Cholesterol goes global

Lipid rafts are involved in regulating important cellular processes — such as signal transduction — and cholesterol has a central role in determining the behaviour of these specialized membrane domains. Depletion of cholesterol disrupts lipid rafts and affects the processes that occur at these platforms, but the global effects of cholesterol depletion have been overlooked. Now, Michael Edidin and colleagues report that membrane-cholesterol depletion causes changes in the actin cytoskeleton, which result in global changes in plasma-membrane function.

The authors grew human fibroblasts and lymphoblasts in a low-density-lipoprotein (LDL)-deficient medium to reduce cholesterol levels and then measured the membrane mobility of human leukocyte antigen 1 (HLA) molecules on the plasma membrane. Lateral diffusion of the HLA molecules was reduced when cholesterol levels were low and, after LDL was added to the medium, it took more than 24 hours to restore mobility to the same level as the controls. By contrast, inhibiting actin polymerization — by adding cytochalasin D to the cholesterol-depleted cells — rapidly restored mobility, so changes in the actin cytoskeleton must accompany the reduced cholesterol levels.

To investigate this further, the authors used laser trapping to capture HLA molecules tagged with 40-nm gold beads and showed that 50% of the tagged HLA molecules on cholesterol-depleted cells underwent elastic recoil, which is a rare event in normal cells. Furthermore, it was easier for the tagged HLA molecules to be pulled perpendicular to the cell membrane in the cholesterol-depleted cells, which indicated that HLA molecules are confined by the elastic membrane cytoskeleton rather than being anchored to it. Moreover, cholesterol depletion altered the activity of actin and actin-modifying proteins, which confirms that cholesterol stabilizes the actin cytoskeleton.

As these observations are similar to those seen when plasma-membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) is lost or redistributed, the authors investigated PtdIns(4,5)P₂ distribution in cholesterol-depleted cells. Plasma-membrane PtdIns(4,5)P₂ levels — measured by a green fluorescent protein (GFP)-tagged plekstrin-homology domain (PH) from phospholipase-C-δ — were reduced after cholesterol depletion. In cells expressing the PH domain, which binds and sequesters PtdIns(4,5)P₂, the lateral diffusion of HLA molecules and their elastic recoil after laser trapping were similar to the diffusion and recoil seen in cholesterol-depleted fibroblasts. So, PtdIns(4,5)P₂ depletion regulates the actin cytoskeleton in a similar manner to cholesterol depletion.

This is therefore the first study that shows how cholesterol levels can globally alter the actin cytoskeleton by affecting the organization of PtdIns(4,5)P₂. It also clearly demonstrates that cellular functions influenced by membrane-cholesterol levels can be independent of lipid rafts.