Effect of synthetic cholesterol derivative, PEG-Chol, on caveolar invagination

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We addressed the effects of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether (PEG-Chol) on the structure/function of clathrin-coated pit and caveolae. Addition of the compound to cultured cells induced progressive smoothenting of the surface. When the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, were inhibited in K562 cells. In A431 cells, not only this activity but also the flattening of caveolae was observed. Opened clathrin-coated pits were accumulated in these cells. We suggest here that the functional clathrin-lattice is much more stiffer than typical phospholipid bilayers. Next, we investigated possible linkage between cortical actin and clathrin coated pits/ lipid-rafts. Since a large body of memebrane dynamics is regulated at the level of protein phosphorylation, we introduced hyper-phosphorylated protein condition in a K562 cell by okadaic acid (OKA). While OKA induced patchy accumulat … More ion of cortical actin, we found subsequent treatement of the cells with biotinylated poly (ethylene glycol) derivative of cholesterol (bPEG-Chol) in conjunction with accumulation via streptavidin (SA) induced numerous phillopodia-like protrusions that are subsequently accumulated and extruded from the main cell body. The actin-rich body was associated with a bundle of vimentin and cluster of mitochondria. Moreover, clathrin-coated pits that were localized by electron microscopy. Moreover, raft-components, CD59 and ganglioside GM1 were also accumulated. Similarly, a raft component Thy-1 was also accumulated in murine T cells but, when non-receptor tyrosine kinase Fyn was knocked-out, such change did not occur. Notably, polarized accmulation was also observed after antibody-mediated accmulation of CD59 and ganglioside GM1. Since Fyn is known to be concentrated in actin-rich domains including coalesced lipid-rafts, we here suggest that certain mechanical stress is transmitted by Fyn to reorganize whole cellular actin and actin-linked proteins into a single mass in OKA-treated cells. Less