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].

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...

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.

Ca 2ϩ is a universal messenger from bacterial to mammalian cells since its concentration gradients across both organelle and plasma membranes can be efficiently used to communicate biological signals. Therefore, the control of the intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i 3 is of crucial importance for the regulation of many cellular functions, including proliferation, contraction, fertilization, motility, apoptosis, and cell death (1). Receptor-mediated Ca 2ϩ influx from the extracellular space is one important mechanism to control [Ca 2ϩ ] i in non-excitable cells, e.g. leukocytes (2). Although the molecular machinery underlying Ca 2ϩ entry is still poorly defined, cation channels of the transient receptor potential (TRP) family that includes several subfamilies (3-5) are likely candidates for Ca 2ϩ entry pathways under the control of membrane receptors. TRPM2 (formerly LTRPC2 and TRPC7) is a member of the TRPM subfamily. TRPM2 forms non-selective Ca 2ϩ -permeable cation channels and is mainly expressed in brain and in cells of the immune system (6 -8). Opening of the channel is induced by intracellular ADP-ribose (ADPR; Refs. 6 and 7) and enhanced by increased cytosolic Ca 2ϩ (9). Whether NAD also activates TRPM2 currents is still controversial (6, 7, 10, 11). The nudix box in the cytosolic C-terminal region of TRPM2, a conserved motif of enzymes with nucleotide pyrophosphatase activity, seems to be responsible for gating of TRPM2 by ADPR and possibly by NAD (6,7,12). An involvement of TRPM2 in cellular signaling processes has been proposed since the expression of TRPM2 confers susceptibility to oxidant-induced cell death (11).A key question in the field relates to the potential role of the NAD metabolite ADPR as a second messenger. A function of NAD or ADPR as the missing link between specific extracellular signals and Ca 2ϩ influx mediated by TRPM2 has been hypothesized (8,13,14), but experimental proofs are missing so far. To test this hypothesis directly, we developed a method to measure intracellular levels of ADPR in Jurkat T cells (15). We report that cytosolic ADPR concentrations are raised in response to concanavalin A (ConA) and induce Ca 2ϩ entry through TRPM2, thereby significantly increasing [Ca 2ϩ ] i . Inhibition of intracellular ADPR formation or gene silencing of TRPM2 efficiently diminished receptor-mediated Ca 2ϩ influx carried by TRPM2. Moreover, blockade of ADPR formation also efficiently blocked ConA-induced cell death. EXPERIMENTAL PROCEDURESElectrophysiology-Membrane currents were recorded in the wholecell configuration of the patch clamp technique (16) or the perforatedpatch configuration with nystatin (17). An EPC9 patch clamp amplifier was used in conjunction with the PULSE stimulation and data acquisition software (HEKA Elektronik, Lamprecht, Germany). The patch electrodes were made from 1.5-mm diameter borosilicate glass capillaries and filled with intracellular solution. Data were low pass-filtered at 1 kHz and compensated for both fast and slow capacity transients. Se...

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.

T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raftlike macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation.Key words: lipid rafts • signal transduction • T cell activation • actin cytoskeleton • raft clustering embrane microdomains/lipid rafts are considered as sites for protein sorting and assembly of signaling complexes. Thus, they allow the spatio-temporal regulation of protein-protein interaction and signal transduction (1, 2). The concept of lipid rafts controlling signal transduction at the plasma membrane has largely evolved from work on lymphocyte activation (3). Lateral segregation of signaling molecules by regulated aggregation of lipid rafts is thought to control the assembly of large immune receptor signaling complexes and to regulate signal strength and duration (4,5). Essential components of the T cell receptor (TCR) signaling complex, including LAT (linker of activated T cells), lck, fyn and the M coreceptors CD4/CD8 acquire raft affinity through palmitoylations. Their targeting to lipid rafts was shown to be crucial for efficient coupling of TCR engagement to downstream effectors (6-9).Specific aspects of lipid rafts, such as dimension and lifetime, are still controversial (10). Moreover, the use of a variety of methods to isolate rafts, as well as different definitions of lipid rafts, raise many open questions (11,12). Recent reports argued that lipid rafts in resting cells are small, dynamic entities unsuitable to act as signaling scaffolds (13,14). Furthermore, different types of lipid microdomains exist on the single cell level, as evidenced by the segregation of GM1-and GM3-enriched membrane domains during T cell polarization and migration (15). Activation of T cells, however, triggers the formation of stable macrodomains/clustered rafts around the engaged TCR complex (3,16,17), as was recently unambiguously shown by visualizing directly the order of the membrane in the vicinity of engaged TCRs using the fluorescent dye Laur...

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The reggies/flotillins were discovered as proteins upregulated during axon regeneration. Here, we show that expression of a trans-negative reggie-1/flotillin-2 deletion mutant, R1EA, which interferes with oligomerization of the reggies/flotillins, inhibited insulin-like growth factor (IGF)-induced neurite outgrowth in N2a neuroblastoma cells and impaired in vitro differentiation of primary rat hippocampal neurons. Cells expressing R1EA formed only short and broad membrane protrusions often with abnormally large growth cones. R1EA expression strongly perturbed the balanced activation of the Rho-family GTPases Rac1 and cdc42. Furthermore, focal adhesion kinase (FAK) activity was also enhanced by R1EA expression, while other signaling pathways like ERK1/2, PKC or PKB signaling were unaffected. These severe signaling defects were caused by an impaired recruitment of the reggie/flotillin-associated adaptor molecule CAP/ponsin to focal contacts at the plasma membrane. Thus, the reggies/flotillins are crucial for coordinated assembly of signaling complexes regulating cytoskeletal remodeling.