Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

TLDR

To investigate whether caveolae participate in constitutive endocytic trafficking, the authors expressed N‑ and C‑terminal GFP‑tagged caveolin‑1 fusion proteins in HeLa, A431, and MDCK cells. They used GFP‑tagged caveolin‑1 expression combined with immunogold labeling, confocal microscopy, and photobleaching to assess caveolae localization and dynamics. Caveolae were found to be highly immobile, requiring cholesterol and an intact actin cytoskeleton; disruption of these components increased their mobility and internalization, and the low mobile fraction (<20%) demonstrates that caveolae are not involved in constitutive endocytosis but constitute a stable plasma‑membrane compartment.

Abstract

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- β-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = ∞) of intracellular, GFP-tagged caveolin- associated structures was 70–90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of &lt;20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.