Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 7;109(1):85-94.
doi: 10.1016/j.bpj.2015.04.042.

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

Affiliations
Free PMC article

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

Asmahan Abu-Arish et al. Biophys J. .
Free PMC article

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

Figures

Figure 1
Figure 1
CFTR distribution on the plasma membrane of primary HBE cells. A fluorescence confocal microscope image of GFP-CFTR in the plasma membrane of a live HBE cell reveals two populations; one comprised of bright clusters that are homogeneously distributed (arrow on right) and another more diffuse population (arrow on left). To see this figure in color, go online.
Figure 2
Figure 2
CFTR CD and DA in the plasma membrane as measured by spatial ICS are strongly cholesterol dependent. CD and DA were normalized to their control values, the CD ratio and the DA ratio, respectively. In the COase condition, cholesterol loss increased the CFTR CD by 30% and decreased the DA by 35% (p < 0.01). Elevating cholesterol in the membrane using CEase reduced the CD by twofold and increased the DA by threefold (p < 0.01). Error bars indicate the mean ± SE.
Figure 3
Figure 3
CFTR distribution in the plasma membrane depends on membrane cholesterol. (a) A confocal microscope image of GFP-CFTR in an ROI on the plasma membrane of live HBE cells under Ctr conditions. (b) After lipid raft disruption (+COase). (c) After membrane cholesterol insertion (+CEase). (d) After acute viral infection (+adenovirus). To see this figure in color, go online.
Figure 4
Figure 4
CFTR macro- and microdynamics and distribution on the plasma membrane are cholesterol dependent. (a) The average macroscale MSD, Dmacroτ, increased linearly as a function of τ for control (Ctr, black) and COase-treated cells (gray). The slope, Dmacro, is the macro diffusion coefficient of unconfined CFTR. (b) The average microscale MSD, Dmicroτ, increased linearly for the first few temporal lags and dramatically changed its slope at a later τ for both conditions. The first three temporal lags were fit with straight lines, as shown, yielding a slope, Dmicro, that describes the mobility of the CFTR population inside confinements. COase treatment resulted in a larger Dmicro compared to the Ctr condition, indicating a weaker trap. (c) The average amplitudes of the macro- (ϕmacro, line) and microcomponents (ϕmicro, solid circles) of the k-space correlation function as a function of τ. For ncell averages, see Table 1. Error bars indicate the mean ± SE.
Figure 5
Figure 5
CFTR macro- and microdynamics and distribution on the plasma membrane are cholesterol dependent. (a) Both Dmacro and Dmicro of CFTR increased significantly after cholesterol depletion from the membrane (+COase) and decreased significantly after cholesterol augmentation (+CEase). (b) Gray bars indicate the unconfined population fraction, fmacro, increased by cholesterol loss (+COase) and decreased after cholesterol insertion (+CEase). Black bars indicate the confined population fraction of CFTR, fmicro, decreased significantly after COase and increased significantly after CEase. Error bars indicate the mean ± SE.
Figure 6
Figure 6
CFTR dynamics and distribution after acute viral infection. (a) Both the Dmacro and Dmicro of CFTR decreased significantly after the infection. (b) Although the unconfined population fraction, fmacro, decreased significantly postinfection (gray), the confined population fraction, fmicro, increased by twofold (black). This indicates recruitment of CFTR to confinement zones during infection. Error bars indicate the mean ± SE.

Similar articles

See all similar articles

Cited by 17 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback