Optogenetic Regulation of Membrane-Associated Proteins

Abstract

Optogenetics has emerged as a powerful tool for controlling cellular processes with light. Two optogenetic approaches were designed using the flavin-containing photoreceptor Cryptochrome 2 (Cry2) from Arabidopsis thaliana that control protein function and target distinct cellular events. These approaches focus on the membrane-bound receptor tyrosine kinase, Eph receptor, and a membrane-associated protein, Inverse Bin, Amphiphysin, and Rvs (I-BAR). Eph receptors are crucial for cell-cell communication, proliferation, and migration. A light-activated EphA1 receptor (EphA1Cry2) was created by fusing Cry2 to the intracellular domains of EphA1 receptor, enabling blue light-dependent oligomerization and auto-phosphorylation of the receptor. Key cell signaling targets were identified and characterized in response to light-stimulated EphA1Cry2 activation. The optogenetic regulation of Eph receptor RTK signaling without the need for external ligand binding promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions. The second developed optogenetic approach involved membrane remodeling light-gated tools called CRY-BARs; this optogenetic system combines the membrane-binding affinity of the I-BAR domain, a negative membrane-curvature generating domain, with the homo-oligomerizing capability of Cry2. Using immortalized cell lines and primary neuron cultures, the CRY-BAR optogenetic tool evokes membrane dynamic changes associated with cellular activity. Moreover, ezrin was shown to act as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of CRY-BARs' switch function. CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells. Overall, these optogenetic approaches offer precise spatial and temporal control over cellular signaling and membrane dynamics, opening up new possibilities for studying cellular functions and developing novel therapeutic strategies.