First published online 19 September 2006
doi: 10.1242/jcs.03181
Journal of Cell Science 119, 4176-4186 (2006)
Published by The Company of Biologists 2006
Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells
Andreas Schmid1,
Ge Bai1,
Nathalie Schmid1,
Manuela Zaccolo2,
Lawrence E. Ostrowski3,
Gregory E. Conner1,4,
Nevis Fregien1,4 and
Matthias Salathe1,*
1 Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, 1600 NW 10th Avenue, RMSB 7063, Miami, FL 33136, USA
2 Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine, Padova, Italy
3 Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC, USA
4 Department of Cell Biology and Anatomy, University of Miami, Miami, FL, USA
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Fig. 1. Estimation of [cAMP]i changes using FRET in NCI-H292 cells. NCI-H292 cells were co-infected with HIV-derived lentiviruses encoding the fluorescently labeled subunits of PKA driven by the CMV promoter. (A) FRET was demonstrated by exciting cells at 436 nm and visualizing CFP emission at 480 nm (left panel, blue) and YFP emission at 535 nm from a pair of cells (middle panel, green). Right panel shows merged image. Bar, 10 µm. (B) Repeated stimulation of the two cells pictured in A (left cell, thin lines; right cell, thick lines) with 20 µM forskolin increased CFP (blue trace) and decreased YFP emission (green), consistent with a reduction in FRET due to separation of the PKA subunits. (C) Ratio data (CFP:YFP fluorescence; left cell: thin line and right cell, thick line) reveal reversible increases in estimated [cAMP]i upon exposure to 20 µM forskolin.
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Fig. 2. Time course of PKA subunit expression using CMV or foxj1 promoter in human airway epithelial cells. Undifferentiated cells were infected and then re-differentiated on T-clear filters at the ALI and imaged every other day for 19 days. Pictures are merged images obtained by first exciting CFP at 436 nm, recording the emitted light at 480 nm, and then exciting YFP at 510 nm, recording the emitted light at 535 nm. (Upper row) CMV promoter. Using this promoter, rapid expression of both fluorescently tagged PKA subunits was seen, reaching a maximal expression around day 5. (Lower row) foxj1 promoter. Using the this promoter, expression of both fluorescently tagged PKA subunits was initially low but increased markedly around day 11 together with the beginning of ciliogenesis as assessed by phase-contrast microscopy (semi-quantitatively shown by the gradient between the rows getting darker from white to black). All images were taken with the same camera settings and a standard excitation time. Bar, 100 µm. Cultures were from a single lung donor and matched with regard to culture and infection conditions.
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Fig. 4. Apical localization of PKA fusion constructs in infected and fully differentiated human airway epithelial cells. Fully differentiated cells were infected in their undifferentiated state with foxj1 promoter-containing lentiviral constructs and stained for cilia with anti-acetylated  tubulin (ac. tubulin) and an Alexa Fluor-555-coupled secondary antibody (red) and with DAPI for nuclei (pseudo-colored in white). (A) Magnified z-axis reconstructions showing expression of both fusion proteins in cilia. (B) Z-axis reconstructions and xy cuts through different levels of the cultures corresponding to cilia, apical and basal cell compartments are shown. Channels are labeled at the bottom including overlay images. Both CFP- and YFP-labeled PKA subunits were mainly localized to the apical compartment of the cells and they can be easily seen inside cilia. Bar, 50 µm.
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Fig. 5. Real-time measurements of simultaneous changes of YFP- and CFP-intensities, FRET-ratio and CBF in single human airway epithelial cells. (A) Dually infected and differentiated cells were basolaterally permeabilized and basolaterally perfused. Intensities of CFP (blue) and YFP (green) emissions were measured during CFP excitation simultaneously with CBF (red). FRET ratio (black) was calculated as quotient of the intensities CFP/YFP. Addition of cAMP to the perfusate is indicated with a bar above the traces. (B) Dually infected and differentiated cells were imaged without permeabilization. Intensities of CFP (blue) and YFP (green) emissions were measured during CFP excitation simultaneously with CBF (red). The FRET ratio (black) was calculated during perfusion of the apical surface with sequential solutions containing 1 µM, 10 µM and 20 µM forskolin.
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Fig. 6. Dose response of FRET ratio and CBF to cAMP and forskolin in permeabilized human airway epithelial cells. FRET ratio (black) and CBF changes (grey) were recorded from a single ciliated cell, in a previously permeabilized culture that was sequentially perfused on the basolateral side with 20 µM, 50 µM and 100 µM cAMP. Subsequent apical perfusion with 1 µM, 10 µM and 20 µM forskolin are shown. At about 7500 seconds ATP was removed from the basal chamber, which caused a rapid fall of CBF, confirming successful permeabilization of the cell. Thus, this last example does not show the delay of the CBF return to baseline compared to baseline with 20 µM forskolin (see Fig. 9).
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Fig. 8. Comparison of the kinetics of FRET ratio and CBF changes during and after stimulation with cAMP or forskolin. Infected and differentiated human airway epithelial cells were either perfused apically with 1 µM, 10 µM or 20 µM forskolin (non-permeabilized cells; n=9) or basolaterally with 20 µM, 50 µM or 100 µM cAMP (permeabilized cells; n=7). The top of the graph shows an example of a permeabilized cell perfused basolaterally with 100 µM cAMP. The left side of the graph depicts the time to increase FRET-ratio (black) or CBF (gray) from baseline to their maxima in seconds, which were statistically indistinguishable (P>0.05). The right side depicts the times for the FRET ratio and CBF to return to baseline from their maxima. In each case, CBF decreased significantly slower than the FRET ratio (P<0.05 for all pairs).
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Fig. 9. Inhibition of FRET ratio and CBF increases with H89 and Rp-8-Br-cAMPS. Non-permeabilized cells were perfused basolaterally with 400 µM Rp-8-Br-cAMPS (inhibitor of RII), or 10 µM H89 (inhibitor of CAT) in the presence of 1 µM, 10 µM or 20 µM forskolin. (A) FRET ratios. Forskolin-mediated dissociation of PKA subunits is prevented by Rp-8-Br-cAMPS (inhibitor of RII), whereas H89 (inhibitor of CAT) did not prevent forskolin-induced PKA subunit dissociation. (B) CBF. H-89 significantly blocks CBF increases, whereas Rp-8-Br-cAMPS allows CBF increases to only 15% above baseline at all forskolin concentrations tested. This partial effect of Rp-Br-cAMPS is likely due to an estimated 70% inhibition of PKA dissociation at the used concentration; the remaining activation of PKA cannot be discerned with FRET but still activates CBF to a certain degree. *P<0.05, comparing groups indicated by brackets.
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© The Company of Biologists Ltd 2006
50% each). Circles indicate fields with little or no cilia signal but with expression of tagged fusion proteins. In cells expressing constructs with (B) the foxj1 promoter, 80-90% of ciliated cells express either the CFP- or YFP-tagged fusion proteins with promoter with almost 100% coexpression. No expression is seen in non-ciliated cells (squares). Bar, 50 µM. Cultures were from a single donor and matched with regard to all culture and infection conditions.
, 20 µM cAMP; 
, 50 µM cAMP; 
, 100 µM cAMP; 
, 1 µM forskolin; 
, 20 µM forskolin. See text for significance levels.