ARPE-19 cells connected by a nanotube containing mitochondria

<p>. (A) The bright field image shows two ARPE-19 cells connected by a membrane nanotube (arrow). (B) The corresponding fluorescence image of (A) shows JC-1 labelled mitochondria of cells (arrow). (C) The overlay of (A) and (B) shows the co-localization of nanotube with fluorescent labelled mitochondria (enlarged box). Scale bar, 20 µm.</p

The presence of membrane nanotubes in live ARPE-19 cells.

<p>(A) Two ARPE-19 cells are directly connected with a straight membrane tube (arrow). Scale bar, 20 µm. (B) High magnification of 3 separate straight membrane tubes (arrows) connecting two ARPE-19 cells. Inset 1 is an overview of the two connected cells. Scale bar, 20 µm. (C) A long nanotube (120 µm, arrow) connects ARPE-19 cells. The inset 2 shows the tube in a higher magnification. Scale bar, 20 µm. (D) A bulge (arrow) locates at the upper end of the tube. In time lapse videos the bulges moved along the tube from one cell to the other. Scale bar, 20 µm. (E) Thin membrane tubes (arrow) form and locate above the confluent layer of ARPE-19 cells after 48 h cultivation. The insets 1 and 2 show the tube in a higher magnification. Scale bar, 20 µm. (F) Scanning electron microscopy (SEM) shows the ultrastructure of a TNT between two ARPE-19 cells. Enlarged views (inset 1 and 2) show smooth membrane between the nanotube and the plasma membrane of the two connected cells. The TNT forms a straight connection between two cells and has a typical diameter of 250 nm. Arrows mark two focal thickenings of 1 µm indicating a possible transport of organelles or vesicles through the nanotube. The picture was taken with 20 kV. Scale bar, 10 µm.</p

Intercellular transfer of small molecules via a TNT of ARPE-19 cells.

<p>(A) The DIC image show a membrane tube (arrow) and two connected ARPE-19 cells (1, 2). (B-C) Cell 1 and Cell 3 were injected with a mixture of Lucifer yellow (green, B) and Texas red Dextran 10,000 (red, C). The fluorescence images taken 10 min after injection show that only Lucifer yellow was transmitted to cell 2 (green, B), while Texas red Dextran was not detected in cell 2 (dashed area in C). A typical result was shown from 5 independent experiments. Scale bar, 20 µm. (D) The overlay of DIC, the Lucifer yellow-positive cells and the Texas red Dextran positive cells, and the membrane tube (arrow in A).</p

Depolarisation signals spread between TNT-connected ARPE-19 cells.

<p>(A) The DIC image shows the mechanically stimulated cell (cell 1), TNT-connected cell (cell 2), TNTs (arrow) and control cells (cell 5 and 6). Scale bar, 20 µm. (B-E) The pseudo-coloured intensity images, generated by subtraction of the image before stimulation, show DiBAC4(3) fluorescence increase of cells in (A) at indicated times after mechanical stimulation. Note that the close associated cells pairs (cell 1 and 3, cell 2 and 4) are also electrically coupled. Colour bar indicates relative level of depolarisation. (F) Quantification of the relative membrane potential changes ( ΔF) of the stimulated cell (cell 1) and the TNT-connected cell (cell 2) as shown in (B-E). (G-I) The presence of Cx43 on TNTs in ARPE-19 cells. Cells were fluorescently labelled using WGA (green; G, I) and anti-Cx43 (red; H, I) and imaged by confocal microscopy. The enlarged images show distinct signals of Cx43 immunolabelling (arrowheads) at one end of a TNT (arrow). Scale bar, 50 µm.</p

The formation of nanotube between migrating ARPE-19 cells.

<p>(A-C) Two separated cells (cell 1 and 2) migrate towards each other and making contact after 32 min. Note the leading front (small arrows) and the trailing end with the nucleus. (D-F) Cell 1 and Cell 2 keep the contact over 60 min and start to diverge in (F). (G-I) Between the two cells a connection (arrow) still remains and becomes longer with the distance of the separating cells. (J-L) The connection between the cells becomes smaller and longer and has now a hair like shape (arrows in J and H). Finally, the connection form a tunnelling nanotube with a size of 70 µm (arrow in L). Scale bar, 20 µm.</p

Intercellular Ca²⁺ flux between TNT connected ARPE-19 cells.

<p>(A-J) After mechanical stimulation of cell 2 (arrow in B), the intracellular Ca²⁺ level increases in cell 2 (B-D). After 13.5 sec of delay, a transmission of Ca²⁺ via a TNT (arrow in A and K) reached the connected cell 1 (E-F). The high level of Ca²⁺ is now clearly seen in cell 1 (G-H) After 45 sec the Ca²⁺ level in both cells recover close to the normal level (I-J) Colour bar indicates relative level of Ca²⁺ measured by the Ca²⁺ indicator Fura-2 AM. Scale bar, 20 µm. (K) DIC image shows the two cells connected via a TNT (arrow). Concentration of Ca²⁺ was measured in the regions of interest (ROIs, circles): stimulated cell (green, ROI 2), TNT (blue, ROI 3), TNT-connected cell (red, ROI 4) and background (black, ROI 1). Scale bar, 20 µm. (L) Quantification of the relative Ca²⁺ concentration (ΔF) within the ROIs in (K) The background level of Ca²⁺ is subtracted in ROIs 2, 3 and 4. The Ca²⁺ level in cell 2 increase directly after manipulation and reaches a peak after 7 sec. At this time point the Ca²⁺ concentration of the TNT (ROI 3) starts to increase and reaches a peak 14 sec after manipulation. After this the Ca²⁺ level in cell 1 increase to a maximum 20 sec after manipulation. Both cells recover to normal level of Ca²⁺ (27–100 sec). A typical result was shown from 5 independent experiments.</p

Transfer of endocytic organelles between ARPE-19 cells.

<p>(A-F) Mixed populations of CTG (green) and DID (red) labelled ARPE-19 cells were cocultured for up to 24 hours and analyzed by fluorescence microscopy. As acceptor population, cells were labelled with CTG (A-B). As an organelle donor population, cells were labelled with DiD leading to red fluorescently labelled endocytic organelles (C-D). After populations were mixed, images were obtained 1 hour (A, C, E) and 24 hours (B, D, F) after seeding. The arrows show identical cells in panels B, D, F, respectively. Double-positive cells are indicative of organelle transfer (arrow in F). Scale bar, 20 μm. (G-H) FACS analyses revealed a significant (p < 0.001, t-test) transfer of 11.04% labelled endocytic organelles after 24 hours cultivation in comparison to 1 hour. A representative dot plot from the flow cytometry analysis is shown in (H).</p

Membrane nanotubes of ARPE -19 cells contained F-actin but not microtubules.

<p>Fluorescence image of ARPE-19 cells stained with phalloidin-TRITC for actin (A, B) anti-ß-tubulin (C, D) and nucleus (DAPI). Images represent the cytoskeleton in control (no treatment A, C, E) and after treatment with 15 µM nocodazole for 24 h (B, D, F). (A, B) Fluorescence image of F-actin. Actin fibres were visible in the cells and clearly seen is a straight connection, representing the TNT structure, between the cells (arrows). (C, D) Fluorescence image of ARPE-19 cells stained with mAb against β-tubulin revealed the lack of β-tubulin in the TNTs (arrows). (E, F) Merged pictures with actin (red), ß-tubulin (green) and nucleus (blue). Arrows mark TNTs. Scale bar, 20 µm.</p

Effect of blue light on Nox-2 and Nox-4 protein expression.

<p><b>A,</b>Western blot analysis showing increased Nox-2 and Nox-4 protein expression in OS following 1 h of blue light exposure (+) or in controls. The blots were first exposed to anti-Nox-2 or anti-Nox-4, respectively and then to anti-beta-Actin antibody as loading control. Images are representative of 5 experiments. <b>B,</b> Bar chart of densitometric analysis of Nox-2 and Nox-4 expression after 1 h and 12 h compared to control beta Actin. Bars represent the mean ± SEM from n = 5 experiments (* shows significance compared to control; *p<0.05 determined by ANOVA, post hoc Bonferroni test).</p

ROS production is reduced by the Nox inhibitor apocynin.

<p>Merged CM-H₂DCFDA fluorescence and bright field microscopy images of 40 µm vibratome sections of retinas are presented. After 1 h of blue light exposure, irradiated explants and respective non-irradiated explants (controls) were loaded with 25 µM CM-H₂DCFDA. In some cases, explants were pretreated with 4 mM apocynin during blue light exposure. The Nox inhibitor apocynin effectively reduced the levels of ROS production in the photoreceptors. The arrowheads mark the assumed border between IS and OS. The images are representative of 3 experiments. OS: outer segments; IS: inner segments; ONL: outer nuclear.</p