Confocal laser scanning micrographs of tCXCR4/CatCh protein in NG108-15 cells.

<p><b>a,b.</b> 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 (similar to CXCR4 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.g002" target="_blank">Fig 2</a>). <b>c.</b> Internalization of anti-CXCR4 antibody after immunoreaction with tCXCR4/CatCh in cells exposed to 50 nM SDF1α. The fluorescence signal of anti-CXCR4 (top, red) and tCXCR4/CatCh (middle, cyan) co-localize to a great extend as can be seen from white areas in the overlay (bottom). Scale bars represent 10 μm.</p

SDF1-mediated CXCR4-internalization in NG108-15 cells.

<p><b>a.</b> Plot of cell capacitance (including stray capacitance) measured in whole cell configuration by patch-clamp technique against the membrane area. Cells were either not treated (red) or treated with 50 nM SDF1α (black) for >40 min. The membrane area was calculated from the measured cell diameter assuming a spherical shape of the cell. The specific cell capacitance (slope of the linear fit) decreases in presence of SDF1α. <b>b.</b> Confocal laser scanning micrographs of NG108-15 cells expressing CXCR4::eYFP in the presence of the inhibitor AMD3100 or the agonist SDF1α as indicated. While strong internalization is observed with SDF1α, only few vesicles are observed in presence of the inhibitor. <b>c.</b> Co-expression of CatCh::mKateA (red) and CXCR4::eYFP (cyan) in presence of 50 nM SDF1α. Note that after binding of SDF1α CXCR4 is internalized while CatCh mainly remains in the plasma membrane and is not directly affected by this growth factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.s003" target="_blank">S3 Fig</a>). Scale bars represent 5 μm.</p

Structure and function of the optochemokine tandem tCXCR4/CatCh.

<p>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. <b>a.</b> The CXCR4-protein was placed at the N-terminus, followed C-terminally by eYFP and the β-subunit of the rat HK-ATPase, to combine the intracellular C-terminus of eYFP with the extracellular N-terminus of ChR2 (aa 1–309). <b>b.</b> Schematic overview of the light-induced intracellular Ca²⁺ signaling mediated by tCXCR4/CatCh in an eukaryotic cell. In this optogenetic application the SDF1/CXCR4 signaling pathway (1–4) is used to induce internalization of tCXCR4/CatCh in endosomes. <b>1.</b> 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. <b>2.</b> 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. <b>3.</b> The endosome is trafficked into intracellular areas of the cell. <b>4.</b> 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.</p

Intracellular, light-induced Ca²⁺-signaling mediated by optochemokine tandem-endosomes.

<p><b>a,b.</b> CLSM micrographs of NG108-15 expressing the tCXCR4/CatCh(D156C) (a, cyan) or CXCR4::eYFP (b, cyan) stained with rhod2 (red). White arrows highlight such endosomes that express the respective membrane protein and are loaded with rhod2. Note that the rhod2 intensity decreases in tCXCR4/CatCh(D156C) to a larger extent than in CXCR4::eYFP endosomes. <b>c.</b> Time course of the rhod2 fluorescence of endosomes exhibiting tCXCR4/CatCh(D156C) (17 cells, mean s.e.m) or CXCR4 (21 cells) as identified by their fluorescence. Upon blue light activation rhod2-fluorescence non-linearly decreases in endosomes with tandem construct, while endosomes containing CXCR4 in their membrane show only moderate, nearly linear decrease of rhod2 fluorescence. <b>d.</b> Time course of rhod2 fluorescence in endosomes (filled symbols) and nuclei (empty symbols) as a measure for small changes in the cytosolic Ca²⁺ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.ref033" target="_blank">33</a>] of cells loaded with the membrane permeable rhod2AM-derivate (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.s011" target="_blank">S11 Fig</a>). 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.</p