Chiara Zurzolo

A Requirement for Caveolin-1 and Associated Kinase Fyn in Integrin Signaling and Anchorage-Dependent Cell Growth

Caveolin-1 functions as a membrane adaptor to link the integrin alpha subunit to the tyrosine kinase Fyn. Upon integrin ligation, Fyn is activated and binds, via its SH3 domain, to Shc. Shc is subsequently phosphorylated at tyrosine 317 and recruits Grb2. This sequence of events is necessary to couple integrins to the Ras-ERK pathway and promote cell cycle progression. These findings reveal an unexpected function of caveolin-1 and Fyn in integrin signaling and anchorage-dependent cell growth.

Prions hijack tunnelling nanotubes for intercellular spread

In variant Creutzfeldt–Jakob disease, prions (PrPSc) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunnelling nanotubes (TNTs) have been identified between cells, both in vitro and in vivo, and may represent a conserved means of cell-to-cell communication. Here we show that TNTs allow transfer of exogenous and endogenous PrPSc between infected and naive neuronal CAD cells. Significantly, transfer of endogenous PrPSc aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrPSc spreading in neuronal cells. In addition, we detected the transfer of labelled PrPSc from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS. We therefore propose that TNTs are involved in the spreading of PrPSc within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.

Lipids as targeting signals: lipid rafts and intracellular trafficking.

Our view of biological membranes has evolved dramatically over the last few decades. In the bilayer model from Singer & Nicholson (Science 1972;175:720–731), both proteins and lipids freely diffuse within the plane of the membrane. Currently, however, membranes are viewed as a mosaic of different compartments or domains maintained by an active cytoskeleton network (Ritchie et al. Mol Membr Biol 2003; 20:13–18). Due to interactions between membrane components, several types of subdomains can form with different characteristics and functions. Lipids are likely to play an important role in the formation of so‐called lipid‐enriched microdomains or lipid rafts, adding another order of complexity to the membrane model. Rafts represent a type of domain wherein lipids of specific chemistry may dynamically associate with each other, to form platforms important for membrane protein sorting and construction of signaling complexes (Simons & Toomre. Nat Rev Mol Cell Biol 2000;1:31–39). Currently, there are several hypotheses concerning the nature of rafts (reviewed in (Edidin. Annu Rev Biophys Biomol Struct 2003;32: 257–283; Zurzolo et al. EMBO Rep 2003;4:1117–1121)). The most commonly cited one, proposed by Kai Simons (Simons & Ikonen. Nature 1997;387:569–572; Pralle et al. J Cell Biol 2000;148:997–1008), suggests that rafts are relatively small structures (∼ 50 nm) enriched in cholesterol and sphingolipids within which associated proteins are likely to be concentrated. Another proposal (Anderson & Jacobson. Science 2002;296:1821–1825) suggests that rafts are constructed of lipid shells. These are small dynamic assemblies wherein ‘raft’ proteins are preferentially associated with certain types of lipids. These ‘shells’ are thermodynamically stable mobile entities in the plane of the membrane that are able to target the protein they encase to preexisting rafts/caveolae domains. In this review we summarize the data suggesting a specific role for lipid domains in intracellular trafficking and sorting and present a modification of the raft model that may help explain the observed phenomena.

Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons

Background: Exogenous, misfolded Tau can be internalized, but details of the mechanism are unknown. Results: Small misfolded Tau species are internalized through endocytosis, anterogradely and retrogradely transported. Conclusion: Tau uptake is dependent on conformation and size of aggregates, and regulated through endocytosis. Significance: Understanding the mechanism by which pathological Tau is internalized provides a foundation for therapeutic approaches targeting uptake and propagation of tauopathy. The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845–12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909–913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein–protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)‐anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1‐DAF, a GPI‐anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1‐DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A‐resistant cells (MDCK‐ConAr) gD1‐DAF was mis‐sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK‐ConAr cells, gD1‐DAF became associated with TX‐100‐insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1‐DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX‐100‐insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1‐DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI‐anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK‐ConAr cells expressed caveolin and clustered GPI‐anchored proteins normally, yet mis‐sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI‐anchored proteins.

Wiring through tunneling nanotubes – from electrical signals to organelle transfer

Tunneling nanotubes (TNTs) represent a subset of F-actin-based transient tubular connections that allow direct communication between distant cells. Recent studies have provided new insights into the existence of TNTs in vivo, and this novel mechanism of intercellular communication is implicated in various essential processes, such as development, immunity, tissue regeneration and transmission of electrical signals. TNTs are versatile structures known to facilitate the transfer of various cargos, such as organelles, plasma membrane components, pathogens and Ca2+. Recently, a new function of TNTs in the long-range transfer of electrical signals that involves gap junctions has been suggested. This indicates that different types of TNTs might exist, and supports the notion that TNTs might not be just passive open conduits but rather are regulated by gating mechanisms. Furthermore, TNTs have been found in different cell lines and are characterized by their diversity in terms of morphology. Here we discuss these novel findings in the context of the two models that have been proposed for TNT formation, and focus on putative proteins that could represent TNT specific markers. We also shed some light on the molecular mechanisms used by TNTs to transfer cargos, as well as chemical and electrical signals.

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC), denoted PrPSc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrPC into PrPSc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrPC and PrPSc and measured PrPSc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans‐Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90–98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.

PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts.

PrPC is a glycosylphosphatidylinositol‐anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrPC remains unknown, a conformational isoform called PrPSc (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrPC to PrPSc conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild‐type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrPC in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol‐anchored proteins, we found that PrPC is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol‐proteins, PrPC associates with detergent‐resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol‐proteins, as well as in the conversion process of PrPC to PrPSc. In order to discriminate whether detergent‐resistant microdomains have a direct role in PrPSc conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent‐resistant microdomain association on PrPC intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol‐linked protein, our data exclude a classical role for detergent‐resistant microdomains in the post‐trans‐Golgi network sorting and transport of PrPC to the plasma membrane.