197 Oral - Techniques and Technology Session
Saturday April 09, 5:15 PM - 5:30 PM

Authors:
Yineng Xu; Bei Wang; Chun han

Affiliation: Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University

Keywords:
q. other (optogenetics); e. intracellular transport

During animal development, the same molecular event can result in drastically different consequences depending on when and where it occurs. Thus, the spatial and temporal properties of signaling inputs and gene activity largely determine the biological outcomes. Spatiotemporal regulation is particularly important for neuronal development and function: Spatially, neurons occupy broad domains and may incur distinct signaling events at different cellular compartments as a result of unique interactions with the surrounding microenvironment. Temporally, neurons develop through distinct stages and require series of molecular events that occur in particular sequences. However, widely used methods for studying gene function, such as mutations, RNAi, and CRISPR, lack the spatiotemporal resolution required for dissecting fine developmental mechanisms. In contrast, light-controllable modules hold great promise for precise and instant manipulation of molecular events in cells. Towards this goal, we developed OptoTrap, a light-dependent protein trapping system, which builds on two light controllable systems—Cry2olig and magnets. This system forms large protein aggregates under blue light and can trap proteins that are tagged by GFP or split GFP. We generated several variants of this system for diverse applications in neurons and epithelial cells and characterized their association and dissociation properties. We demonstrate that this system can effectively trap GFP-tagged endogenous proteins of diverse sizes, subcellular locations, and functions. Functionally, light-dependent protein trapping of the septate junction protein Nrg in epithelial cells caused gain-of-function (GOF) and cell shrinkage, suggesting Nrg activation through clustering. In contrast, optogenetic trapping of kinesin heave chain (Khc) in somatosensory neurons caused effective loss-of-function (LOF), resulting in disruption of microtubule-dependent transport and dendrite reduction. Similarly, trapping of Nmnat in neurons caused dendrite reduction, likely due to disruptions of Nmnat’s chaperon function. OptoTrap allows us to fine tune protein manipulation for graded phenotypes by changing illumination conditions, and thus offers great flexibility for dissecting protein functions in neuronal development. This toolkit should be applicable to broader developmental stages and diverse cell types for studying development mechanisms.