Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid

Hypertonic conditions trigger transient plasmolysis, growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.52.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating. © 2011 Informa UK, Ltd