Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in Յ0.1-m plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after crosslinking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.

Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl‐phosphatidylinositol (GPI)‐anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie‐1 and ‐2 (80% identical to goldfish reggie proteins) shows that reggie‐2 is practically identical to mouse flotillin‐1. Flotillin‐1 and epidermal surface antigen (ESA) (flotillin‐2) are suggested to represent possible membrane proteins in caveolae. Rat reggie‐1 is 99% homologous to ESA in overlapping sequences but has a 49‐amino‐acid N‐terminus not present in ESA. Antibodies (ABs) which recognize reggie‐1 or ‐2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC‐12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti‐caveolin negative) neurons and show anti‐reggie‐1 immunogold‐labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI‐anchored cell adhesion molecules (CAMs) F3 and Thy‐1 are applied to live DRGs, the GPI‐linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti‐reggie antibodies. Thus, reggie‐1 and reggie‐2 identify sites where activated GPI‐linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains). © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 502–523, 1998

Abstract. Reggie/flotillin proteins are considered to be components of lipid rafts and are commonly used as marker proteins for lipid microdomains. Yet almost a decade after their discovery, the function of reggies/ flotillins is still enigmatic. In this review we summarize the present state of knowledge on reggie/flotillin structure, localization and function, and discuss the role of the proteins in development and disease. Based on insights into reggie/flotillin function and by comparison with related proteins of the so-called SPFH (Stomatin/Prohibitin/ CMLS, Cell. Mol. Life Sci. 62 (2005) 2228-2240 1420-682X/05/202228-13 DOI 10.1007/s00018-005-5166-4 © Birkhäuser Verlag, Basel, 2005 Flotillin/HflK/C) protein family, including stomatin, podocin and prohibitin, we propose the existence of specific types of protein-defined microdomains which are sculpt by the clustering of individual SPFH proteins. As 'specialized rafts' similar to caveolae, these membrane domains provide platforms for the recruitment of multiprotein complexes. Since, under certain circumstances, reggie-2/flotillin-1 translocates to the nucleus, reggie/ flotillin microdomains are not only stable scaffolds but also dynamic units with their own regulatory functions.Key words. Lipid raft microdomains; signaling scaffolds; actin cytoskeleton; SPFH domain; stomatin; podocin; prohibitin; caveolae. Discovery of reggie/flotillins'Third time is a charm' the saying goes, and accordingly, reggie/flotillin proteins were independently 'discovered' three times. Madeleine Duvic and co-workers were the very first to discover part of one of the proteins during a screen for the antigen of the monoclonal antibody ECS-1 in 1994. They identified a complementary DNA (cDNA) coding for a N-terminally truncated version of reggie-1/flotillin-2 of 42 kDa (see below), which they called epidermal surface antigen (ESA) [1]. This name was later abandoned, as it was shown that this protein is not the true antigen recognized by ECS-1 [2]. In a screen for proteins upregulated in retinal ganglion cells during axon regeneration after optic nerve lesion in goldfish, we identified in 1997 two proteins of 47 kDa which we called reggie-1 and -2 [3,4]. In the same year Michael Lisanti's group identified two proteins associated with the 'floating' lipid raft fraction from mouse lung tissue which they called flotillin-1 (= reggie-2) and flotillin-2 (= reggie-1) [5]. Although flotillins became the more commonly used name for the proteins, we will stick with reggie-1 and -2, since we believe that our names and numbers are more appropriate in light of the physiological relevance of the two proteins. Structure of reggiesReggie proteins are highly conserved, with about 64% homology between fly and man [6,7]. Reggie-like proteins even exist in some bacteria, plants and fungi [8,9].

Developing retinal axons in the zebrafish embryo were stained with HRP or with the fluorescent dyes dil and diO to study the formation of the retinotectal projection. Retinal axons leave the eye at 34-36 hr postfertilization (PF), invade the tectum at 46-48 hr PF, and innervate the tectal neuropil at 70-72 hr PF. Dorsal and ventral axons occupy separate aspects of the optic nerve and tract and pass into their retinotopically appropriate ventral and dorsal hemitectum, respectively. Nasal and temporal axons are segregated in the nerve, mixed in the tract, and are coextensive over the rostral half of tectum until 56 hr PF. They then segregate again, due to the progression of nasal axons into the open caudal tectum. Thus, at 70-72 hr PF, dorsal and ventral as well as temporal and nasal axons occupy their retinotopically appropriate tectal quadrants. After ablation of the temporal retina prior to the time of axonal outgrowth, the nasal axons bypass the vacant rostral tectum to terminate in the caudal tectal half. Temporal axons in the absence of nasal axons remain restricted to their appropriate rostral tectal half, suggesting that nasal and temporal axons possess a preference for their retinotopically appropriate tectal domains. Measurements of individual terminal arbors and the tectal areas in embryos and in adult zebrafish showed that individual arbors are large with respect to the embryonic tectum but are about 14-15 times smaller than in the adult. However, the proportion of tectum covered by embryonic arbors is about 7 times larger than in the adult, suggesting that a higher precision of the adult projection is achieved as a result of a greater enlargement of the tectum than of the arbors.

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

The cellular prion protein (PrP c ) resides in lipid rafts, yet the type of raft and the physiological function of PrP c are unclear. We show here that cross-linking of PrP c with specific antibodies leads to 1) PrP c capping in Jurkat and human peripheral blood T cells; 2) to cocapping with the intracellular lipid raft proteins reggie-1 and reggie-2; 3) to signal transduction as seen by MAP kinase phosphorylation and an elevation of the intracellular Ca 2+ concentration; 4) to the recruitment of Thy-1, TCR/CD3, fyn, lck and LAT into the cap along with local tyrosine phosphorylation and F-actin polymerization, and later, internalization of PrP c together with the reggies into limp-2 positive lysosomes. Thus, PrP c association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that promote the focal concentration of PrP c itself by guiding activated PrP c into preformed reggie caps and then to the recruitment of important interacting signaling molecules. ) is a glycosylated glycosylphosphatidyl inositol (GPI-) anchored protein that is mostly expressed on the surface of neurons and immune cells (1−4). PrP c has gained considerable attention due to the conversion of α helix to β sheet structures leading to the protease-resistant conformer designated PrP scrapie (PrP sc ) and the spreading of prion disease (1,4,5). The physiological function of PrP c is still under debate: PrP c has been implicated in cell adhesion, differentiation, copper binding (6), neuroprotection against oxidative stress (7,8), apoptosis (9), and transmembrane signaling via a lipid raft-based mechanism (10).Clearly, PrP c resides in plasma membrane lipid rafts/microdomains (2,11,12). Lipid rafts are discussed as platforms for proteins involved in signal transduction, allowing for example GPIanchored proteins to signal across the plasma membrane (13,14 by natural ligands or antibodies (Abs) leads to so-called clustered rafts, ~100−200 nm in size (15, 16) that can be visualized at the light microscopic (LM) level. In fact, an activation of the nonreceptor Src kinase fyn was reported to occur in neurites of a neuroectodermal cell line in a caveolin-1-dependent manner using AB-induced PrP c cross-linking (10).Conflicting views exist, however, concerning the association of PrP c with caveolin-1 and caveolae as opposed to its association with noncaveolar lipid rafts (16), particularly as neurons and lymphocytes lack caveolin-1 and caveolae (14,17,18).The existence of noncaveolar lipid raft microdomains is clearly revealed by the pattern of the two proteins reggie-1 and reggie-2 (18,19,20), also known as flotillin-2 and flotillin-1 (22). In lymphocytes, the reggie proteins exhibit a strikingly polarized expression known as "capping" (20, 23, 24). AB-mediated sequestration of GPI-anchored proteins such as Thy-1 results in Thy-1 capping and cocapping with the reggies (20) and seems to involve transmission of signals into the cell (13, 25, 26, 27, reviewed in 14). Signaling leading to full T cell activati...