Figure 2. GIV and S245-phosphorylated GIV localize to cell-cell junctions.

(A) MDCK cells were grown on glass cover slips, exposed or not to energetic stress (glucose deprivation for 30 and 120 min), and subsequently fixed and stained for total (t) GIV (red) and DAPI (blue; nuclei) and analyzed by confocal microscopy. Representative confocal images are shown. In domed monolayers at basal condition, GIV is absent from cell-cell junctions. However, GIV is detected at cell-cell junctions (arrowheads) after energetic stress induced by glucose deprivation. Scale bar = 10 μm. (B) MDCK cells were grown on glass cover slips, at various stages during their growth phase: from single-cells (top), to non-confluent monolayers (middle), to confluent domed monolayers (bottom). They were fixed and stained for Occludin (a TJ marker; green), pS245-GIV (red) and DAPI (blue; nuclei) and analyzed by confocal microscopy. Confluent monolayers were either maintained in complete medium in the presence of glucose (+ Glucose), or subjected to energetic stress by glucose deprivation (No Glucose) or calcium deprivation (EGTA). Representative fields are shown on the left. RGB plots, generated using ImageJ on the right assess the degree of colocalization between pS245-GIV and Occludin along the lines in the corresponding images during cell polarization are shown in the middle. Schematics summarizing the staining pattern in each condition are shown on the right. Scale bar = 25 μm.

DOI: http://dx.doi.org/10.7554/eLife.20795.003

Figure 2—figure supplement 1. Active AMPK localizes to specialized regions of the TJs called tricellular TJs exclusively after energetic stress.

Fully polarized domed monolayers of type II MDCK cells at steady-state were exposed to energetic stress induced by glucose deprivation for indicated duration of time prior to fixation. Fixed cells were stained for phospho(p) AMPK (red), Occludin (green) and DAPI (nuclei, blue) and analyzed by confocal microscopy. Representative images are displayed. Active (phospho)-AMPK was not detected at steady-state (upper left); however, when domed monolayers are subjected to 6 hr of energetic stress active, AMPK was detected exclusively at the TJs of tricellular contact (tTJs), as determined by colocalization with Occludin where three or more cells come in contact (Furuse et al., 2014). With prolonged energetic stress (i.e., 12 hr of glucose starvation), tTJs were the first to disassemble and were associated with a blush of active AMPK. Longer duration of glucose starvation was associated with loss of signal for active AMPK (not shown).

DOI: http://dx.doi.org/10.7554/eLife.20795.004

Figure 2—figure supplement 2. AMPK is activated when type II MDCK cells are exposed to energetic stress or low-calcium growth conditions.

Type II MDCK cells grown to domed confluence on culture dishes in the presence of normal calcium and glucose (left lane), were exposed to low calcium [+ EGTA] but normal glucose conditions (middle lane), or to energetic stress by depriving them of glucose (-) while maintaining normal calcium (right lane) for 6 hr prior to lysis. Equal aliquots of whole cell lysates were analyzed for total (t) and phospho (p) AMPK and actin (loading control) by immunoblotting. Representative blots are shown (n = 5–7 times for EGTA and glucose starvation, respectively, alongside various experiments using these stimuli during the course of this project).

DOI: http://dx.doi.org/10.7554/eLife.20795.005