Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

Abstract

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.

FIGURE 1.

Overview of fluorescent proteins. Two raft-associated proteins are shown: apically sorted GFP-FR containing the GPI signal attachment of the folate receptor and basolaterally sorted GFP-PrP containing the GPI signal attachment of the prion protein. Two transmembrane non-raft-associated proteins are shown: apically sorted P75-GFP containing P75 neurotrophin receptor and basolaterally sorted GFP-PIT containing PIT (truncated form of low density lipoprotein receptor deleted of its extracellular domain). All of these proteins are fused to the GFP (green cylinder) and expressed independently in MDCK cells.

FIGURE 2.

Cholesterol plays a major role in the organization of proteins in apical and basolateral domains of polarized MDCK cells. A, apparent diffusion coefficients (D) of the four studied proteins at steady state (colored bars) and upon depletion of cholesterol synthesis by treatment with mevinolin (white bars). B, confocal pictures of filipin staining at apical pole (panels a and b) and basolateral pole (panels c and d) of polarized MDCK cells in control conditions (panels a and c) and upon loading of cholesterol (panels b and d). Bar, 10 μm. C, quantification of the filipin staining in polarized MDCK cells loaded with cholesterol. Intensity fluorescence is expressed as percentage of control cells. The experiments were performed three independent times, and the error bars are the mean ± S.D. D, apparent diffusion coefficient (D) of proteins of interest at steady state (colored bars) and upon addition of cholesterol (white bars). Fitted data are shown from at least three independent experiments (for A and D) with n > 15. The error bars are the means ± S.D. ^*, p < 0.0001; ^**, p < 0.005.

FIGURE 3.

Involvement of the actin cytoskeleton in the organization of apical pole of polarized MDCK cells. A, two apical transmembrane raft-associated proteins fused to the GFP, LAT-WT-GFP, and LAT-TMD-GFP (deleted of the cytoplasmic domain) were considered for this FRAP analysis. B, apparent diffusion coefficients of the studied proteins at steady state (colored bars) and after 5 min of latrunculin A treatment (white bars) within the first 20 min after removal of the drug because its effect is reversible (35). The experiments were performed at least three independent times, n > 15. ^*, p < 0.0001; ^**, p < 0.001. C, apical (panels a, c, and e) and basolateral (panels b, d, and f) confocal images of actin immunostained with phalloidin-rhodamine in polarized MDCK cells at steady state (panels a and b) or upon 5 min (panels c and d) or 60 min (panels e and f) treatment with latrunculin A. Bar, 10 μm.

FIGURE 4.

Diffusional mobilities of raft- and non-raft-associated proteins at the level of the Golgi compartment in polarized MDCK cells. A, apparent diffusion coefficients of our studied proteins at steady state (colored bars) and upon depletion of cholesterol by inhibition of HMG-CoA reductase (white bars). B, apparent diffusion coefficients of our studied proteins at steady state (colored bars) and upon loading of cholesterol (white bars). These experiments have been performed at least three independent times, n > 15. The error bars are the means ± S.D. with ^*, p < 0.0001. C, analysis by velocity gradient of oligomerization status of basolaterally sorted proteins (GFP-PrP and GFP-PIT) at steady state or upon loading of cholesterol.

FIGURE 5.

Contribution of the actin cytoskeleton in the organization of membranes in the Golgi complex in polarized and nonpolarized MDCK cells. A, apparent diffusion coefficients of our studied proteins at steady state (white boxes) and upon 5 min latruncuclin A treatment (colored bars) in Golgi membranes of polarized MDCK cells. B, apparent diffusion coefficients of apical raft-associated protein (GFP-FR) and non-raft-associated protein (P75-GFP) at steady state (colored bars) and upon 5 min (white bars) treatment with latrunculin A in Golgi membranes in polarized and nonpolarized MDCK cells. The experiments were performed at least three independent times, n > 15. ^*, p < 0.0001; ^**, p < 0.05.

FIGURE 6.

Model of raft- and non-raft-associated proteins organization at the level of the polarized plasma membrane (A) and of the Golgi apparatus (B) in polarized MDCK cells. A, raft-associated proteins (GFP-FR, GFP-LAT-TMD, and GFP-LAT-WT) are surrounded by orange lipids, whereas non-raft-associated P75-GFP is represented surrounded by a different membrane environment in dark gray. At the basolateral membrane GFP-PrP raft-associated is also surrounded by a dark orange membrane environment, whereas the non-raft-associated protein GFP-PIT is surrounded by a light gray membrane environment. Raft domains appear different between the apical and the basolateral membrane (dark orange/orange) because they have a different sensitivity to cholesterol depletion. Non-raft domains are also distinct (dark gray and light gray) because only the apical non-raft domain is modified upon cholesterol loading. Only transmembrane proteins with a cytosolic domain and independently of their raft association seem to be connected to the actin meshwork. B, in the Golgi, apical and basolateral proteins already appear to be in a different environment, which does not show the same sensitivity to cholesterol depletion and actin rearrangement. In both of these environments raft- and non-raft-associated proteins are found (orange/dark orange for raft-associated proteins and gray/dark gray for non-raft-associated proteins).