Reggie/flotillin and the targeted delivery of cargo

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Reggie/flotillin and the targeted de livery of cargo

Claudia A. O. Stue1111er

Department ofBiology, University ofKonstanz, Konstanz, Germany

Abstract

The two proteins reggis-1/f1otillin-2 and reggie-21llötillin-1 lorm microdomains at the plasma membrane and at intracellular compartments where src tyrosine kinases associate with them. Specilic GPI-anchored proteins, in particular prion protein and Thy-1, co-cluster with reggie microdomains at the plasma membrane and elicit signal transduc- ti on in association with reggies which regulates the activation 01 several GTPases involved in the recruitment 01 specilic membrane .proteins Irom intracellular carriers to target sites 01 the cell membrane in a cell type-specific manner. For example, prion protein and reggieregulate the . recruitment and targeted delivery 01 the T cell receptor

Evidence is accumulating that the reggie proteins are involved in trafficking and the targeted delivery ofmembrane and membrane pro teins to very specific sites in a cell type- specific manner (Solis et al. 20 I 0; Stuermer 2010). This review discusses evidence from selected publications and results from a spectrum of cells which is put together Iike pieces of a puzzle to develop this new view on reggie function.

Reggies form microdomains

~gie-land reggie-2 (f1otillin-2 and - I) were discovered as two proteins being up-regulated during axon ~eneration in retinal ganglion cells after lesioning the optic nerve in goldfish (Schulte et al. 1997) - which explains their name - and independently as constituents of the so-ca lied lipid raft floating fraction after solubilisation of membranes in Triton- X-100 at 4

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and sucrose density centrifugation which led to the name f10tillins (Bickel et al. 1997). Flotillins were considered as residents of caveolae, a view which, however, was incompatible with the notion that neurons and Iympho- cytes which express reggie, are devoid of caveolae (Fra et al.

1994; Lang et al. 1998). Double immuno electron micros- copy (Stuermer et al. 2001) then demonstrated that reggies

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complex to the T cell cap, of E-cadherin to cell-cell contact sites in epithelial cells, and of bulk membrane and growth receptors to the, growth cone in developing neurons. Evi- dence is accumulating that reggies are involved in guiding the celHype-specific membrane proteins lrom the intracellular compartments to their target sites at the cell membrane, a function required in all cells which explains why reggies are expressed in many,or all cells in invertebrates and vertebrates.

Keywords: flotillin, lipid rafts, PrP, reggie, src tyrosine kinases, Thy-1 .

define their own microdomains indeed with no overlap with caveolae.

Since their discovery, antibodies against f10tillin are widely used in biochemical experiments as diagnostic tools for lipid raft fractions - reflecting reggie's least exciting property - and leaving the question aside wh ich function the reggielflotillin proteins might actually subserve.

Reggies reside at the cytoplasmic face of the plasma membrane and at membran es of intracellular compartments (Lang et al. 1998; Stuermer et al. 2001). They have been shown to associate with the plasma membrane by myristoyl and palmitoyl residues and a hydrophobic stretch of amino acids in their head domain (Morrow et al. 2002; Neumann- Giesen et al. 2003). The reggie tail domain is predicted to adopt an alpha-helical coiled-coil structure (Schulte et al.

1997) and is involved in the formation of reggie homo-and

Address corrcspondence and reprint requests to Claudia A. O.

Stuerlllcr, Departlllent of Biology, University of Konstanz, 78457 Kon- stanz, GerIllany. E-mail: c1audia.stucrmer@uni-konstanz.de

Abbreviatiol1s used: CAP, c-cbl-associated protein; GPI, glycosyl- phosphatidyl inositol; Glut4, glucose transporter; PrP, prion protein;

TCR, T cell receptor.

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-145990

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heterooligomers (Solis et al. 2007). Clusters formed by reggie oligomers can be recognised by immunofluorescence and double immunogold labelling at the electron microscope as microdomains ofroughly ~ 100 nm (Stuermer et al. 200 I, 2004; Rivera-Milla et al. 2005) appearing as rows of puncta along the plasma membrane by immunofluorescence in which reggie-I and reggie-2 are co-Iocalised (Lang et al.

1998). Jt was then observed that specific glycosylpho- sphatidyl inositol (GPI)-anchored proteins at the cell surface and src tyrosine kinases co-c1ustered with reggie so that reggie microdomains were proposed to represent centers for the communication of GPI-anchored proteins with intracellular signal transduction molecules thus promoting the cross- talk of the cells with their environment (Stuermer and Plattner 2004).

Reggies are involved in cell-type-specific functions

But how to arrive at a conclusive explanation for the role that reggies might play when considering the extremely different cell types in which the reggies were detected:

adipocytes (Baumann et al. 2000), hepatocytes (Ismair et al.

2009), neurons (Munderloh et al. 2009), epithelial cells (Schrock et al. 2009), neutrophils (Rossy et al. 2009) and T Iymphocytes (Stuermer et al. 2001, 2004)? In fact, reggies have meanwhile been identified in basically every cell type analysed and in species as distant as fly and man (Rivera- Milla et al. 2005; Katanaev et al. 2008); and an evolutionary connection with reggie-Iike proteins in plants (Borner et al.

2005) and bacteria (Hinderhofer et al. 2009) has also been established. In other words, reggies are ubiquitous and evolutionarily highly conserved predicting that their function(s) is crucial for all cells (Stuermer 2010). On the other hand, however, reggies were found in neurons in the context ofaxon regeneration (Schulte et al. 1997; Lang et al. 1998), in adipocytes implicated in the transfer of the glucose transporter (Glut4) from intracellular stores to the cell membrane (Bau mann et al. 2000; Kioka et al. 2002) and in T cells in the recruitment of the T cell receptor (TCR) (Stuermer et al. 2004) - to name a few examples - indicating that reggies might participate in each cell in a cell type-specific rather than general function. Results obtained in adipocytes, studied in the context of type 2 diabetes (Kioka et al. 2002; Chang et al. 2007; Chen et al.

2007) were the first to indicate that reggies participate in trafficking.

In adipocytes, the delivery of Glut4 to the cell membrane is initiated by the activation of the insulin receptor and includes the reggielflotillin-binding adapter protein CAP (c-cbl-associated proteinlponsin) and signaling to the Rho- family GTPase TCIO (Kioka et al. 2002; Chen et al.

2007). CAP and its relatives vinexin and ArgBP2, in turn, were discussed in a different context based on the fact that these adaptor proteins communicate with regulators of the

actin cytoskeleton (Kioka et al. 2002), such as vinculin, talin and afadin. Accordingly, CAP and relatives were implicated in the regulation of cell-cell and cell-substrate interactions as they lie in the cadherin and integrin signaling pathways.

This indicated a connection between reggies and cell-cell and cell-substrate adhesion which was, in turn, in agreement with observations showing that reggies reliably cluster at cell contact sites, and with later findings showing their involvement in focal adhesion formation and focal adhesion kinase signaling (Stuermer et al. 2004; Schrock et al. 2009). New ideas were spurred by the discovery ofthe unique expression pattern of reggies in T cells and their association with GPI- anchored proteins.

The preformed reggie cap and its functional association with the GPI-anchored proteins prion protein and Thy-l

In the Jurkat T cell line and in primary T Iyrnphocytes from human blood, the reggies accumulate at one pole even in quiescent cells. This region is known as the T cell cap (Rajendran et al. 2003; Stuermer et al. 2004) to which the TCR complex is recruited when the cells are stimulated (Nel 2002).

It was known that activation of the GPI-anchored protein Thy-I by antibody-mediated cross-Iinking is sufficient for capping (Friedrichson and Kurzchalia 1998; Si mons and Ehehalt 2002). As Thy-I and GPI-anchored proteins in general reside preferentially in lipid rafts (Simons and Toomre 2000; Goswami et al. 2008), Thy-I dependent capping provided evidence for raft-dependent signaling (Simons and Ehehalt 2002). The fact that reggies are 'lipid raft' proteins and scaffolds of microdomains in the cap (Rajendran et al. 2003) strongly suggested that they should participate in c1ustering and signaling of Thy-I and other GPI-anchored proteins. Indeed, when Thy-I or the cellular prion protein (PrP) were activated by antibody-mediated cross-linking, the GPI-anchored proteins accumulated in the cap and co-c1ustered with reggies (Stuermer et al. 200 I, 2004). Members ofthe src tyrosine kinase family such as f)'ll and lek, as weil as actin were enriched in the cap and recruited the TCR to the cap region. Importantly, however, capping of the TCR did not automatically lead to TCR activation (Stuermer et al. 2004). TCR activation requires contact between the T cell and an antigen presenting cell, or cross-Iinking of the TCR directly by antibodies against the TCR (Nel 2002).

That TCR capping provoked by PrP or Thy-I crosslinking differed from full T cell activation was also recognised at the level of the signal transduction pathways.

PrP capping and co-capping with reggies resulted in Ca²+ signaling and phosphorylation (activation) of the MAPK extracellular-signal-regulated kinase I, 2. But extracellular-

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signal-regulated kinase phosphorylation was weaker and the Ca²+ signal was brief and smaller in amplitude than signals genera ted by TCR activation which is two times stronger and longlasting (Nel 2002; Stuetmer and Plattner 2005). To clarify which response may come from PrP and Thy-I crosslinking, the PrP-(Thy-l) induced Ca²+ signal was blocked by the membrane permeable Ca²+ chelator BABTA AM. Under these conditions, PrP as weil as Thy-I did not undergo capping nor did the TCR and other T cell stimulating components assemble in the cap. Thus, PrP activation prornotes its own selective accumulation and that of Thy-I in the reggie cap and recmits the TCR to the cap. Therefore, PrP-reggie-mediated signaling is evidently involved in the assembly of the TCR and the relevant partner proteins in the cap but does not induce the full TCR activation.

These results suggested the new view that reggies in close association with GPI-anchored proteins such as PrP and Thy-

I participate in signaling leading to the recmitment of TCR components. The emphasis here lies on recmitment. In more general terms, reggies cluster anq form larger assemblies at strategically important sites, and the function of reggies, in turn, are needed where-ever they cluster and co-cluster with GPI-anchored proteins. Signal transduction emanating, for example, from PrP-reggie interaction leads to the recmitment of important membrane proteins to strategically important sites in a cell type specific manner (Fig. I).

Reggies during axon growth and regeneration

Neurons dynamically regldate reggies in the context ofaxon growth and exhibit elevated expression levels during growth cone elongation in development and during axon regeneration after lesion (Schulte et al. 1997; Lang et a/. 1998).

TCR complex

Immunostainings with antibodies show that reggies and PrP are enriched in the growth cones (Stuermer 20 I 0). Moreover, both reggie and PrP are localised to focal adhesions, the contacts of cells and axons with the extracellular matrix (Schrock et al. 2009) suggesting a role of the reggies in axon elongation and migration. If so then reggie down-regulation should negatively affect axon growth and regeneration.

It is possible in fish to down-regldate specific proteins, such as reggies, by morpholino- (a modified siRNA) mediated knock down in vivo. The morpholinos are applied to the lesioned axons in the optic nerve and are retrogradely transported to the parent neurons in the retina where they block reggie protein production. This approach led to a 70%

reduction of the nu mb er of regenerating axons showing that reggies are indeed necessary for axon regrowth (Langhorst et al. 2008b; Munderloh et a/. 2009). Down-regulation of reggies in mouse hippocampal neurons in culture allowed to determine the consequences of the loss of reggie on a single cell level: neuronal differentiation was massively impaired and outgrowth ofaxons and dendrites blocked so that neurons were unable to develop. The devastating effect that down-regulation of reggie had on the developing neurons suggested furthermore, that reggies regldate so me very basic function connected to trafficking and directed transport of membrane material and proteins needed for process formation and neuronal morphology (Stuermer 20 I 0). These functions depend on the coordinated activation of GTPases and regulation of cytoskeletal dynamics. Indeed, down- regulation as weil as misregulation of reggie-I caused a dismption of the normally balanced activation state of the Rho-GTPases Rac, Rho and cdc42, and also affected MAP- kinase and Ras activation (Langhorst et al. 2008a; Munder- loh et a/. 2009). Besides regulating cytoskeletal dynamics

Integrin

• GTPases

I

Integrin

PrP

. . . . Reggie microdomain Src-related kinases

A I

ECM Crosslinking antibody

Fig. 1 Schematised drawing illustrating the trafficking pathway regulated by reggies.

(a) Antibody-mediated cross-linking of PrP in the reggie cap of T cells leads to signal transduction towards compartments to recruit the TCR complex to the cap. (b) PrP-PrP trans-interaction at cell-cell contact sites leads to co-'clustering of PrP with reggie and leads to signal transduction towards intracellular compartments and recruits E-cadherin to the contacts. (c) At cell substrate contacts, PrP-reggie co-clustering and interaction leads to the recruitment of integrins to focal adhesions . The targeted delivery of bulk membrane and growth receptors such as integrins to the growth cone is also crucial for axon growth and regeneration.

, E-cadherin

T cell receptor

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via GTPase activation, reggie-I can also bind to actin directly. Thus, regulation of neurite extension is congruent with an effect of reggie on GTPases and other signaling molecules.

Reggies at intracellular compartments and their role in trafficking

The hypothesis that reggies might participate in cargo trafficking was supported by observations that reggies reside at many intracellular compartments including lysosomes, recycling endosomes (but not early endosome antigen 1- positive early endosomes) (Langhorst et al. 2008a), post- Golgi vesicles (Morrow et al. 2002; Langhorst et al. 2008a), lipid bodies (Reuter et al. 2004) and phagosomes in macro- phages (Dermine et al. 200 I). Moreover, it has been demonstrated that reggies interact with actin (Langhorst et al. 2007) in addition to communicating with the Rho-type GTPases and their effectors for the regulation of actin dynamics (Langhorst et al. 2008a; Munderloh et al. 2009).

Total intemal reflection fluorescence microscopy and life imaging of enhanced green fluorescent protein-Iabeled reggie in HeIa cells revealed the abundance and dynamics of reggie vesicles in the cell, showing furthermore, that these vesicles shuttle between the plasma membrane, with which they occasionally merge, and the nearby cytoplasm (Langhorst et al. 2007, 2008b). Studies from other labs suggested that flotillins (reggies) would control clathrin-, caveolin- and dynamin-independent endocytosis of GPI-anchored proteins (Glebov et al. 2006) which points in the same direction that reggies contribute to trafficking. However, we and others were unable to confirm that reggies would participate directly in endocytosis of cargo (Langhorst et al. 2008a; Schneider et al. 2008) as published by Glebov et al. (2006). Reggies are not at early endosome antigen I-positive early endosomes, however, are associated with recycling endosomes.

This is consistent with findings from Kawase et al. (2006) showing that reggies function in a pathway involving the exocyst that deli vers specific cargo to specific sites in a cell type-dependent mann er. It has recently been shown that TC I 0 and the exocyst subunit exo 70 form a complex which is essential for membrane expansion and axon growth (Dupraz et al. 2009). In addition, the GTPase TCIO was shown to be downstream of insulin signaling and the reggie- CAP signaling pathway (Kioka et al. 2002; Chen et al.

2007), and to participate in the exocyst-mediated transport of Glut4 to the plasma membrane. Taken together, these data strongly speak for a function of the reggies in the control of the targeted delivery of cargo being indispensible for axon growth, regeneration and process extension in general, and also being essential in all situations in which cells specify particular regions such as cell contact sites, the T cell cap and neurites which depend on supply of site- and situation- specific molecules.

PrP and its association with reggies is necessary for the recruitment of E-cadherin to cell contact sites

To obtain experimental support for the hypothesis presented above, we studied which molecular mechanism reggie and PrP might subserve during cell contact formation. Ongoing research on the function of PrP and reggie in zebrafish showed that both proteins are expressed very early in the embryo (von Philipsbom et al. 2004; Rivera-Milla et al.

2005). PrP was clustered at contact sites between blasto- cytes at the onset of embryonic development (Malaga-Trillo et al. 2009) and throughout gastrulation and epiboli. When PrP-I was down-regulated by morpholinos, the embryos failed to develop properly and died showing that PrP-I subserves some very important function. Microscopic analysis of embryos after PrP down-regulation and before death showed that cells had lost adhesive contacts with neighboring cells indicating that PrP-PrP trans-interaction is necessary for tissue integrity. It is known that a similar function is performed by the bona-fide cell adhesion proteins of the cadherin superfamily, and in the early embryo specifically by E-cadherin. In fact, the phenotypes after PrP and E-cadherin down-regulation were surprisingly similar suggesting that both proteins lie in the same signaling pathway. Indeed, thorough analysis at the subcel- lular level revealed that PrP-PrP trans-interaction was necessary for the recruitment of E-cadherin to the cell-cell contact sites (Malaga-Trillo et al. 2009). Loss of PrP led to the transfer of E-cadherin into Rab II recycling compartment whereas the direction of E-cadherin trafficking was reversed in the presence of PrP and directed to the cell surface at contact sites where E-cadherin became co- localised with PrP-1. The embryonic cell-cell contact sites were also labelIed by reggies.

This co-assembly between PrP, reggie and cadherins at cell contacts was confirmed in several other mammalian epithelial and neuronal cell types such as in N2a, PC 12, HeIa and the A43 I cell lines suggesting that the three proteins typically interact in all these types of cells and across species when cell contacts need to be formed. This predicts that reggie down-regulation should impair the formation of intercellular contacts. Indeed, manipulation of the reggie expression levels was recently discovered to perturb PrP and cadherin-dependent cell contact formation (Solis et al., manuscript submitted for publication).

A hypothesis of reggie function: reggies and the delivery of cargo to specific sites

When taking into account that reggies are involved in the recruitment of the transporter protein Glut4 from intemal stores to the plasma membrane in adipocytes (Bau mann et al. 2000; Chen et al. 2007), in conjunction with PrP in

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the recruitment (i) of cadherin to the cell membrane of embryonic and epithelial cells, (ii) of the TCR to the T cell cap in Iymphocytes, and (iii) by inference, of integrins to focal adhesions (Solis et al. 20 I 0), it becomes reasonable to propose that the reggies participate in guiding specific membrane proteins from internal compartments to very specific regions of the plasma membrane. The many reggie-decorated vesicles in the cells moving towards and away from the cell membrane (Langhorst et al. 2008a) seem to represent vesicular carriers of cargo originating from post-Golgi compartments and the Rabll recycling endosome. In differentiating neurons the role of reggies appears to consist in targeting growth receptors and bulk membrane to the axon and growth cone for process extension and elongation (Munderloh

e t

al. 2009). Direc- tional cues for the delivery of the membrane proteins might co me from the GPI-anchored proteins which co-cluster with the reggies upon their activation. PrP-PrP trans-interaction and association with reggies at cell contact sites, for instance, provides a spatial c1ue for the recruitment of E-cadherin to these sites (Fig. I). It has been recognised that E-cadherin- dependent adhesion is a dynamic process (Wirtz-Peitz and Zallen 2009) involving the continuous recycling of E-cadherin between the plasma membrane at the contact sites and the recycling compartment. This would explain why PrP and reggie are always together at cell contact sites; they are the 'motor' driving E-cadherin turnover. This mechanistic concept on reggielPrP fimction could Iikewise be applied to focal adhesion formation and turnover of focal adhesion components during cell migration, to the growth receptors and adhesion and guidance molecules in the growth cones and to the recruitment of the TCR to the cap. The delivery of Glut4 from internal stores to the cell membrane seems to be regulated in a similar manner although information on the potential involvement of specific GPI-anchored proteins in demarcating the site of Glut4 deposition is not available.

It has, however, been reported that Glut4 delivery depends on the exocyst (Chen et al. 2007), and this is consistent with the observation that reggie signals to the Rho-GTPases including TCIO (Baumann et al. 2000; Munderloh et al.

2009) which are involved in exocyst-mediated cargo transport and delivery (Feig 2003; Kawase et al. 2006;

Chang et al. 2007). Whether the exocyst is involved in the other reggie-dependent delivery pro ces ses discussed herein has not yet been explicitly analysed but is expected as the exocyst is known to participate in growth receptor delivery, in E-cadherin delivery and process elongation in neurons (Dupraz et al. 2009), and is expressed in T Jurkat cells (our own unpublished observation). The hypothesis of reggie functions, presented here (Fig. I) can, in fact, be experi- mentally tested and refined as research progresses and knowledge increases and is consistent with the evolutionary conservation of reggies and their widespread expression across cell types.

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