Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway

Abstract

General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs.cytoskeleton ͉ photoswitch ͉ phytochrome ͉ signal transduction ͉ protein engineering B iological processes occur over a wide range of scales in length and time. The development of organisms depends on the formation of macroscopic tissues that grow over a period of days. Micrometer-size cells divide and differentiate on a timescale of minutes to hours. Nanometer scale signaling assemblies reorganize in seconds after cell stimulation. To understand biology, tools are required that enable observation and perturbation of processes on these different length-and timescales.

Visualizing dynamic interaction between calmodulin and calmodulin-related kinases via a monitoring method in live mammalian cells

“…1E, F). These results show clearly that constitutively active Cdc42 binds the CRIB domain of WASP, whereas a dominant-negative form of Cdc42 has very low binding-affinity (22,23). Thus, we confirmed that ECLIPSE represents an effective method for monitoring constitutive P-P interactions in live mammalian cells.…”

Algal photoreceptors: in vivo functions and potential applications

“…Fusion of an Arabidopsis cryptochrome with a DNA damage checkpoint protein even enabled the activation of the DNA damage signaling pathway in the absence of DNA damage (Ozkan-Dagliyan et al 2013). Also the light-inducible interactions between phytochrome and the phytochrome interaction factor 3 (PIF3) (Leung et al 2008;Tyszkiewicz and Muir 2008) or the LOV-containing F-box proteins FKF1 and its partner GIGANTEA (Yazawa et al 2009) have been used to regulate various cellular activities. A light-switchable gene promoter system should also be mentioned, which was constructed using an Arabidopsis phytochrome; this system allows for reversible control of gene expression with red and far-red light (Shimizu-Sato et al 2002).…”

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

“…Precise control and perturbation over cellular fate and activities such as neuronal firing, 19,20 activation of specific signaling cascades, [21][22][23] gene transcription, 24 protein production, 25 and modulation of proteinprotein interactions 26 in living cells and organisms have been achieved using light signals. These light-based methods can modulate individual cell behaviors at the subcellular level with high time resolution, allowing for quantitative dynamic information on cellular processes to be Knowing that the co-expression of YFP-Parkin and KillerRed-dMito is well tolerated by cells, we then tested the use of KillerRed protein for spatiallyencoded ROS generation in living cells using a well-established ROS sensor: 2,7-Dichlorodihydrofluorescein diacetate (DCF).…”