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Title: Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites
Abstract

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein‐protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light‐activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light‐tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC.

This article was corrected on 25 July 2022. See the end of the full text for details.

Basic Protocol 1: Genetic engineering strategy for the generation of modular light‐activated protein dimerization units

Support Protocol 1: Molecular cloning

Basic Protocol 2: Cell culture and transfection

Support Protocol 2: Production of dark containers for optogenetic samples

Basic Protocol 3: Confocal microscopy and light‐dependent activation of the dimerization system

Alternate Protocol 1: Protein recruitment to intracellular compartments

Alternate Protocol 2: Induction of organelles’ membrane tethering

Alternate Protocol 3: Optogenetic reconstitution of protein function

Basic Protocol 4: Image analysis

Support Protocol 3: Analysis of apparent on‐ and off‐kinetics

Support Protocol 4: Analysis of changes in organelle overlap over time

 
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NSF-PAR ID:
10228392
Author(s) / Creator(s):
 
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
1
Issue:
3
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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