CHAPTER FOUR

Annexins and Endosomal Signaling Francesc Tebar*, Mariona Gelabert-Baldrich*, Monira Hoque†, Rose Cairns†, Carles Rentero*, Albert Pol*,{, Thomas Grewal†, Carlos Enrich*,1

*Departament de Biologia Cellular, Immunologia i Neurocie`ncies, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain † Faculty of Pharmacy, University of Sydney, Sydney, New South Wales, Australia { ICREA, Institucio´ Catalana de Recerca Avanc¸ada, Barcelona, Spain 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Isolation of Endocytic Compartments 2.1 Isolation of endocytic fractions from rat liver 2.2 Isolation of endocytic fractions from CHO cells 3. Targeting Raf-1 Signaling to Early Endosomes 3.1 Expression of CFP–2xFYVE-Raf-1 and Raf-1 activation analysis 3.2 Raf-1 immunoprecipitation and kinase assay 4. Monitoring Endosomal Signaling by Fluorescence Resonance Energy Transfer Microscopy 4.1 FRET acceptor photobleaching 5. Targeting Annexins to Endosomes and Other Cellular Compartments 6. Summary Acknowledgments References

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Abstract Cell signaling and endocytosis are intimately linked in eukaryotic cells. Signaling receptors at the cell surface enter the endocytic pathway and continue to activate downstream effectors in endosomal compartments. This spatiotemporal regulation of signal transduction provides opportunity for signal diversity and a cell-specific machinery of scaffolding/targeting proteins contributes to establish compartment-specific signaling complexes. Members of the annexin (Anx) protein family, in particular AnxA1, AnxA2, and AnxA6, appear to target their interaction partners to specific membrane microdomains to contribute to the formation of compartment-specific signaling platforms along the endocytic pathway. A major challenge to understand the impact of scaffolding/targeting proteins on spatiotemporal signal transduction along endocytic pathways is the identification, isolation, and functional analysis of low-abundance Methods in Enzymology, Volume 535 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397925-4.00004-3

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2014 Elsevier Inc. All rights reserved.

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signal-transducing protein complexes in endocytic compartments. Here, we describe methods to isolate endosomes and to target signaling molecules to endosomes. Applying these methodologies to suitable animal or cell models will enable the dissection of signal transduction in the endocytic compartment in the presence or absence of annexins.

1. INTRODUCTION After internalization, endocytosed molecules are routed to early endosomes (EEs). In this compartment, the internalized material can be recycled back to the plasma membrane (PM) or remain there during their maturation to late endosomes (LEs) or multivesicular bodies (MVBs). LE/MVBs eventually fuse with lysosomes, where endocytosed molecules are degraded. The organelles of the endocytic pathway are endowed with SNARE and motor proteins, Rab GTPases, and phosphoinositides that confer spatiotemporal functionality to the system. Endosomal membranes are enriched in compartment-specific molecular markers such as Rab5, early endosome antigen 1 (EEA1), and phosphatidylinositol 3-phosphate (PI3P) in EE, Rab7, and phosphatidylinositol 3, 5-bisphosphate (PI3, 5P2) in LE or LAMP proteins in lysosomes (Sigismund et al., 2012). The endocytic compartment represents a functional continuity of the PM, which allows signaling events to continue once generated at the cell surface, making EE and LE to represent specific signaling platforms. By interaction with the cytoskeleton, endosomes allow the traffic of signaling molecules to different cellular locations and enable the degradation of signaling complexes through directing them to lysosomes. Over the last decade, the importance of endosomal signaling for growth factor/tyrosine kinase receptors (EGFR, TGFbR, Met, Notch, and TNFR) and G-protein-coupled receptors has been well established. Receptor signaling in endosomal compartments is associated with the presence of small Ras GTPases to control cellular outputs like growth, proliferation, and cell mobility (Flinn, Yan, Goswami, Parker, & Backer, 2010; Gould & Lippincott-Schwartz, 2009; Kermorgant & Parker, 2008; Lobert & Stenmark, 2011; Ohashi et al., 2011; Palamidessi et al., 2008; Platta & Stenmark, 2011; Sorkin & von Zastrow, 2009; Taub, Teis, Ebner, Hess, & Huber, 2007). Along these lines, we and others have studied the EGFR signaling pathway initiated at the PM and active in endosomes. Accordingly, many

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activated signaling proteins of the EGFR pathway have been detected in endosomes, for example, Shc, Grb2, Sos, Ras, and the kinases Raf-1, Mek, and Erk1/2 (Balbis, Parmar, Wang, Baquiran, & Posner, 2007; Lu et al., 2009; Moreto et al., 2008; Pol, Calvo, & Enrich, 1998; Sorkin & von Zastrow, 2009; Teis et al., 2006). It is generally believed that this reflects sustained signaling of internalized receptors (Mor & Philips, 2006), but more importantly, the multiple and differential location of signal-transducing molecules is crucial to diversify cellular signaling output (Calvo, AgudoIbanez, & Crespo, 2010). Despite the improved knowledge on compartment-specific signaling, there are still fundamental questions about the role and specificity of signaling molecules in endosomes. Alternatively, besides providing a signaling continuity derived from receptor activation at the cell surface, targeting of signaling molecules specifically to endosomes could elucidate its activation and signaling exclusively from endosomes and discriminate this from signals derived from the PM. In this context, scaffolding/targeting proteins are essential to organize and stabilize the formation of signal-transducing complexes in distinct cellular compartments (Kheifets & Mochly-Rosen, 2007; Palfy, Remenyi, & Korcsmaros, 2012; White, Erdemir, & Sacks, 2012). Their ability to specifically interact with certain signaling molecules provides a platform to recruit regulators/effectors and modulate signaling amplitude and kinetics in a celland stimulus-specific manner. Scaffolding/targeting proteins regulating signaling and trafficking of activated growth factor receptors in EE and LE include several annexins, in particular AnxA1, AnxA2, AnxA6, and AnxA8 (Grewal & Enrich, 2009). Annexins are a dynamic, multifunctional, and evolutionary conserved superfamily of proteins and are characterized by their ability to interact with biological membranes in a calcium-dependent manner (Gerke, Creutz, & Moss, 2005). The current understanding of the contribution of these annexins in the regulation of spatiotemporal signaling along the endocytic pathway is mostly derived from studies examining the EGFR/Ras/MAPK signaling cascade. It would go beyond the scope of this chapter to recapitulate all these findings and we refer the reader to reviews from our laboratory and others summarizing the role of annexins in cellular signaling and the endocytic pathway in more detail (Futter & White, 2007; Gerke et al., 2005; Grewal & Enrich, 2009). In brief, AnxA1 was one the first EGFR tyrosine kinase substrates identified and regulates EGF-induced MVB biogenesis to facilitate lysosomal degradation of EGFR (White, Bailey, Aghakhani, Moss, & Futter, 2006).

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The role of AnxA2 is more complex and involves the sorting of EGFR to recycling endosomes or MVB (Morel, Parton, & Gruenberg, 2009; Zobiack, Rescher, Ludwig, Zeuschner, & Gerke, 2003), possibly via interaction with cholesterol- and PI(4,5)P2-rich domains and the actin cytoskeleton and interaction and modulation of signaling output of regulators/ effectors such as src, PKC, SHP2, Pyk2, cdc42, and Rho (Hayes, Rescher, Gerke, & Moss, 2004). AnxA8 is found in LE/MVB and its ability to bind phosphatidylinositols and F-actin might affect final steps in EGFR degradation (Goebeler, Poeter, Zeuschner, Gerke, & Rescher, 2008). Our laboratory has focused on the role of AnxA6, which is a targeting protein for p120GAP and PKCa, both negative regulators of EGFR and Ras (Enrich et al., 2011). The ability of annexins to act as membrane organizers and scaffolding proteins is exemplified by AnxA6, which is a highly dynamic protein involved in several cellular events linked to membrane transport. This includes the reorganization of the actin cytoskeleton at the PM (Monastyrskaya et al., 2009), the delivery of cholesterol from LE to the Golgi and PM (Cubells et al., 2007), the cholesterol-dependent recruitment of cytosolic phospholipase A2 to the Golgi for Golgi vesiculation (Cubells et al., 2008), and t-SNARE trafficking and assembly in the secretory pathway (Reverter et al., 2011). AnxA6 is synthesized as a cytosolic protein but is predominantly targeted to the PM upon cell activation. This possibly includes the association of AnxA6 with cholesterol-enriched membrane microdomains (lipid rafts, both caveolae and noncaveolae). Furthermore, AnxA6 is a major component of rat liver endosomes (Jackle et al., 1994; Pol, Ortega, & Enrich, 1997). Together with cell culture studies, these findings linked AnxA6 with low-density lipoprotein (LDL) receptor-mediated endocytosis (Grewal et al., 2000; Kamal, Ying, & Anderson, 1998) and the delivery of LDLcontaining LE to lysosomes for degradation (Pons et al., 2000). LDLinduced translocation of AnxA6 to LE (Grewal et al., 2000) correlates with the recruitment of AnxA6 to cholesterol-enriched LE, establishing a cholesterol-dependent membrane-binding capacity of AnxA6 in LE (de Diego et al., 2002). In addition, AnxA6 interacts with EGFR, Ras, Raf-1, MAPK, and effectors/regulators of the EGFR/Ras pathway at the cell surface and endosomes (Grewal & Enrich, 2006; Koese et al., 2012; Vila de Muga et al., 2009). In line with studies on other scaffolding proteins, expression levels and subcellular localization of annexins seem to modulate membrane transport and signal complex formation to differentially determine signaling output at the PM or endosomes.

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One of the challenges in dissecting signaling events in different subcellular compartments is the identification and isolation of low-abundance signal protein complexes and to provide material for further functional assays. In the following sections, we describe methods to isolate endosomes and to target signaling molecules to endosomes. In combination with suitable animal and cell models, this can serve to dissect the endocytic compartment and determine the contribution of annexins in the signaling output of intracellular signaling pathways.

2. ISOLATION OF ENDOCYTIC COMPARTMENTS In this section, we describe procedures to isolate endosomes from rat or mice liver and cell cultures, such as Chinese hamster ovary (CHO) cells, respectively. A number of methods have been developed for the isolation of endosomes largely free of contamination from other intracellular membranes. The similarity in the equilibrium densities of endocytic vesicles, Golgi membranes, and other smooth membranes has led to the development of methods for specifically modifying the density of endocytic vesicles based on their ligand content (Belcher et al., 1987; Bergeron, Searle, Khan, & Posner, 1986; Debanne, Evans, Flint, & Regoeczi, 1982; Evans & Flint, 1985; Mueller & Hubbard, 1986). For rat or mice liver, we used the method developed by Belcher et al. (1987), which distinguishes three endosomal fractions on the basis of their distinct morphologies and their association with, or absence of, various radioligands at different time points with “early” (compartment of uncoupling receptors and ligands (CURLs)) and “late” (MVB) endosomes and a third endosomal fraction, the receptor-recycling compartment (RRC), representing recycling and transcytotic structures (Enrich, Jackle, & Havel, 1996). Although this procedure is based on the shift density that endocytic structures undergo after loading with LDL in estradioltreated rats, these fractions have been proven most valuable to characterize signaling platforms in endosomes. Indeed, one- and two-dimensional gel electrophoresis to investigate the protein composition of the three isolated endosome fractions identified a differential distribution of various receptors and ligands, including growth factor receptors and signaling proteins. In summary, rat and mouse liver endocytic compartments consist of specific and defined stations, with CURL being the central and most important location for sorting and RRC being involved in recycling and transcytosis (Enrich, Pol, Calvo, Pons, & Jackle, 1999; Pol et al., 1997).

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2.1. Isolation of endocytic fractions from rat liver Three rats are treated for 3 days with 17-a-ethinyl estradiol, dissolved in propylene glycol (1 mg/ml) and injected (5 mg/kg) subcutaneously, to induce a high rate of uptake of LDL into the liver. Rats are then anesthetized with 4% isoflurane, and human LDL (5 mg of protein) is injected into the femoral vein (Belcher et al., 1987). 1. After 15 min, livers are flushed via the portal vein with 0.15 M NaCl and 0.1% EDTA. Then, the livers are removed, minced with scalpels, and using a Dounce homogenizer, homogenized (1:3 wt/vol) in 0.25 M sucrose with protease inhibitors (1 mM PMSF, 100 mM leupeptin, 150 mM aprotinin, and 1 mM pepstatin). 2. The homogenate is centrifuged for 10 min at 500
g in an SW28 rotor (Beckman Coulter). 3. The supernatant is centrifuged at 4800
g for 20 min in an SW28 rotor (Beckman Coulter). 4. Finally, the supernatant is centrifuged at 16,600
g for 20 min in a type 30 rotor (Beckman Coulter). 5. This third supernatant (64 ml) is diluted with 30 ml of isotonic Percoll solution at pH 7.4 (27 ml Percoll plus 3 ml 2.5 M sucrose, 9:1 vol/vol), layered in tubes each containing a marker density of 1.062 g/ml (Pharmacia, Uppsala, Sweden) and centrifuged for 45 min at 40,700
g in a type 30 rotor (Beckman Coulter). 6. The fraction over the marker density is collected, 2 volumes of 0.9% NaCl are added, and the fraction is then layered on 2 ml of 2.5 M sucrose and centrifuged at 24,250
g for 45 min in an SW28 rotor (Beckman Coulter). 7. The white crude endosome band is harvested and 2.5 M sucrose is added to a final density of 1.15 g/ml. The 1.15 g/ml portion is loaded at the bottom of the tubes containing a discontinuous sucrose gradient of densities 1.032, 1.074, 1.11, and 1.139 g/ml. Tubes are centrifuged for 170 min at 140,000
g in an SW28 rotor (Beckman Coulter). Three distinct populations are obtained at the density interfaces: MVB at 1.032/ 1.074 g/ml, CURL at 1.074/1.110 g/ml, and RRC at 1.110/ 1.130 g/ml. 8. Each fraction is collected and ice-cold water is added to generate isotonic fractions. Finally, the isotonic fractions are pelleted by centrifugation at 71,000
g for 30 min in a 50 Ti rotor (Beckman Coulter), resuspended in 0.9% NaCl, and stored at 80  C.

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170 -

Dissection of early sorting endosomes

RRC

CURL

B MVB

A

Sucrose density

EGFR

19 21 23 25 27 29

(% w/v)

45 -

Gαi

14 -

VAMP3

25 -

Ras

70 -

Raf-1

70 -

Raf-1

68 -

Annexin A6

45 -

Mek-P

28 -

Rab4

74 -

Mek-32 P

36 -

Annexin A2

Figure 4.1 Signaling in isolated rat liver endosomes. (A) Differential distribution of signaling proteins (EGFR, Ras, Raf-1, and Mek) and G-proteins (Gai) in three endosomal fractions (MVB, CURL, and RRC) from rat liver by Western blotting. The electrophoretical mobility of Raf-1 differs in the various fractions suggesting posttranslational modification. The last panel shows Raf-1 kinase activity as judged by GST-Mek phosphorylation from Raf-1 immunoprecipitates purified from MVB, CURL, and RRC (see Section 3.2). (B) Biochemical dissection of CURL. Isolated early sorting endosomes (CURL) were loaded at the bottom of a multistep sucrose gradient (from 19% to 29% w/v) and centrifuged for 2 h and 50 min at 78,000
g in an SW28 rotor. Samples from this gradient (2 ml) were pelleted, resuspended in 0.9% NaCl, TCA-precipitated, electrophoresed (4 mg/lane), and transferred to Immobilon-P membranes. Western blotting was used to analyze the distribution of proteins along the gradient.

If further separation is needed, the interface corresponding to CURL or RRC can be mixed with heavy sucrose (2.5 M) and loaded at the bottom of a discontinuous sucrose gradient with 19%, 21%, 23%, 25%, 27%, and 29% sucrose (w/v) for CURL or 29%, 31%, 33%, 35%, 37%, and 39% for RRC. Then, the tubes are centrifuged at 136,000
g for 2 h 50 min in a Beckman SW28 rotor. Following centrifugation, the interfaces are collected, pelleted, resuspended in 0.5 ml 0.9% NaCl, and stored at 80  C. Figure 4.1A shows a representative Western blot profile of signaling proteins in the three endosomal fractions. Further subfractionation of the CURL fraction is shown in Fig. 4.1B. 2.1.1 Characterization of the recycling endocytic compartment (RRC) In view of the complexity of the endocytic compartment, it is not surprising that isolation and biochemical characterization of this pleiomorphic organelle from tissue homogenates has been a major task. In respect to compartmentalized signaling, recycling endosomes have received increased attention in recent years, as growth factor receptors and signaling proteins can be

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diverted to this compartment for transport back to the cell surface. For instance, EGFR recycling was previously considered a default pathway, but we showed that this involves a complex and PKCd-dependent regulation of actin dynamics (Llado et al., 2004, 2008). It was assumed that tubules contained in RRC have their origin in the membrane appendages emanating from CURL and MVB, as structures involved in the recycling of receptors (Belcher et al., 1987; Geuze et al., 1984). In principle, RRC is a subcellular fraction that comprises all those morphological-described endocytic structures, tubules, and vesicles, which do not contain LDL (after LDL loading) and therefore float at the same density in the sucrose gradient (d ¼ 1.13 g/ml, 35.5% sucrose). However, these endocytic membranes possibly originate from different cellular compartments. Thus, whereas RRCr (recycling) and RRCt (transcytosis) might come from different regions of the membranous tubular extensions of CURL (and MVB), RRCc (caveolae) originates from the caveolae-enriched PM domains at the sinusoidal PM (a subcellular well-characterized fraction denominated caveolin-enriched PM fraction) (Pol, Calvo, Lu, & Enrich, 1999).

2.2. Isolation of endocytic fractions from CHO cells Several methods have been developed for the isolation of endocytic fractions from cells in culture and even procedures to obtain enriched populations of early and late endocytic structures. However, none of these procedures allow the degree of enrichment obtained compared to those prepared from the liver, at least in terms of morphology of the isolated fractions. Also, and very importantly, neither of the methods using cells in culture are able to obtain a reasonably pure and enriched fraction representative of the recycling endocytic compartment (RRC). In the following sections, we describe those fractionation procedures optimized for CHO cells. Over the years, this protocol has also been used to isolate endocytic fractions from other cell lines, such as baby hamster kidney cells (Jost, Zeuschner, Seemann, Weber, & Gerke, 1997; Morel et al., 2009). However, given the cellspecific differences in endosome morphology and endocytic transport, detailed analysis and characterization of isolated fractions using established early/late and lysosomal markers is required. 2.2.1 Early and late endosomes Subcellular endosomal fractions are prepared according to the protocol described by Gorvel, Chavrier, Zerial, and Gruenberg (1991).

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1. CHO cells are grown in Ham’s F-12 containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. 4–6
107 CHO cells are used for each gradient. If needed, increased amounts of enlarged LE can be obtained upon treatment with U18666A (4.5 mg/ml for 16 h) (Cubells et al., 2007; de Diego et al., 2002) or bafilomycin A1 (50 nM for 16 h) (Lu et al., 2009). U18666A treatment or LDL loading (0.5 mg/ml for 120 min) can be utilized to recruit or increase AnxA6, and possibly other annexins, in LE fractions (de Diego et al., 2002; Grewal et al., 2000). 2. Cells are put on ice, washed two times with cold PBS, and collected in 5 ml of homogenization buffer (250 mM sucrose, 3 mM imidazole, pH 7.4, and protease inhibitors: 1 mM PMSF, 1 mM aprotinin, 20 mM leupeptin, and 5 mM Na3VO4). 3. Cells are pelleted at 500
g in a bench centrifuge, resuspended in 2 ml of homogenization buffer, and homogenized by 10 passages through a 22 gauge needle. Complete homogenization is confirmed under the phase microscope. 4. The homogenate is centrifuged for 15 min at 4  C at 1000
g in a bench centrifuge. 5. The postnuclear supernatant (PNS) is brought to a final 40.2% sucrose (w/v) concentration by adding 62% sucrose (3 mM imidazole, pH 7.4) and loaded (4 ml) at the bottom of an SW41 centrifugation tube (Beckman Ultraclear). Then, 35% sucrose (3 ml), 25% sucrose (3 ml), and finally 3 ml of homogenization buffer are poured stepwise on top of the PNS. 6. The gradient is centrifuged for 90 min at 210,000
g and 4  C in a swing out Beckman SW41 Ti rotor. 7. After centrifugation, 1 ml fractions are collected from top to bottom. Aliquots of each fraction are trichloroacetic acid-precipitated to determine the distribution of markers by Western blotting.

3. TARGETING Raf-1 SIGNALING TO EARLY ENDOSOMES EGFR activates different signaling pathways that can trigger a variety of cellular responses. The Ras/Raf-1/Mek/Erk (MAPK) pathway is an excellent example and regulates fundamental processes such as proliferation, differentiation, migration, and apoptosis. Upon ligand binding, EGFR activates Ras GTPases, which transmit the extracellular signals to Raf kinases. Raf-1 plays a central role in EGFR-mediated activation of the MAPK

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pathway and is recruited to the PM through binding to activated Ras. Once translocated to the PM, Raf-1 is activated by sequential dephosphorylation and phosphorylation events. Several phosphatases, kinases, and scaffold proteins have now been identified to finely coordinate and guarantee the proper spatiotemporal activation of Raf-1 and subsequently Mek and ERK (McKay & Morrison, 2007). In particular, the phosphorylation of Ser338 strongly correlates with the activated state of Raf-1. As outlined earlier, Ras/Raf-1 and MAPK signaling has also been observed in EE, but the low abundance of these signaling proteins in endocytic compartments has yet limited the analysis of endosomal Raf-1 signaling. To analyze Raf-1 activation specifically in EE, we have developed a method to target Raf-1 to this compartment. Proteins like EEA1 or Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) are recruited to endosomes through their FYVE domains, which interact specifically with PI3P on early endosomal membranes. This was demonstrated in elegant studies by the Stenmark laboratory, showing that the fusion of 2xFYVE domains with the green fluorescent protein (GFP) can serve as an in vivo marker of the EE compartment (Gillooly et al., 2000; Pattni, Jepson, Stenmark, & Banting, 2001; Petiot, Faure, Stenmark, & Gruenberg, 2003). Figure 4.2 shows GFP–2xFYVE localization in EEA1-positive endosomes (EE), but not in LBPA-positive LE. Based on the ability of the FYVE domain to target EE and to overcome the technical difficulty to analyze Raf-1 signaling in this compartment, we therefore cloned an expression vector encoding Raf-1 fused to CFP–2xFYVE. To avoid interference of CFP– 2xFYVE with the signaling capacity of Raf-1, the fluorescently tagged FYVE domain was fused to the Raf-1 N-terminus. Therefore, the human Raf-1 cDNA (kindly provided by Dr. Richard Marais, Institute of Cancer Research, London, United Kingdom) was subcloned into a CFP-containing Living Colors expression vector (Clontech). Then, the PCR-amplified 2xFYVE motif was cloned in between CFP and Raf-1 using BsrG1 restriction sites, generating a fusion protein with CFP N-terminal of the chimeric 2xFYVE-Raf-1 protein.

3.1. Expression of CFP–2xFYVE-Raf-1 and Raf-1 activation analysis 1. COS-1 cells are grown in DMEM, containing 10% FBS, 1 mM pyruvic acid, antibiotic (50 U/ml penicillin and 50 mg/ml streptomycin), 2 mM glutamine, and 1% nonessential amino acids. Cells are plated on coverslips up to 50% confluence for transient transfections.

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GFP–2xFYVE

GFP–2xFYVE

EEA-1

LBPA

Figure 4.2 GFP–2xFYVE localizes in early endosome compartment. COS-1 cells grown on coverslips were transfected with GFP–2xFYVE. After 24 h, cells were fixed with 4% paraformaldehyde, and immunocytochemistry with monoclonal anti-EEA1 (BD Transduction Laboratories) (upper panels) or monoclonal anti-LBPA (Echelon Inc.) (lower panels) and the corresponding Alexa555 (Molecular Probes, Invitrogen) secondary antibody was performed. Images were acquired through GFP and CY3 channels with the epifluorescence Axiovert 200M microscope (Zeiss). Arrows in panel A indicate colocalization between GFP–2xFYVE and EEA1. Arrows in panel B show no colocalization between GFP–2xFYVE and LBPA.

2. Cells are transfected with CFP–2xFYVE-Raf-1 using the Effectene kit (Qiagen) and analyzed 24 h thereafter. 3. Cells are starved for 2 h and then stimulated with EGF (100 ng/ml) in DMEM, 0.1% BSA for 0, 5, and 15 min. 4. Cells are washed twice in PBS and fixed with freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min at RT. 5. Detection of Ser338-phosphorylated Raf-1 (activated Raf-1) in endosomes can be performed using immunofluorescence microscopy with the anti-PSer338-Raf-1 monoclonal antibody (Upstate, Millipore). Therefore, fixed cells are mildly permeabilized with PBS, 0.1% BSA, and 0.1% saponin, for 5 min. After two washes with PBS, cells are incubated with blocking solution (PBS and 1% BSA) for 5 min at RT. Then, coverslips are incubated with primary antibody (anti-PSer338-Raf-1) diluted in PBS, 0.1% BSA, and 0.02% saponin for 1 h, washed extensively, and thereafter incubated with the adequate Cy5-labeled secondary antibody (Jackson, ImmunoResearch; Europe Ltd.) for 30 min. After

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A

B 5ⴕ EGF

15ⴕ EGF PSer338/CFP–2xFYVE-Raf-1

PSer338-Raf-1 2xFYVE-Raf-1

0ⴕ EGF

1.0

*

0.8 0.6 0.4 0.2 0.0

0

5 Time (min)

15

Figure 4.3 Raf-1 targeted to early endosomes is activated by EGF. (A) COS-1 cells expressing CFP–2xFYVE-Raf1 were serum-starved for 2 h and then treated with EGF (100 ng/ml) for 0–15 min as indicated. Fixation and immunocytochemistry with the anti-PSer338Raf-1 monoclonal antibody followed by the corresponding Cy5 secondary antibody was performed as described (see Section 3.1). Images were acquired through the cyan and Cy5 channels with the epifluorescence Axiovert 200M microscope (Zeiss) (arrows indicate endosomal Raf-1 localization). (B) Graph showing the ratio of active PSer338Cy5-Raf-1 (Cy5) and total CFP–2xFYVE-Raf-1 (cyan) in endosomes after 0–15 min EGF treatment. Statistical significances of differences between control and EGF treatment were determined using the Student’s t test. Data are means  SEM; *p 5 cells for each group. Image analysis can be performed using the Image Processing Leica Confocal Software (FRET wizard) and ImageJ (Schneider, Rasband, & Eliceiri, 2012; Vila de Muga et al., 2009) or similar.

5. TARGETING ANNEXINS TO ENDOSOMES AND OTHER CELLULAR COMPARTMENTS Every annexin is generally found in multiple locations in each cell, making it difficult to specifically analyze the contribution or participation of endosomal annexins in signaling events and protein–protein

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interactions in this compartment. In addition, the transient and reversible nature of the Ca2þ-dependent membrane association of annexins complicates investigations aiming to address their scaffolding function in certain cellular sites. We therefore recently developed a model system for a constitutive membrane association of AnxA6 and A1 (Monastyrskaya et al., 2009). Using the PM-anchoring sequences of H- and K-Ras, we targeted both annexins to the PM independently of Ca2þ. Similarly, other researchers have developed other PM anchors or ER- and Golgitargeting sequences (Matallanas et al., 2006). As shown for CFP– 2FYVE-Raf-1 (see the preceding text), the fusion of the 2FYVE domain to fluorescently tagged AnxA1, A2, A6, and A8 could serve as an approach to target annexins to the endosomal compartment. Given the low abundance of annexins in these compartments, even upon cell stimulation, these experimental approaches will ultimately help to improve our understanding of the contribution of scaffold/targeting proteins in compartmentalized signaling.

6. SUMMARY Although originally regarded as a route for the degradation or recycling of membrane receptors, endosomal structures are now credited to be a crucial site for signal transduction. Studies using isolated fractions, in situ probes that identify activities in life cells or by targeting specific molecules, will provide new insights into the signaling dynamics of the endocytic compartment. The techniques presented in this chapter should be useful in future investigations to identify and characterize new details of the machinery along the vesicle and tubulovesicular membranes of the endocytic compartment.

ACKNOWLEDGMENTS We would like to thank all members of our laboratories, past and present, for their invaluable contributions. C. R., A. P., and C. E. acknowledge funding from Consolider-Ingenio (CSD2009-00016); the work in this laboratory is supported by grants from Plan Nacional from the Spanish Ministerio de Economı´a y Competitividad (BFU2012-36272 to C. E., BFU2011-23745 to A. P. and BFU2012-38259 to F. T.), Fundacio´ Marato´ TV3, and Generalitat de Catalunya (AGAUR). T. G. is supported by the National Health and Medical Research Council of Australia (NHMRC) and the University of Sydney (201002681). M. G.-B. is thankful to IDIBAPS fellowship.

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