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Abstract

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.

Recruitment of cellular prion protein to mitochondrial raft-like microdomains contributes to apoptosis execution

“…In contrast, an evident relocalization of PrP C to the mitochondrial compartment was detectable in anti-Fas–treated cells (as revealed by yellow staining in the merge picture of Figure 1A, second row). Two different experimental controls were performed in order to confirm the specificity of this redistribution: the first deals with GM1, a marker known to colocalize with PrP C at the plasma membrane (Mattei et al ., 2002), and the second with CD71, the transferrin receptor, a plasma membrane PrP C -unrelated marker. We found that after anti–CD95/Fas treatment, the association GM1/PrP C at the cell surface was decreased (Supplemental Figure S1), whereas no overlapping fluorescence CD71/mitochondria was detected (Supplemental Figure S2), indicating that in our experimental conditions the transferrin receptor localization was unaffected by proapoptotic stimulation.…”

Co-localization of CD3 and prion protein in Jurkat lymphocytes after hypothermal stimulation

“…To study the distribution of lipid rafts before, during and after hypothermal activation, we used fluorescent labeled CT-B, which binds to the GM1 ganglioside in the outer leaflet of the plasma membrane. Besides being useful markers for lipid-raft membrane microdomains, gangliosides such as GM1 or GM3 have been described in several cell lines to be co-localized with PrP [27][28][29]. In addition, raft association has been shown to be essential for membrane protein redistribution in polarized T cells [30].…”

Trafficking of PrPcto mitochondrial raft-like microdomains during cell apoptosis

“…PrP is present in body fluids and in the plasma membrane of neural and lymphocytic cells. 4 PrP has been also described as a component of plasma membrane-derived microvesicles, suggesting that microvesicles may contribute both to the intercellular mechanism(s) of PrP diffusion and signaling, as well as prion spread and neuroinvasion. 5 The cellular form of PrP (PrP C ) is a highly conserved cell surface GPIanchored glycoprotein that was first identified in vitro as a molecule able to bind Cu 2+ , 6 in neurons and other cells, including lymphocytes.…”

“…14 Usually, PrP C is reported to be a plasma membrane protein, however several studies have revealed the presence of endogenous PrP C as an interacting protein mainly with the membrane/organelles, 15 as well as with cytoskeleton network. Indeed, lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named lipid raft-like microdomains, with relatively low concentrations the neural plasma membrane and lymphocytes, 4,7,8 which are also enriched in several cytoplasmic proteins, including tyrosine kinases. 9 The association with these specialized portions of the cell plasma membrane is required for conversion of PrP C to the transmissible spongiform encephalopathy-associated protease-resistant isoform.…”