Optically inducible membrane recruitment and signaling systems

Abstract

Optical induction of intracellular signaling by membrane-associated and integral membrane proteins allows spatiotemporally precise control over second messenger signaling and cytoskeletal rearrangements that are important to cell migration, development, and proliferation. Optogenetic membrane recruitment of a protein-of-interest to control its signaling by altering subcellular localization is a versatile means to these ends. Here, we summarize the signaling characteristics and underlying structure-function of RGS-LOV photoreceptors as single-component membrane recruitment tools that rapidly, reversibly, and efficiently carry protein cargo from the cytoplasm to the plasma membrane by a light-regulated electrostatic interaction with the membrane itself. We place the technology-relevant features of these recently described natural photosensory proteins in context of summarized protein engineering and design strategies for optically controlling membrane protein signaling.

Figure 1.. Fully genetically encoded strategies for optically inducible intracellular signaling by integral membrane and membrane-associated proteins in mammalian cells.

Examples schematized are underlined. Abbreviations: POI = protein of interest. BD = binding domain, CIBN = cryptochrome interacting binding partner, CRY2 = cryptochrome, ER = endoplasmic reticulum, Gq* = activated Gα_q, IP₃ = inositol triphosphate, IP₃R = IP₃ receptor, LOV = light-oxygen-voltage, PLC = phospholipase C, Rec = membrane receptor.

Figure 2.. BcLOV4, a membrane-interacting photoreceptor and single-component system for dynamic membrane recruitment across diverse model systems.

(a) Schematized signaltransmission mode of membrane recruitment by a directly light-regulated and high-affinity electrostatic interaction with anionic phospholipids, which are largely enriched in the plasma membrane in mammalian cells. The interaction is inhibited by an N-terminal RGS (Regulator of G-protein signaling). A critical membrane binding site exists in a polybasic amphipathic helix in the linker region between the LOV sensor and a C-terminal DUF (domain of unidentified function). Inset: Schematized helix-membrane interaction and amphipathic helix sequence from BcLOV4, with known membrane binding motifs underlined. Blue = hydrophobic. Red = Basic. Green = Polar. (b) Pymol rendered model of predicted DUF structure by de novo energy minimization modeling in Rosetta. The DUF has a PAS-like mixed α-helix / anti-parallel β-sheet topology, suggesting that the LOV-DUF interaction is an evolutionary conserved PAS-PAS interaction. (c-e) Fluorescence micrographs of light-activated membrane recruitment in diverse contexts: (c) Expressed as FLAG epitope-tagged protein in mammalian HEK cells, fixed in the dark/light, and visualized by immunocytochemistry with Alexa488-conjugated anti-FLAG antibody. Scale = 10um. (d) Expressed in fungal S. cerevisiae cells and visualized by mCherry-fusion tag. Scale = 5 um. (e) In vitro as purified recombinant mCherry-tagged protein that binds lipid-stabilized water-in-oil emulsion interfaces. BcLOV4 does not bind purely zwitterionic interfaces of phosphatidylcholine (PC), but binds anionic interfaces with 20% phosphatidylserine (PS) that emulates mammalian plasma membrane inner leaflets. Scale = 25 um. Images modified from Reference 7. Copyright 2018 National Academy of Sciences.

Figure 3.. Spatially localized and optically induced cytoskeletal rearrangements by BcLOV4-derived opto-DHPH.

(a) Schematic overview of spatially precise opto-DHPH membrane recruitment and consequent activation of Cdc42 by the DHPH domain of the Intersectin GEF, which drives downstream actin polymerization. Cdc42 = Cell division control protein 42. DHPH = Diffuse B-cell lymphoma homology, Pleckstrin homology domain. GEF = Guanine exchange factor. Arp2/3 = Actin-related protein-2/3. WASp = Wiskott-Aldrich Syndrome protein. (b) Optically induced filopodia formation in HEK cells visualized by fluorescence imaging of a C-terminal mCherry tag. Only the blue light-illuminated (rectangle) regions show pronounced protusions, which are induced with very little stimulation (duty cycle = 0.8% = 0.5 sec per minute, λ = 450nm, 15 mW/cm²; spatially patterned by a digital micromirror device). Post-illumination times (i) 300 sec, (ii, iii) 500 sec Supplementary Movie 1 corresponds to cell (i).