PLoS One. 2016 Oct 21;11(10):e0165344. doi: 10.1371/journal.pone.0165344.

Optochemokine Tandem for Light-Control of Intracellular Ca2+.

Feldbauer K1, Schlegel J2, Weissbecker J1, Sauer F2, Wood PG1, Bamberg E1,3, Terpitz U2.

1 Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt (Main), Germany.

2 Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University, Wuerzburg, Germany.

3 Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University, Frankfurt (Main), Germany.

 

Abstract

An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

PMID: 27768773

 

Supplement

The optochemokine tandem

Ca2+ is a key signal in cell regulation, modulating the activity of a plenitude of sensitive proteins. Nevertheless, there is no report on intracellularly acting light-gated calcium switches. In this study an optochemokine tandem was developed to control the release of Ca2+ from endosomes into the cytosol by light and beside that, to analyse the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology (Fig. 1).

The structural similarity of GPCRs and rhodopsins both exhibiting 7-transmembrane domains allowed for re-engineering a previously constructed rhodopsin tandem [1] to combine the light-gated Ca2+-permeable cation channel ChR2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. Within the tandem protein the membrane proteins are linked by a yellow fluorescent protein (YFP) and the beta-subunit of the H,K-ATPase. The optochemokine tandem tCXCR4/CatCh was expressed in different mammalian cell lines and YFP fluorescence was localized in the cytoplasmic membrane for both mammalian cell types. Importantly, the protein was expressed in one entity and not fragmented during the expression/trafficking process.

 

 

Fig 1. Structure and function of the optochemokine tandem tCXCR4/CatCh. This optophysiological tool combines two features in one functional unit, the internalization after the binding of stromal cell-derived factor 1 (SDF1) and the light-gated influx of cations, including Ca2+, via CatCh upon illumination. a. The CXCR4-protein was placed at the N-terminus, followed C-terminally by eYFP and the β-subunit of the rat H+,K+-ATPase, to combine the intracellular C-terminus of eYFP with the extracellular N-terminus of ChR2 (aa 1-309). b. Schematic overview of the light-induced intracellular Ca2+ signaling mediated by tCXCR4/CatCh in a eukaryotic cell. In this optogenetic application the SDF1/CXCR4 signaling pathway (1-4) is used to induce internalization of tCXCR4/CatCh in endosomes. 1. tCXCR4/CatCh is expressed heterologously in the mammalian cell and trafficked towards and integrated into the plasma membrane. There, the chemokine receptor CXCR4 will be activated by its endogenous ligand SDF1. 2. Upon SDF1-activation tCXCR4/CatCh is internalized. As the Ca2+ concentration in the cell environment is four magnitudes higher than within the cell, also the calcium concentration in these endosomes is considerably higher than in the cytosol. 3. The endosome is trafficked into intracellular areas of the cell. 4. Upon illumination with blue light, CatCh will open and release Ca2+ ions into the cytosol, resulting in a local intracellular increase of Ca2+. This transient Ca2+ signal might be used for triggering Ca2+-dependent physiological processes by light. N = nucleus, M = mitochondrion.

 

The optochemokine tandem preserves protein function and is internalised via the endocytic SDF1/CXCR4 signalling pathway

CXCR4 was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. The ability of the tandem to be internalized upon addition of SDF1α was shown by CLSM and Patch-clamp analysis (Fig. 2). The kinetics of internalization was followed electrophysiologically via the current amplitude of the CatCh signal. In addition using a CXCR4 specific antibody labelled with red-fluorescent dye it was shown that heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signalling pathway. Immediately after the treatment, immuno-labelled CXCR4 was only found in the cytoplasmic membrane. After several hours of incubation it was also localized in a number of intracellularly located vesicles. These intracellular vesicles represent endosomes as shown by staining with endosomal marker Rab5a-RFP and Alexa647-labeled transferrin.

 

 

Fig. 2. Preservation of protein function in the optochemokine tandem a,b. Confocal laser scanning micrographs of tCXCR4/CatCh protein in NG108-15 cells. Z-stack overlay of a typical tCXCR4/CatCh cell either treated with 50 nM SDF1α for 45 min (a) or with AMD3100 (b). The tandem construct is internalized by the action of SDF1α. Scale bars represent 10 μm. c,d. Patch-clamp investigation of the optochemokine tandem in cell attached configuration at 34°C. The pipette solution was supplemented with 50 nM SDF1α. c. Typical current trace recorded at an applied membrane potential of -100 mV showing the light-dependent signal of tCXCR4/CatCh directly after the sealing process and 30 min later. During that time the cell was illuminated every two minutes for 100 ms. d. Time course of the relative tCXCR4/CatCh current in presence of SDF1α (square, 5 cells) or SDF1α and the inhibitor AMD3100 (triangle, 3 cells), and CatCh in presence of SDF1α (circle, 4 cells). Mean values are given, bars represent the standard error.

 

Optochemokine tandem endosomes release calcium into the cytosol upon illumination

The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dye rhod2(AM) showing an increase of intracellular Ca2+ in response to light (Fig. 3). In the Ca2+ imaging experiments a variant of tCXCR4/CatCh was used, referred to as tCXCR4/CatCh(D156C). This mutant provides prolonged open state [2] and allowed for triggering tCXCR4/CatCh(D156C) with very short light pulses with a scanning laser during CLSM measurements. In order to discover whether a release of Ca2+ from the endosomes could notably change the Ca2+ concentration in the cytosol, imaging experiments were performed with NG108-15 cells either expressing CXCR4::eYFP or tCXCR4/CatCh(D156C) tandem loaded with 2 ÂµM rhod2-AM. Indeed, in tCXCR4/CatCh(D156C) endosomes a decrease of fluorescence was observed whereas the fluorescence in the nucleus increased. In contrast, the CXCR4 cells exhibited a decrease of fluorescence in nucleus as well as endosomes.

 

 

Fig 3. Intracellular, light-induced Ca2+-signaling mediated by optochemokine tandem-endosomes. a. CLSM micrographs of NG108-15 expressing the tCXCR4/CatCh(D156C) stained with rhod2 (red). White arrows highlight such endosomes that express the optochemokine tandem and are loaded with rhod2. b. Time course of rhod2 fluorescence in endosomes (filled symbols) and nuclei (empty symbols) as a measure for small changes in the cytosolic Ca2+ of cells loaded with the membrane permeable rhod2AM-derivate. Note that in tCXCR4/CatCh(D156C) cells (red, n = 7, mean + s.e.m) an increase in cytosolic Ca2+ concentration was observed while only fluorescence decrease was observed in CXCR4-expressing cells (black, n = 8, mean + s.e.m.). All rhod2-experiments were performed in absence of extracellular Ca2+. Scale bars represent 10 μm.

 

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