Small Fraction of Caveolae may become Constitutively Internalized

Clearly, the possibility cannot be excluded that a few caveolae (or clusters of cav-eolae) pinch off occasionally and therefore participate in the delivery of CT and other molecules to intracellular compartments, although this must - in terms of endocytic activity - be relatively insignificant. Recent studies conducted by Kirk-ham et al. [76] showed that a very small fraction (or a subpopulation) of caveolae is actually internalized constitutively. These authors developed an ultrastructural assay which made it possible to quantitate free, budded caveolae versus surface-connected caveolae. For this, they used CT-B-HRP as an endocytic marker, and quenched the HRP reaction of labeled structures connected to the cell surface with ascorbic acid, which is membrane-impermeable. This was combined with an im-munolabeling protocol that allowed them to show that truly free caveolae-like vesicles containing the HRP reaction product were in fact caveolae. In this way, it was shown that about 2% of the total population of caveolae bud off per minute.

Interestingly, the internalization of prion proteins that are GPI-anchored also appears to be a rather slow or inefficient process that can be mediated by cav-eolae [89]. Thus, in an immunogold labeling pulse-chase experiment it was found that approximately 90% of the caveolae at the plasma membrane that were labeled after a 10-min period were still labeled after a 50-min chase; only about 10% of all caveolae were internalized per hour. This slow internalization may be constitutive, although it cannot be excluded that it is somehow stimulated by the prion proteins because the caveolae-mediated pathway was not observed for several other GPI-anchored proteins [89].

In our FRAP experiments in which the interior of the cells were bleached, only leaving a narrow, fluorescent zone of GFP-tagged caveolin structures, the fluorescence disappeared only very slowly from the peripheral zone (see Fig. 5t in Ref. [31]). Based on extrapolations of such data, the fluorescence would disappear completely from the periphery within about 3 h. Taken together, the described EM and FRAP data are consistent with a very slow, constitutive internalization of caveolae.

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Essentials of Human Physiology

Essentials of Human Physiology

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