The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein.

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

Thy-1 immunolabeled thymocyte microdomains studied with the atomic force microscope and the electron microscope.

The atomic force microscope (AFM) and the transmission electron microscope (TEM) have been used to study the morphology of isolated mouse thymocyte microdomains and Thy-1 antigen distribution at the surface of these structures. AFM images were recorded in air in the contact mode on membrane vesicles deposited on previously heated tissue culture plastic sheets and indirectly immunolabeled for Thy-1 expression with colloidal gold-conjugated secondary antibodies. AFM images of untreated plastic plates showed a very characteristic network of streaks 20-200 nm wide. Heating the plastic removed the streaks and provided flat surfaces (r.m.s. 1 nm). This substrate allowed strong adsorption and homogeneous spreading of the vesicles and easy manipulations during immunolabeling experiments. Vesicles flattened on the substrate without losing their morphology. The 10-nm membrane-bound gold beads were reproducibly imaged without degradation by repeated tip scanning. The observed microdomains had a mean diameter of 184 +/- 76 nm, and 65% of them were specifically labeled. Images obtained with the TEM on the same vesicles, deposited on carbon-coated grids and negatively stained, confirmed the AFM observations. The size distribution of the microdomains was quite similar, but the number of beads per vesicle was significantly higher, and 76% of the vesicles were labeled. The difference may be explained 1) by removal of beads from the vesicles in the additional washing step with water, which was necessary for the AFM; 2) by tip-sample convolution; and 3) by statistical fluctuations.

Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells.

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.