Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26 - PubMed (original) (raw)

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

K Paemeleire et al. Mol Biol Cell. 2000 May.

Free PMC article

Abstract

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

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Figures

Figure 1

A schematic of the digital high-speed microscope. The specimen is either excited with 488 or 514 nm wavelength light from an argon–krypton laser (visible laser) or by 386 nm wavelength light from an argon laser (UV laser). The laser beams pass through beam expansion and collection optics to provide wide-field illumination. Shutters operated by a Pentium PC control the exposure to the lasers. The excitation beams are directed to the specimen by two dichroic mirrors (DM1 400 nm; DM2 505 nm). The specimen is viewed with a 60× NA 1.4 oil objective lens with the numerical aperture set at 1.4. The fluorescence emitted from the specimen is filtered by a 510 nm long-pass emission filter and is captured by a high-speed, 128 × 128 pixel frame transfer CCD camera. The 12-bit output is read out through four ports and stored on two high-speed data capture boards, each with 4 megabytes of dual-ported RAM. When the RAM is filled with 200 images, the data are transferred to the hard disk in a Pentium PC. This PC also controls the advancement of the piezo-electric translator (PZT) when images at multiple focal planes throughout the specimen are acquired. Images are transferred from the Pentium PC to a Silicon Graphics Imaging (SGI) facility for image deconvolution and analysis.

Figure 2

Cellular localization of Cx43–GFP observed by confocal microscopy. (A) A culture of Cx43–GFP-transfected HeLa cells was excited with 450–490 nm light and viewed at 510–520 nm to reveal the location of GFP. Discrete fluorescence plaques, characteristic of gap junctional staining, were observed along the boundary between two cells (arrows). Fluorescence was also detected in the perinuclear region and most likely represents Cx–GFP being processed through the ER. (B) The same area of cells as shown in A stained with Cy3-conjugated antibodies to Cx43 and illuminated at 540–550 nm and viewed at 580–590 nm. A similar staining pattern was observed. (C) The images in A and B were digitally superimposed. The combination of green and red fluorescence results in yellow fluorescence and demonstrates that the membrane plaques and cytoplasmic areas represent sites of colocalization of GFP and Cy3 fluorescence and confirms that GFP fluorescence indicates the location of expressed Cxs. The plaques in C have a green–yellow appearance caused by the larger contribution of green as compared with red. The cells shown were selected for the large number of plaques expressed. For Ca2+ experiments, cells with fewer plaques were used. Image width = 60 μm.

Figure 3

Western blot analysis of Cx43–GFP expressed by mammalian cells. HeLa and COS-7 cells were transiently transfected with 10 μg Cx43–GFP or wild-type Cx43 cDNA, respectively, and were harvested. Cells were dissolved in SDS and analyzed by SDS-PAGE and Western blotting using an antibody to either Cx43 (Gap33) or GFP. Lane 1: control (mock-transfected HeLa cells). Lane 2: HeLa cells expressing Cx43–GFP stained with GFP antibodies. Lane 3: HeLa cells expressing Cx43–GFP stained with Cx43 antibodies. Two major bands are visible. The band at ∼71 kDa represents the Cx43–GFP (lanes 2 and 3), whereas the band at ∼65 kDa (lane 3) probably represents proteolytic products of Cx43. Lane 4: COS-7 cells expressing wild-type Cx43 stained with Cx43 antibodies. A broad band representing phosphorylated isoforms of Cx43 is evident at ∼48–50 kDa. The numbers on the right represent the location and molecular weight (kilodalton) markers.

Figure 4

Electrical coupling between isolated pairs of Cx43–GFP-transfected HeLa cells. The left image illustrates a pair of HeLa Cx43–GFP cells impaled with microelectrodes (M) with two gap junctional plaques (white structures or dots) that formed between these cells (white arrows). The image was generated by digitally combining the phase-contrast image of the cells with the fluorescence image of the Cx43–GFP. Cell 1 (C1) was injected with 1 nA square current pulses of 200 ms duration at a frequency of 2Hz (I1). The resulting changes in membrane potential in cell 1 (V1) are synchronous with the changes in membrane potential (V2) of cell 2 (C2), indicating electrical coupling between both cells. The coupling ratio (V2/V1) is ∼5%.

Figure 5

The effects of mechanical stimulation in HeLa control cells using digital video microscopy. The morphology of the cell culture is indicated by the fluorescence of cells loaded with fura-2 (left), and the changes in [Ca2+]i are indicated by the pseudocolored images (right) (A) A single cell (∗, white arrow) was gently stimulated with a glass micropipette. Although this stimulus produced Ca2+ changes in the stimulated cell, it did not induce propagated Ca2+ changes. (B) Stronger mechanical stimulation of another cell in a different area, induced by distorting the cell to a great extent, initiated propagated Ca2+ changes that took the form of a radiating Ca2+ wave. (C) The propagating Ca2+ changes induced by strong mechanical stimulation were abolished by the presence of 40 IU/ml apyrase in the extracellular solution. The time that each image was recorded after mechanical stimulation is shown in the lower right of each image. Changes of fura-2 fluorescence corresponding to changes in [Ca2+]i are represented according to a pseudocolor scale bar in arbitrary values.

Figure 6

Intracellular Ca2+ changes of HeLa control cells in response to ATP. (A) The dose–response curve of the number of HeLa control cells showing an increase in [Ca2+]i in response to a range of ATP concentrations. Each data point represents the mean (±SE) of three separate experiments and >200 cells. (B) The relative changes in [Ca2+]i of a HeLa control cell represented by fluo-3 fluorescence in a 2.3 × 2.3 μm (7 × 7 pixels) perinuclear area with respect to time during exposure to 10 μM ATP, which was applied by solution exchange. Before global [Ca2+]i increases, a much smaller and transient [Ca2+]i change or Ca2+ puff (black arrow) could be observed with digital high-speed microscopy. The Ca2+ puff represents a ∼10% change in fluo-3 fluorescence.

Figure 7

The effect of extracellular apyrase on changes of [Ca2+]i of HeLa control cells in response to agonists. (A) In the absence of apyrase, fura-2–loaded HeLa control cells respond to 50 μM ATP, added at t = 0 and present throughout the experiment, with an initial Ca2+ increase followed by Ca2+ oscillations. Time scale bar is indicated at the bottom of the figure. (B) In the presence of 40 IU/ml apyrase, the [Ca2+]i increase in response to 50 μM ATP is completely abolished. (C) In the absence of apyrase, HeLa control cells also respond to the continuous presence of 100 μM histamine with an initial [Ca2+]i peak followed by oscillatory changes of [Ca2+]i. (D) In the presence of 40 IU/ml apyrase, the [Ca2+]i responses to 100 μM histamine are unchanged. The changes in [Ca2+]i are represented in arbitrary values.

Figure 8

Digital high-speed microscopy of Ca2+ waves mediated by an extracellular messenger in HeLa control cells. A and B show the reconstructed ER-Tracker signal (green) in 1-μm-thick slices through the (A) top of the cells and the (B) middle of the cell. The ER surrounds the dark nucleus. No GFP-like fluorescence was detected in these untransfected cells. (C1–C6) A Ca2+ wave was evoked, in the absence of apyrase, by mechanically stimulating the cell (∗) on the left (B). The propagation of [Ca2+]i changes in the neighboring cell was initiated by an increase in [Ca2+]i (white arrow) that was correlated to the supranuclear ER (A, arrow). These initial Ca2+ changes were followed by an intracellular Ca2+ wave that propagated toward the stimulated cell. Recording times after mechanical stimulation are indicated in the bottom right of each image. The white dotted line represents the cell boundary. Changes of fluo-3 fluorescence at 488 nm excitation, corresponding to changes in [Ca2+]i, are represented in pseudocolor, according to a scale bar. The color scale was chosen to enhance the differentiation of small Ca2+ changes. The color scale numbers represent the minimal, saturating, and maximal percentage changes.

Figure 9

Digital video microscopy of a mechanically induced intercellular Ca2+ wave in HeLa cells expressing Cx43–GFP in the presence of 40 IU/ml apyrase. (A) A phase-contrast image of Cx43–GFP HeLa cells superimposed with the corresponding fluorescent image of Cx43–GFP showing plaques of GFP-labeled gap junctions (arrows, white patches) that formed between individual cells. To discard irrelevant background information and before superimposition, the fluorescent GFP image was processed to set the pixel values to black if less than a selected threshold value. (B) An outline of the cell boundaries (gray lines) as well as the positions of the Cx43–GFP plaques (red dots, arrows) were mapped from the phase-contrast and fluorescence images. (C and D) Digital video microscopy of the sequential changes in [Ca2+]i induced by mechanically stimulating a single Cx43–GFP HeLa cell (white arrow). The time each image was recorded is indicated at the bottom right. The changes in [Ca2+]i are mapped onto the cell outlines (the expanding gray shaded zone) to correlate the propagation route of the intercellular Ca2+ wave with the location of the Cx43–GFP. Close inspection reveals that a Ca2+ wave only propagates at sites where two cells are coupled by a gap junction (red dots). Pseudocolor bar represents changes in [Ca2+]i, as measured from the changes in fura-2 fluorescence, in arbitrary values.

Figure 10

Digital high-speed microscopy of intercellular Ca2+ waves in Cx43–GFP HeLa cells. The left top panel shows the distribution of the endoplasmic reticulum and gap junctions reconstructed from ER-Tracker fluorescence (green) and the Cx43–GFP signal (red), respectively, in a 1-μm-thick slice through the cells. A white dotted line represents the cell boundary. The pseudocolored time series of images (right), numbered 1–6, represent the relative changes in fluo-3 fluorescence at the times indicated (bottom right) after mechanical stimulation of a single cell (∗) in the presence of extracellular apyrase (40 IU/ml). (1, 2) The Ca2+ wave propagates to fill the entire cell in the top left. (3) A small change in [Ca2+]i is observed in the adjacent cell at the site of the Cx–GFP gap junction. (4–6) The changes in Ca2+ continue to spread out from the gap junction. The gap junction remains blue because the GFP fluorescence dominates any Ca2+-based fluorescence changes, and therefore the ratio of ΔF/F does not change. The color scale was chosen to enhance the visualization of small [Ca2+]i changes, and numbers represent minimal, saturating, and maximal percentage changes.

Figure 11

Digital high-speed microscopy of a Ca2+ wave induced by mechanical stimulation of a single cell in Cx26–GFP HeLa cells. (A) Ca2+ changes in two neighboring cells are analyzed in the vicinity of two gap junctions. The locations of the gap junctions are indicated by the arrows and are derived from B. The color scale was chosen to enhance the visualization of small [Ca2+]i changes, and the numbers represent minimal, saturating, and maximal percentage changes. (B) The corresponding fluorescent Cx26–GFP image indicates the location of the two gap junctions and the location of the four analysis points (marked with +, area = 2.3 × 2.3 μm) that were used to follow changes in [Ca2+]i shown in C. The superimposed white line indicates the location of the adjacent plasma membranes. (C) The time course of [Ca2+]i changes, represented as percentage of relative change of fluo-3 fluorescence, on either side of the two gap junctions at locations 1–4 indicated in B. These traces indicate that the delay times at the site of the gap junctions are very small. The [Ca2+]i increase on the distal side of the gap junction begins immediately as the [Ca2+]i is elevated on the near side of the gap junction but takes several seconds to reach a maximal level.

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Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26 - PubMed (original) (raw)