MICROSCOPY RESEARCH AND TECHNIQUE 68:290–295 (2005)
GABAB Receptor Intracellular Trafficking After Internalization in Paramecium PAOLA RAMOINO,1* CESARE USAI,2 FRANCESCO BELTRAME,3 MARCO FATO,3 LORENZO GALLUS,1,4 GRAZIA TAGLIAFIERRO,4 RAFFAELLA MAGRASSI,5,6 AND ALBERTO DIASPRO5,6 1
Department for the Study of Territory and its Resources (DIP.TE.RIS.), University of Genoa, 16132 Genoa, Italy Institute of Biophysics, CNR Genoa, 16149 Genoa, Italy 3 Department of Communication, Computer and System Sciences (DIST), University of Genoa, 16145 Genoa, Italy 4 Department of Biology, University of Genoa, 16132 Genova, Italy 5 LAMBS-MicroScoBio and IFOM Research Center, Department of Physics, University of Genoa, 16146 Genova, Italy 6 INFM/CNR Research Unit, Department of Physics, University of Genoa, 16146 Genoa, Italy 2
KEY WORDS
GABA receptors; receptor trafficking; rab proteins; immunofluorescence; confocal microscopy; protozoa
ABSTRACT The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome–lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively. Microsc. Res. Tech. 68:290–295, 2005. V 2005 Wiley-Liss, Inc. C
INTRODUCTION We have previously shown in Paramecium, a motile single-celled organism, that GABAB receptors are internalized by clathrin-dependent and clathrin-independent endocytosis (Ramoino et al., 2003). The present article is focused on the mechanisms for GABAB receptor intracellular trafficking after internalization. Although a great deal has been learned about the mechanism mediating the initial endocytosis of certain G protein-coupled receptors (GPCRs) from the plasma membrane, relatively little is known about the mechanisms that determine the specificity of GPCR trafficking after endocytosis. Endocytosis of receptors can contribute to functional resensitization of signal transduction by promoting dephosphorylation and recycling of receptors to the plasma membrane (Ferguson et al., 1998), as well as by the down-regulation of receptors, a process that leads to functional desensitization of signal transduction by reducing the number of receptors present in the plasma membrane and promoting degradation of receptors in lysosomes (Kallal et al., 1998; Hoxie et al., 1993; Roettger et al., 1995). These processes of receptor regulation are thought to involve membrane trafficking of receptors via distinct recycling or degradative pathC V
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ways that can mediate opposite effects on the regulation of functional signal transduction (reviewed by Bohm et al., 1997; Ferguson, 2001). Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled back to the plasma membrane, either directly or through the recycling endosomes (Yamashiro and Maxfield, 1984; Hopkins et al., 1994; Ullrich et al., 1996). These processes are mediated by a continuous traffic of vesicular and tubular intermediates, which need to be coordinated to ensure proper progression of cargo through the different compartments. Several rab family members have been localized to distinct compartments of the endocytic pathway and play different roles in endocytosis and recycling (Chavrier et al., 1990; van der Sluijs et al., 1991; Olkkonen et al., 1993; Ullrich et al., 1996).
*Correspondence to: Paola Ramoino, DIP.TE.RIS., University of Genoa, Corso Europa 26, 16132 Genoa. E-mail:
[email protected] Received 1 January 2005; accepted in revised form 23 August 2005 Contract grant sponsor: IFOM, Milan, Italy DOI 10.1002/jemt.20250 Published online in Wiley InterScience (www.interscience.wiley.com).
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Rab5 and rab4 are both localized to early endosomes but exert opposite effects on the uptake of membranebound proteins. Rab5 plays a role in the formation of clathrin-coated vesicles at the plasma membrane (McLauchlan et al., 1998), their subsequent fusion with early endosomes, in the homotypic fusion between early endosomes (Gorvel et al., 1991; Bucci et al., 1992) and in the interaction of early endosomes with microtubules (Nielsen et al., 1999). Rab4 has been implicated in the regulation of membrane recycling from the early endosomes to the recycling endosomes or directly to the plasma membrane (Daro et al., 1996). In accordance with its functional diversity, rab5 lies at the center of a complex machinery made up of several effector proteins (Christoforidis et al., 1999). Of these proteins, EEA1 was identified as a core component of the homotypic endosome docking and fusion machinery and was shown to play a role in the docking/tethering of the endosome membranes (Christoforidis et al., 1999). EEA1 is predominantly localized to early endosomes and is regarded as a specific marker of this compartment. Because of this localization and given its function in endosome membrane docking (Christoforidis et al., 1999), it has been proposed that EEA1 may confer directionality to rab5-dependent vesicular transport to the early endosomes. Another effector protein for rab5 is rabaptin-5. Rabaptin-5 binds directly to the GTP-bound form of rab5 and is recruited to early endosomes by rab5 in a GTP-dependent manner (Stenmark et al., 1995). It stabilizes rab5 in the GTP-bound active form by down-regulating the GTP hydrolysis (Rybin et al., 1996) and, finally it is required for the homotypic fusion between early endosomes as well as for the heterotypic fusion of clathrincoated vesicles with early endosomes in vitro (Stenmark et al., 1995; Horiuchi et al., 1997). Rabaptin-5 also interacts with GTP-bound rab4 (via a distinct structural unrelated N-terminal rab-binding domain), but does not appear to interact with rab11 (Vitale et al., 1998), a GTPase that is highly present on the recycling endosome and whose activity is required for receptor recycling through this compartment (Ullrich et al., 1996). Thus the same effector interacts with the two rab proteins, which act sequentially in transport through the early endosomes (Vitale et al., 1998). In this study we demonstrate, by immunofluorescence and confocal microscopy, the presence in the unicellular eukaryote, Paramecium, the immunoreactivity for several proteins implicated in the regulation of membrane internalization, recycling, and degradation in mammalian cells. MATERIALS AND METHODS Cell Cultivation Paramecium primaurelia (P. primaurelia) stock 90 was grown at 258C in lettuce medium (pH 6.9) bacterized with Enterobacter aerogenes. Cells were harvested in mid-log phase of growth. Antibodies Monoclonal antibodies rab4, clone 7 (0.625 lg/mL); rab5, clone 15 (2.50 lg/mL); rab11, clone 47 (0.625 lg/ mL); rabaptin-5, clone 42 (1.25 lg/mL); EEA1, clone 14
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(0.25 lg/mL); LAMP1, clone 25 (2.50 lg/mL); and TGN38, clone 2 (2.50 lg/mL) were purchased from Transduction Laboratory (BD Biosciences, San Jose, CA, USA). The polyclonal guinea pig anti-GABAB R1 from Sigma (St Louis, MO, USA), and the antiguinea pig Alexa Fluor 594 and antimouse Alexa Fluor 488 secondary antibodies from Molecular Probes (Eugene, OR, USA). Immunolabeling Cells were fixed in 4% paraformaldehyde in PBS buffer (0.01 M, pH 7.4) for 30 min, washed three times with PBS, and incubated for 60 min with 3% bovine serum albumin (BSA) and 1% Triton X-100 in PBS for 1 h at room temperature (23–248C). Afterward, the sections were incubated overnight at 48C in the polyclonal antibody against the GABAB R1 receptor (1:2000), together with one of the above-mentioned monoclonal antibodies. After three washes in PBS containing 1% BSA and 0.1% Triton X-100 for 10 min each, a cocktail of the secondary antibodies Alexa Fluor 594 antiguinea pig IgG (1:300) and Alexa Fluor 488 antimouse IgG (1:300) was applied for 2 h at 378C. After repeated washes in PBS the cells were mounted in glycerol/buffer. In control experiments, the absence of cross-reactivity was verified by omission of either primary antibodies, which resulted in a complete lack of immunolabeling. Image Acquisition Images (1024 3 1024 3 8 bit) were acquired by a confocal laser scanning microscope Nikon C1 (Nikon Instruments, Florence, Italy), mounted on an inverted optical microscope Nikon Eclipse TE 300. An argon-ion laser (488 nm, 514 nm) and a He-Ne laser (543.5 nm) provided the excitation beams. Emission was observed through the standard filter sets for fluorescein fluorescence (515/530 nm) and Texas red fluorescence (emission, 620 nm). Using a laser power of 1.5 mW (with illumination attenuated by a 3% transmission neutral density filter to reduce photobleaching), a 20 lm pinhole diameter, and an oil immersion objective 1003/ NA ¼ 1.3, serial optical sections were taken through the cell at a z-step of 0.5 lm. The software EZC1 (Coord, Amsterdam, NL) was used for image acquisition, storage, and analysis. Images are representative of observations of an average of 30 cells in each sample. Illustrations were prepared using PhotoShopPro 7. Alterations to the original images included correction for contrast and removing dust and scratches. Image Analysis The spatial colocalization was analyzed through 2D correlation cytofluorograms accomplished by routines integrated in the ImageJ 1.31_03 software (Wayne Rasband, Nat. Inst. of Health, USA). Two pixels are considered as colocalized: (a) if their respective intensity levels (0–255, 8 bit) are higher than the threshold of their channels, set to 40 for both green and red channels, and (b) if the ratio of their intensity levels is strictly higher than 50%.
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RESULTS Intracellular GABAB Receptors Colocalize With Endosomal Markers We examined the distribution of GABAB-like receptor immunoreactivity in P. primaurelia and compared this immunolabeling with those obtained with the endocytic pathway marker antibodies rab5, EEA1, and rabaptin 5. Small and large GABAB-like receptor immunostained vesicles showed a punctate distributive pattern throughout the cytoplasm; however were more densely packed at the periphery of the cell. GABAB receptor antibody strongly labeled early endosomes, identified by antibody immunostaining against the molecular markers EEA1 and rab 5 (Clague, 1998). Both GABAB-like receptor and rab5-like immunoreactivities were localized in vesicles showing a clustered distribution near the cell membrane and in the most inner portion of the cytoplasm; with the double immunolabeling method most of the peripheral vesicle clusters appeared yellow because GABAB-like receptors and rab5-like immunoreactivities partly colocalized (Fig. 1a). A similar colocalization pattern was obtained using, together with the anti-GABABR1 antibody, either the monoclonal antibodies anti-EEA1 (Fig. 1b) or antirabaptin-5 (Fig. 1c). Colocalization percentages of GABAB-like receptor with rab5-like, EEA1-like, and rabaptin-5-like proteins were reported in Table 1.
Retrieval of GABAB Receptor by Recycling Endosomes Internalized receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment were characterized by the presence of rab4 and rab11 proteins. To visualize GABAB receptor retrieval, the cells were double labeled with anti-GABABR1 and, either antirab4 or antirab11 as primary antibodies, and visualized with antiguinea pig Alexa Fluor 594 and antimouse Alexa Fluor 488 secondary antibodies, respectively. All immunostained vesicles showed a clustered distribution near the cell membrane and inside the cytoplasm; the yellow immunofluorescent vesicles, due to the colocalization of the GABAB-like receptor with either rab4-like or rab11-like immunofluorescence, distributed in the peripheral zone is visible in Figures 1d and 1e. Rab 11 also controls the traffic to the trans-Golgi network (TGN), which is generally identified through the marker TGN38. Moreover, TGN38 protein was shown to undergo constitutive cycling through the cell surface, bypassing late endosomes as it moves back to the TGN (Mallet and Maxfield, 1999). Evidence was given for the presence of GABAB receptors in the TGN by the colocalization of GABAB-like with TGN38-like immunoreactivity; the yellow fluorescence was detected in a small number of vesicles located in the cytoplasm (Fig. 1f). Indeed, the Golgi apparatus of Paramecium is decentralized and occurs as hundreds of small stacks of three or so highly cisternae scattered throughout the cytoplasm (Esteve, 1972). Colocalization percentages of GABAB-like receptor with rab4-like, rab11-like, and TGN38-like proteins were reported in Table 1.
GABAB Receptors are Degraded by Lysosomes In addition to receptor recycling, another possible fate for sequestered receptors is their degradation. Receptors destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome–lysosome pathway (for review, see Barnes, 2000). We have shown that in Paramecium most of the GABAB-like receptor immunostained vesicles were also immunolabeled with the lysosomal marker LAMP1 antibody (Fig. 1g). Colocalization percentage of GABABlike receptor with LAMP1-like protein was reported in Table 1. DISCUSSION We have previously demonstrated, using immunocytochemical and ultrastructural methods, that GABAB receptors are internalized in Paramecium via clathrindependent and -independent endocytosis and via a dynamin-dependent mechanism (Ramoino et al., 2003). In fact, surface GABAB receptors can cluster on clathrin-coated pits, which invaginate during the process of endocytosis, and some intracellular GABAB receptors are found on clathrin-coated vesicles. Yet, GABAB receptors also cluster with caveolin 1, a caveolae marker protein. A clathrin- and dynamin-dependent mechanism in the adrenergic receptor internalization has been already shown in Paramecium (Wiejak et al., 2004a,b). Here we have studied how GABAB receptor trafficking in Paramecium fits into the conception of general membrane trafficking. As illustrated in Figure 1h, the majority of membrane proteins that undergo endocytic trafficking are initially internalized to the rab5-positive sorting endosome. Once in the sorting endosome, membrane proteins can be targeted to the late endosome–lysosome pathway or enter the endosomal recycling cycle and undergo exocytosis back to the cell surface. Fitting our data into the known model of endocytic trafficking, we hypothesize that constitutive trafficking of GABAB receptors could occur in Paramecium in the following manner. After internalization by rab5- and clathrin-dependent buddings and by caveolae, GABAB receptors are first delivered to early endosomes, characterized by the EEA1 and rab5 markers. Receptors are then partly recycled back to cell membrane and partly degraded. The recycling of GABAB receptors is seen by the overlapping of their immunolocalization with both rab4-like and rab11-like immunostaining. Rab4 controls the rapid recycling of cargo proteins directly back to the cell surface from rab4/ rab5 positive endosomal structures, and the slow recycling of cargo via rab11 positive recycling endosomes (So¨nnichsen et al., 2000). Rab11 is ideally localized to early endosomes, perinuclear recycling endosomes, as well as to the TGN and is considered to control slow endosomal recycling, as well as traffic to the Golgi apparatus (Ullrich et al., 1996; Chen et al., 1998). The traffic of GABAB receptors to Golgi apparatus is seen by the colocalization of GABAB-like with TGN38-like immunoreactivity. The communication between contiguous rab-domains and thereby the sequential transport of receptors from one intracellular compartment to another is regulated by rab effector rabaptin-5. Furthermore a fraction of GABAB receptors seemed to be
GABAB RECEPTOR TRAFFICKING IN PARAMECIUM
Fig. 1. (a–g) Colocalization of GABAB receptor (red) in Paramecium primaurelia with proteins involved in the intracellular trafficking (green): (a) rab5-like, (b) EEA1-like, (c) rabaptin5-like, (d) rab4-like, (e) rab11-like, (f) TGN38-like, and (g) LAMP1-like proteins. In all cases examined most receptors aggregate in clusters and their
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fluorescence is localized in spots, varying in size and number, distributed on the plasma membrane and throughout the cytoplasm. Optical sections collected in the middle plane (a, b, c, f, g) and in the peripheral zone of the cell (d–e) are shown. Bar, 20 lm. (h) Schematic drawing of the endocytic pathway in mammalian cells.
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TABLE 1. Colocalization of GABAB receptor labeling with various endocytic protein labeling Red GABAB receptor GABAB receptor GABAB receptor GABAB receptor GABAB receptor GABAB receptor GABAB receptor
Green
Red/green (%)
Green/red (%)
Rab5 EEA1 Rabadaptin-5 Rab4 Rab11 LAMP1 TGN38
37 6 5 45 6 6 38 6 5 22 6 4 24 6 3 34 6 4 16 6 3
39 6 6 35 6 4 38 6 5 23 6 4 33 6 5 28 6 5 17 6 4
Every colocalization value is the average from four optical sections of five cells. Data were calculated as means 6 standard errors.
directed to lysosomes for degradation, as shown by GABAB-like and LAMP1-like immunocolocalization. For rab4 and rab11 proteins, sequences are cloned from Paramecium (International Paramecium genomic project, Dessen et al., 2001). Also rab7, a positive regulator of homotypic fusion between late endosomes, was cloned and sequenced in Paramecium and revealed 79% identity to N-terminal region of human rab7 (Surmacz et al., 2003). The confocal laser scanning microscopy revealed its immunoanalogue localized to the phagosomal membrane (Surmacz et al., 2003). Therefore, plasma membrane components are internalized by endosomes, which are first localized in the cortical region of the cell, transported in the most internal cytoplasmic portion, and fused with other endosomal compartments until their content is transferred to the phagosomes (Ramoino et al., 2001). With respect to other GPCRs, it was shown that the adrenergic receptors traffic to rab5 positive early endosomes following agonist-stimulated internalization (Moore et al., 1995; Seachrist et al., 2000). Furthermore, endothelin receptors rapidly localize with the rab5 proteins shortly after internalization, despite the fact that the endothelin A receptor is recycled to the cell surface, whereas the endothelin B receptor is targeted to lysosomes (Bremnes et al., 2005). A rapid recycling of the adrenergic receptors appears to occur directly from the early endosomal compartment (Seachrist et al., 2000), whereas other GPCRs undergo slower recycling, in which they traffic from early endosomes through the pericentriolar recycling endosomal compartment and then back to the plasma membrane (Trischler et al., 1999). Upon chronic agonist stimulation, several GPCRs undergo lysosomal trafficking and subsequent proteolytic degradation (reviewed by Ferguson, 2001). In fact, a significant fraction of adrenergic receptors specifically traffic to lysosomes via rab5 positive early endosomes (Moore et al., 1999). The dopioid receptor is also targeted to lysosomes and its degradation is blocked by inhibitors of lysosomal proteolysis (Tsao and von Zastrow, 2000). In conclusion, using standard immunomarkers for early endosomes, recycling vesicles, and lysosomes, and comparing our data with those obtained in mammalian cells relating to the internalization and recycling of other GPCRs, we may assume that in Paramecium GABAB receptors are partly recycled to cell plasma membrane and partly degraded into lysosomes. Moreover, using immunohistochemical methods we show that in the single-celled organism Paramecium, as in mammalian cells, rab-like proteins are involved in the vesicle transport from one compartment to another.
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