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. 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.

Keywords: light-dependent dimerization; optogenetics; organelle contacts; protein reconstitution; protein-protein interaction.

Figure 1:. Optical dimerization systems for the light-dependent induction of protein heterodimerization used in mammalian cells and their characteristics.

Light-dependent protein heterodimerization pairs can be activated by wavelengths of light that span all the visible spectrum including ultraviolet, blue, cyan, red and far-red wavelengths. Every pair has different switch on- and off-kinetics that can be as fast as milliseconds or last several hours. Abbreviations: UV Resistance locus 8 (UVR8), Constitutively Photomorphogenic 1 (COP1), Flavin Kelch-repeat F-box1 (FKF1), GIGANTEA (GI), Cryptochrome 2 (CRY2), Cryptochrome Interacting Basic helix–loop–helix1 (CIB1), Tunable light-controlled interacting protein tags (TULIPs), improved Light Inducible Dimer (iLID), Phytochrome B (PhyB), Phytochrome Interacting Factor (PIF), Bacteriophytochrome P1 (BphP1), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), phycocyanobilin (PCB).

Figure 2:. Domain organization of soluble or membrane anchored proteins containing components of light-sensitive dimerization pairs.

A) In soluble protein, the component of the dimerization pair (dimerization component, DC) of choice can be fused either at the N- or C-terminus of the protein of interest (POI) and the fluorescent protein (FP) selected to visualize the construct. It is often preferable to insert the component of the dimerization pair between the fluorescent protein and the protein of interest in order to localize the protein of interest in closer proximity to the binding partner upon light-dependent dimerization. In order to preserve the full functionality of some proteins, N- or C-termini cannot be tagged. In this case it is recommended to introduce the component of the dimerization pair in an intracellular loop (example not illustrated). B) Domain organization of membrane anchored proteins. Organelle-targeting sequences (OTS) are protein sequences encoding transmembrane segments or protein domain that are post-translationally modified with lipid anchors and therefore capable of positioning fusion proteins on the cytosolic surface of subcellular compartments. Each OTS must be specifically encoded at the N- or C-terminus of the protein and followed by either dimerization component or fluorescent protein. In both, soluble and membrane anchored proteins, linker regions between each domain must be optimized choosing flexible or stiff linkers of the appropriate length.

Figure 3:. Dark containers for optogenetic samples

Frontal (A) and top (B) view of the light-proof, gas-permeable, double walled, black, plastic jar with a dome lid used to store the cells after transfection with light-sensitive proteins.

Figure 4:. Soluble protein recruitment to intracellular organelles

A. Schematic representation of the method used to induce soluble protein recruitment to intracellular membranes. One of the dimerization components (DC) of the optical dimerization pair is anchored to the membrane of an intracellular compartment (bait) with an appropriate organelle targeting sequence (OTS) and fused to a fluorescent protein (FP) to monitor its intracellular distribution. The other dimerization component is cytosolic (prey) before light stimulation (pre-excitation). This protein is recruited to the membrane of the organelle where the bait is expressed upon blue-light irradiation and subsequently released when the activating light is turned off (recovery). B. Representative example of reversible light-dependent recruitment of eMagB-TagRFP-T (prey) to mitochondria in HeLa cells expressing the mitochondrial Mito-EGFP-eMagA (bait). Confocal images. Scale bar: 10 μm. C. Representative example of reversible light-dependent recruitment of eMagB-TagRFP-T (prey) to the endoplasmic reticulum in U-2 OS cells expressing the bait ER-eMagA-EGFP. Confocal images. Scale bar: 2 μm.

Figure 5:. Light-dependent induction of organelles contacts

A. Graphical representation of the strategy used to elicit contacts formation/expansion between membranes of intracellular organelles. Constructs encoding both components of the dimerization pair (DC) were fused to a fluorescent protein (FP) and to an organelle-targeting sequence (OTS) to drive expression in specific organelles (Organelle A or B). The dimerization components in the lit state can interact one to each other bringing the membranes of the two organelles in close proximity. HeLa cells expressing the ER-Mitochondria (B) or ER-Lysosomes (C) light-dependent tethering systems, respectively. Cells shown before, during, and after blue-light illumination. The overlap between the membranes of the two organelles increased during illumination, as illustrated by the white color in the fluorescence micrographs. Scale bar: 2 μm (B), 5 μm (C).

Figure 6:. Light-dependent reconstitution of protein function

A. VAP is an integral membrane protein composed of a cytosolic major sperm protein (MSP) domain (which binds FFAT motif-containing proteins), a coiled-coil domain (CCD), and a C-terminal membrane anchor (TM). OSBP1 has an N-terminal PH domain that preferentially binds PI4P, an internal FFAT motif, and a C-terminal ORD domain which binds in a competitive way PI4P and cholesterol. In the experiment shown in the figure, OSBP represents the endogenous protein. B. Schematic representation of reconstitution of a split VAP on the ER membranes using eMags (Opto-VAP). FP: fluorescent protein tags, DC: dimerization component. The N-terminal portion of VAPB (VAPB(1–218)) fused to TagRFP-T and to eMagB (prey) was expressed together with ER-anchored eMagA fused to EGFP (bait) and with the PI4P reporter iRFP-P4C in HeLa cells. Upon blue-light illumination, eMags heterodimer formation results in reconstitution of the tether, allowing the ORD domain of endogenous OSBP to transfer PI4P to the ER for degradation, leading to PI4P loss from Golgi apparatus membranes. C. The image shows the perinuclear area of a HeLa cell (where the Golgi apparatus is localized) expressing TagRFP-T-MSP(VAPB(1–218))-eMagB, ER-EGFP-eMagA and the PI4P reporter iRFP-P4C, showing that blue-light dependent Opto-VAP reconstitution results in the recruitment of the prey to the ER and concomitant dissociation of iRFP-P4C from the Golgi apparatus, reflecting PI4P loss. Scale bar: 3 μm.

Figure 7:. Analysis of cytosolic fluorescence redistribution and apparent on- and off-kinetics

A. Representative example of reversible light-dependent recruitment of eMagB-TagRFP-T (prey) to the endoplasmic reticulum in HeLa cells expressing ER-eMagA-EGFP (bait). The red circle illustrates a representative ROI drawn in a cytosolic portion of the cell in the fluorescence channel corresponding to eMagB-TagRFP-T. Confocal images. Scale bar: 2 μm. B. Graph showing the depletion of cytosolic pool of prey from the cytosol due to its recruitment to the ER (n = 13 cells, from 3 independent experiments). C. Isolated recruitment and plateau with single exponential fit to quantify the apparent on-kinetic of the dimerization system. D. Isolated recovery and plateau with single exponential fit to quantify the apparent off-kinetic of the dimerization system.

Figure 8:. Analysis of changes in organelles overlap over time

A. HeLa cells expressing ER-mCherry-eMagA (green) and eMagB-iRFP-Mito (magenta) before, during, and after blue-light illumination. The lower panels show the superimposition of regions of interest comprising the mitochondrial structure superimposed to the ER fluorescent channel. The overlap between the membranes of the two organelles increased during illumination, as illustrated by the white color in the fluorescence micrographs. Scale bar: 2 μm. B. Graph showing changes in ER and mitochondria overlap upon light-dependent induction of contacts between the two organelles (n = 5 cells). Scale bar: 2 μm.