Curated Optogenetic Publication Database

Abstract: TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Abstract: Chronic pain is a prevalent problem that plagues modern society, and better understanding its mechanisms is critical for developing effective therapeutics. Nerve growth factor (NGF) and its primary receptor, Tropomyosin receptor kinase A (TrkA), are known to be potent mediators of chronic pain, but there is a lack of established methods for precisely perturbing the NGF/TrkA signaling pathway in the study of pain and nociception. Optobiological tools that leverage light-induced protein-protein interactions allow for precise spatial and temporal control of receptor signaling. Previously, our lab reported a blue light-activated version of TrkA generated using light-induced dimerization of the intracellular TrkA domain, opto-iTrkA. In this work, we show that opto-iTrkA activation is able to activate endogenous ERK and Akt signaling pathways and causes the retrograde transduction of phospho-ERK signals in dorsal root ganglion (DRG) neurons. Opto-iTrkA activation also sensitizes the transient receptor potential vanilloid 1 (TRPV1) channel in cellular models, further corroborating the physiological relevance of the optobiological stimulus. Finally, we show that opto-iTrkA enables light-inducible potentiation of mechanical sensitization in mice. Light illumination enables nontraumatic and reversible (<2 days) sensitization of mechanical pain in mice transduced with opto-iTrkA, which provides a platform for dissecting TrkA pathways for nociception in vitro and in vivo.

Abstract: Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.

Abstract: Receptor tyrosine kinases (RTKs) play crucial roles in human health, and their misregulation is implicated in disorders ranging from neurodegenerative diseases to cancers. The highly conserved mechanism of activation of RTKs makes them especially appealing candidates for control via optogenetic dimerization methods. This work offers a strategy for using the improved Light-Induced Dimer (iLID) system with a constructed tandem-dimer of its binding partner nano (tdnano) to build light-activatable versions of RTKs. In the absence of light, the iLID-RTK is cytosolic, monomeric and inactive. Under blue light, the iLID + tdnano system recruits two copies of iLID-RTK to tdnano, dimerizing and activating the RTK. We demonstrate that iLID opto-iTrkA and opto-iTrkB are capable of reproducing downstream ERK and Akt signaling only in the presence of tdnano. We further show with our opto-iTrkA that the system is compatible with multi-day and population-level activation of TrkA in PC12 cells. By leveraging genetic targeting of tdnano, we achieve RTK activation at a specific subcellular location even with whole-cell illumination, allowing us to confidently probe the impact of context on signaling outcome.

Abstract: Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of BAR domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.