observed cellular responses, we modulated the amplitude of each OS flux by applying light stimulation different frequencies, as previously described 12 . In marked contrast to lamellipodia induction which was apparent for all frequencies tested (from 33 to 2 mHz, Fig.…”

Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis

“…Currently, a few photoactivatable variants of Rho-family small GTPases are available: these optogenetic tools function either by promoting small GTPase oligomerization [44], or by activating or localizing GTPases or GTPase-specific GEFs to the plasma membrane 28, 29, 45, 46, 47, 48. These tools could allow us to understand where and when these proteins are required during cell locomotion.…”