The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS
2008; Springer Nature; Volume: 27; Issue: 7 Linguagem: Inglês
10.1038/emboj.2008.59
ISSN1460-2075
AutoresPrisca Liberali, Elina Kakkonen, Gabriele Turacchio, Carmen Valente, Alexander Spaar, Giuseppe Perinetti, Rainer A. Böckmann, Daniela Corda, Antonino Colanzi, Varpu Marjomäki, Alberto Luini,
Tópico(s)Genetic Syndromes and Imprinting
ResumoArticle20 March 2008free access The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS Prisca Liberali Prisca Liberali Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Elina Kakkonen Elina Kakkonen Department of Biological and Environmental Science, Nanoscience Centre, University of Jyvaskyla, Jyvaskyla, Finland Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Carmen Valente Carmen Valente Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Alexander Spaar Alexander Spaar Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Giuseppe Perinetti Giuseppe Perinetti Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Rainer A Böckmann Rainer A Böckmann Theoretical and Computational Membrane Biology, Centre for Bioinformatics, Saarland University, Saarbruecken, Germany Search for more papers by this author Daniela Corda Daniela Corda Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Antonino Colanzi Corresponding Author Antonino Colanzi Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Varpu Marjomaki Varpu Marjomaki Department of Biological and Environmental Science, Nanoscience Centre, University of Jyvaskyla, Jyvaskyla, Finland Search for more papers by this author Alberto Luini Corresponding Author Alberto Luini Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Prisca Liberali Prisca Liberali Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Elina Kakkonen Elina Kakkonen Department of Biological and Environmental Science, Nanoscience Centre, University of Jyvaskyla, Jyvaskyla, Finland Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Carmen Valente Carmen Valente Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Alexander Spaar Alexander Spaar Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Giuseppe Perinetti Giuseppe Perinetti Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Rainer A Böckmann Rainer A Böckmann Theoretical and Computational Membrane Biology, Centre for Bioinformatics, Saarland University, Saarbruecken, Germany Search for more papers by this author Daniela Corda Daniela Corda Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Antonino Colanzi Corresponding Author Antonino Colanzi Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Varpu Marjomaki Varpu Marjomaki Department of Biological and Environmental Science, Nanoscience Centre, University of Jyvaskyla, Jyvaskyla, Finland Search for more papers by this author Alberto Luini Corresponding Author Alberto Luini Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy Search for more papers by this author Author Information Prisca Liberali1, Elina Kakkonen2, Gabriele Turacchio3, Carmen Valente1, Alexander Spaar3, Giuseppe Perinetti1, Rainer A Böckmann4, Daniela Corda1, Antonino Colanzi 1, Varpu Marjomaki2 and Alberto Luini 3 1Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy 2Department of Biological and Environmental Science, Nanoscience Centre, University of Jyvaskyla, Jyvaskyla, Finland 3Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy 4Theoretical and Computational Membrane Biology, Centre for Bioinformatics, Saarland University, Saarbruecken, Germany *Corresponding authors: Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy. Tel.: +39 0872 570310; Fax: +39 0872 570412; E-mail: [email protected] Laboratory of Membrane Traffic, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy. Tel.: +39 0872 570355; Fax: +39 0872 570412; E-mail: [email protected] The EMBO Journal (2008)27:970-981https://doi.org/10.1038/emboj.2008.59 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS-induced fission to human viral infection. Introduction Membrane fission is an essential step in different cellular processes, including the formation of membrane traffic carriers and the partitioning of the Golgi complex during mitosis (Corda et al, 2006; McNiven and Thompson, 2006; Colanzi et al, 2007). While numerous fissioning and trafficking events involve the well-characterized large GTPase dynamin (Schmid et al, 1998; Conner and Schmid, 2003; Pelkmans and Helenius, 2003; Praefcke and McMahon, 2004; Newton et al, 2006; Ferguson et al, 2007), others have been shown to be dynamin independent (Sabharanjak et al, 2002; Glebov et al, 2006) and to require, at least in some cases, the C-terminal-binding protein-1/brefeldin A-ADP ribosylated substrate (CtBP1/BARS; Hidalgo Carcedo et al, 2004; Bonazzi et al, 2005; Yang et al, 2005; Corda et al, 2006). CtBP1/BARS belongs to a dual-function protein family that is known to be involved in both membrane fission and gene transcription (Chinnadurai, 2003; Corda et al, 2006). As a nuclear transcription factor, CtBP1/BARS regulates numerous cellular functions, including epithelial differentiation, tumorigenesis and apoptosis (Chinnadurai, 2002; Grooteclaes et al, 2003). In the cytoplasm, CtBP1/BARS controls the fission machinery that is involved in the formation of post-Golgi carriers, endocytic fluid-phase carriers (Bonazzi et al, 2005) and COP1-coated vesicles (Yang et al, 2005); CtBP1/BARS is also involved in mitotic Golgi partitioning (Hidalgo Carcedo et al, 2004; Colanzi et al, 2007). Whether and how the nuclear and cytoplasmic functions of CtBP1/BARS are related remains so far unclear. The precise mechanism of action of CtBP1/BARS, and whether CtBP1/BARS has a direct mechanistic or regulatory role in membrane fission, also remains unclear. Thus, the phrase CtBP1/BARS-dependent fission will be used in this study to refer to the whole fissioning process, including its regulatory stages. To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied macropinocytosis, a dynamin-independent (and hence possibly CtBP1/BARS dependent) endocytic pathway (Meier et al, 2002; Pelkmans and Helenius, 2003; Kirkham and Parton, 2005). While macropinocytosis is constitutive in specialized antigen-presenting cells (Steinman and Swanson, 1995), it can also be rapidly and synchronously induced by growth factors in other cell types (Haigler et al, 1979; Mellstrom et al, 1988; West et al, 1989). This is advantageous for mechanistic studies, in that it can help to reveal fission-related CtBP1/BARS modifications and interactions that occur during stimulation. Macropinocytosis is an essential aspect of normal cell function that contributes to a number of cellular processes, such as antigen sampling (Steinman and Swanson, 1995) and cell motility (Ahram et al, 2000). It results in the formation of large endocytic vesicles that originate from actin ruffles at the plasma membrane. This ruffling is followed by the invagination of the plasma membrane and the formation of a macropinocytic cup, which is then closed via the fissioning of its junction with the plasma membrane (its ‘neck’) (Swanson and Watts, 1995). Here, we show that CtBP1/BARS has an essential role in the fission of the macropinosome neck during epidermal growth factor (EGF)-mediated macropinocytosis, and we analyse the mechanisms leading to CtBP1/BARS activation in this context. We thus show that, upon EGF receptor engagement, CtBP1/BARS is translocated to the macropinocytic cup and its surrounding membrane. In addition, during macropinocytosis, CtBP1/BARS is phosphorylated on a specific serine that is known to be a substrate of p21-activated kinase (Pak1), and this phosphorylation is essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for the macropinocytic internalization and infection of echovirus 1 (EV1). Our study thus opens the way for further investigations into the molecular mechanisms of action of CtBP1/BARS in membrane fission and demonstrates the relevance of this process in human disease. Results CtBP1/BARS is required for EGF-stimulated macropinocytosis To determine the role of CtBP1/BARS in macropinocytosis, we used an established in-vivo assay for EGF-stimulated macropinocytosis in human A431 epidermoid carcinoma cells (Hewlett et al, 1994; Hamasaki et al, 2004). The cells were incubated in serum-free buffer and then stimulated with EGF in the presence of TRITC-labelled dextran, to monitor macropinosome formation. In the absence of EGF, dextran was internalized into a few endosomes of variable sizes. The addition of EGF induced actin ruffling (Supplementary Figure 1A) and a strong stimulation of dextran uptake (Figure 1A), which increased with time and reached a plateau 8–10 min after EGF addition (Figure 1B). To establish whether this uptake was due to bona-fide macropinocytosis, we investigated the effects of the following macropinocytosis inhibitors: amiloride, a Na+/H+ exchange inhibitor (West et al, 1989); cytochalasin D, an actin depolymerizing agent (Amyere et al, 2002); wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor (Amyere et al, 2002); Go6976 and Ro31-8220, two protein kinase C (PKC) inhibitors (Swanson and Watts, 1995); BAPTA-AM, a cell-permeant calcium chelator (Falcone et al, 2006); and U73122, a phospholipase C (PLC) inhibitor (Amyere et al, 2002). In cells separately pretreated with each of these agents, dextran uptake was strongly inhibited (Supplementary Figure 1B). This inhibitory pattern is considered diagnostic of macropinocytosis (Schnatwinkel et al, 2004). Figure 1.CtBP1/BARS is required for EGF-stimulated macropinocytosis in A431 cells. (A) Cells were incubated for 1 h in serum-free Ringer's buffer and for 8 min in Ringer's buffer containing TRITC-conjugated dextran in the absence (w/o) or presence (+) of 50 ng/ml EGF. After fixing, the cells were examined under confocal microscopy for macropinocytosis. (B) Quantification of macropinocytosing cells treated as in (A), as indicated (see Materials and methods). (C) Cells were transfected for 72 h with non-targeting siRNAs or siRNAs targeted to CtBP1/BARS, and injected with GST or wild-type CtBP1-S/BARS–GST, as indicated. One hour later, they were incubated for 1 h in serum-free Ringer's buffer and then for 8 min in Ringer's buffer containing 50 ng/ml EGF and TRITC-conjugated dextran. Injected cells are outlined. (D) Quantification of macropinocytosing cells treated as in (C), as indicated (see Materials and methods). (E) Cells were microinjected with GST, NBD or SBD, as indicated, and processed as in (C). (F) Quantification of macropinocytosing cells treated as in (E), as indicated (see Materials and methods). (G) Cells were either untreated (not shown), or injected with IgG or the blocking p50-2 anti-CtBP1/BARS antibody (CtBP1/BARS Ab), as indicated, and 1 h later they were processed as in (C). (H) Quantification of macropinocytosing cells treated as in (G), as indicated (see Materials and methods). More than 100 cells were analysed under each experimental condition, and the data are means±s.d. from three independent experiments. Scale bars: 10 μm. Download figure Download PowerPoint We then examined whether CtBP1/BARS is involved in macropinocytosis by using multiple approaches to inhibit CtBP1/BARS function. First, we targeted CtBP1/BARS by RNA interference in A431 cells. Notably, CtBP1/BARS exists as two splice variants that differ only in the absence of the first 11 amino acids from the N-terminus of the long isoform, CtBP1-L/BARS, which results in the short isoform, CtBP1-S/BARS. Of these, CtBP1-S/BARS has so far been characterized in membrane fission assays, whereas CtBP1-L/BARS has been characterized as a transcription factor (Chinnadurai, 2003; Corda et al, 2006), although their functions are in fact very likely to overlap (Corda et al, 2006). Our small-interfering RNAs (siRNAs) were designed to target both forms. They strongly reduced the overall CtBP1/BARS levels (Supplementary Figure 1C) and markedly decreased macropinocytosis (Figure 1C and D) without affecting cell viability. To ascertain the specificity of these siRNAs, we sought to rescue macropinocytosis by first injecting each of the recombinant CtBP1/BARS–GST forms into CtBP1/BARS-depleted cells 1 h before the macropinocytosis assay. Both CtBP1-S/BARS–GST (Figure 1C and D) and CtBP1-L/BARS–GST (data not shown) substantially restored EGF-stimulated macropinocytosis. Moreover, the two isoforms were equally active in other CtBP1/BARS-dependent fissioning events (Corda et al, 2006) (data not shown). From hereafter, therefore, we will refer to both proteins as CtBP1/BARS, unless specified otherwise. Second, we inhibited CtBP1/BARS acutely, by injecting cells with two recombinant deletion mutants of CtBP1/BARS that have previously been shown to act as dominant-negative (DN) mutants, that is, the CtBP1/BARS nucleotide-binding domain (NBD, which blocks cargo export from the Golgi complex; Bonazzi et al, 2005) and the substrate-binding domain (SBD, which blocks mitotic Golgi partitioning, but has no effects on Golgi trafficking; Hidalgo Carcedo et al, 2004). Both of these DN mutants strongly inhibited the formation of macropinosomes (Figure 1E and F), indicating that CtBP1/BARS is required for EGF-stimulated macropinocytosis. Also of note here is that, first, the effects of the CtBP1/BARS DN SBD mutant on macropinocytosis may not follow changes in trafficking out of the Golgi complex, as SBD has no inhibitory effect on Golgi export (Bonazzi et al, 2005), and second, the inhibitory action of the DN mutants in microinjection experiments is too rapid to be mediated by the transcriptional effects of these CtBP1/BARS mutants (less than 1 h). In addition, we injected cells with a previously characterized anti-CtBP1/BARS blocking antibody that recognizes both CtBP1/BARS isoforms (Hidalgo Carcedo et al, 2004; Bonazzi et al, 2005). Again, this resulted in strong inhibition of macropinocytosis, as compared to IgG-injected cells (Figure 1G and H). In parallel, we carried out a series of control experiments. First, as macropinocytosis originates from ruffling, we examined whether the inhibition/depletion of CtBP1/BARS by each of the above treatments affects the actin cytoskeleton: none of them showed detectable effects on actin, either in the absence or presence of EGF (Supplementary Figure 1D). Second, we examined the effects of acutely enhancing the cellular CtBP1/BARS levels by microinjecting native CtBP1/BARS into control cells: this did not increase or otherwise alter the macropinocytic response upon EGF stimulation, indicating that endogenous CtBP1/BARS is sufficient (i.e., non-rate-limiting) for a full macropinocytic response (Figure 1C and D). Finally, we observed that long exposures (24 h) of the cells to enhanced levels of CtBP1/BARS achieved by microinjection of recombinant CtBP1-S/BARS–GST or overexpression of CtBP1-S/BARS–YFP (see Supplementary Figure 1E) partially reduced the macropinocytic response to EGF (Supplementary Figure 1E), similar to the inhibitory effect seen previously for secretory trafficking (Bonazzi et al, 2005). Upon EGF stimulation, CtBP1/BARS is recruited to actin-rich membrane ruffles and to the macropinocytic cup To analyse the role of CtBP1/BARS in macropinocytosis, we performed video microscopy for both actin and CtBP1/BARS. The actin dynamics in living cells were monitored using the actin-binding domain of filamin fused to GFP (ABD-filamin–GFP) (Figure 2A). This marker confirmed the effects of EGF on the actin cytoskeleton, and also allowed visualization of forming macropinocytic cups: segments of the membrane in the ruffling area retracted (invaginated) towards the cell interior to form cup-shaped structures (0.5–2.0 μm in diameter) that persisted for variable times (60–90 s) (Figure 2A, arrowheads). These appeared to narrow around their orifice and then to close (Figure 2A, arrow). Simultaneously, the internalized macropinosomes lost their fluorescent actin marker (Figure 2A, lower panel). This is similar to previous descriptions, and it confirms that actin localization at the macropinocytic cup is transient, lasting only until the closure of the macropinosome (Lee and Knecht, 2002; Schnatwinkel et al, 2004). Figure 2.Effects of EGF stimulation on CtBP1/BARS localization in A431 cells. (A) Cells were transfected with the actin-binding domain of filamin–GFP (ABD-filamin–GFP), incubated for 1 h in serum-free Ringer's buffer and stimulated with 50 ng/ml EGF. Representative frames of time-lapse imaging for ABD-filamin–GFP are shown. Arrowheads, cup-shaped macropinocytic invaginations; arrow, closing macropinocytic cup. (B) Immunofluorescence analyses of endogenous CtBP1/BARS and F-actin localization in cells incubated for 1 h in serum-free Ringer's buffer and then for 8 min in Ringer's buffer in the absence (control) or presence (+) of 50 ng/ml EGF. The cells were then fixed and stained with the p50-2 anti-CtBP1/BARS antibody (red) and 488-conjugated phalloidin to reveal actin organization (F-actin), as indicated. The merged signals are shown in the lower panels. White arrows, macropinocytic cups seen among plasma membrane ruffles. (C) Cells were transfected with CtBP1-S/BARS–YFP (6 h of overexpression), incubated for 1 h in serum-free Ringer's buffer and stimulated with 50 ng/ml EGF in the presence of TRITC-conjugated dextran. Representative frames of time-lapse imaging for CtBP1-S/BARS–YFP (green) and TRITC-dextran (red) and merged signals are shown, as indicated. Upper panels, 90–200 s: white arrow, closing macropinocytic cup and CtBP1-S/BARS enrichment at the neck; black arrow, disappearance of CtBP1-S/BARS–YFP from the base of the macropinocytic cup. Middle panels, 200–300 s: arrowhead, internalized macropinosome. (D) Immuno-EM analysis of CtBP1-S/BARS–YFP localization in cells prepared and stimulated with 50 ng/ml EGF for 8 min, as in (A). The cells were then fixed, stained with an anti-YFP antibody and prepared for EM (see Materials and methods). MR, membrane ruffles; PM, plasma membrane. Black dots, enrichment of CtBP1-S/BARS–YFP at plasma membrane ruffles (arrows). More than 50 cells were analysed under each experimental condition, in three independent experiments. Scale bars: (A, C) 1 μm; (B) 10 μm; (D) 600 nm. Download figure Download PowerPoint We then examined the localization of CtBP1/BARS during EGF stimulation, by immunofluorescence. In quiescent cells, CtBP1/BARS was both nuclear and cytoplasmic (Figure 2B, control). Upon addition of EGF, CtBP1/BARS moved to the forming membrane ruffles (Figure 2B), which were rich in F-actin, and also to round structures within the ruffling areas that were reminiscent of macropinocytic cups (Figure 2B, arrows). Notably, the overlap of the actin ruffles with CtBP1/BARS at the plasma membrane was nearly perfect at all times after EGF stimulation, indicating that CtBP1/BARS is recruited on these ruffles as soon as they form. To monitor the living dynamics of CtBP1-S/BARS, we used a fluorescent-tagged construct (CtBP1-S/BARS–YFP) and examined the cells under the confocal microscope 6 h after transfection. CtBP1-S/BARS–YFP showed a localization that was similar to that of the endogenous protein in both quiescent and EGF-stimulated cells (not shown). Upon EGF stimulation, CtBP1-S/BARS–YFP translocated onto the membrane ruffles and the cup-shaped structures that developed from the ruffles (see Supplementary Movies 1 and 2). These structures, and the associated CtBP1-S/BARS–YFP, persisted for less than 90 s before closing (Figure 2C, white arrows, 90–200 s), with this coinciding with the detachment of CtBP1-S/BARS–YFP from the macropinocytic cup. The large round macropinosomes that formed from these cups then moved rapidly into the cell (confirming the macropinocytic nature of the cups) (Figure 2C, arrowheads, 200–300 s). A detailed inspection of this closure event revealed that the detachment of CtBP1/BARS–YFP initiated at the base of the cup (Figure 2C, black arrows, 120–200 s) and coincided with its transient enrichment at the neck, where the fissioning of the macropinosome occurs (Figure 2C, white arrows, 180–200 s). To confirm this enrichment on the plasma-membrane ruffles and macropinocytic cups, the localization of CtBP1-S/BARS–YFP in EGF-stimulated cells was analysed by immuno-electron microscopy (EM). As shown in Figure 2D, CtBP1/BARS–YFP was strongly enriched in ruffling areas of the plasma membrane, as compared to other areas (Figure 2D, arrows). CtBP1/BARS is specifically required for the fission of the macropinocytic cup Having established an essential role for CtBP1/BARS in macropinocytosis, we next sought to determine its site of action. In principle, CtBP1/BARS could act at the following steps: (i) formation of actin-dependent membrane ruffles; (ii) invagination of the macropinocytic cup; and/or (iii) fission of the macropinosome neck. The first possibility can be excluded, as under all conditions investigated the inhibition or ablation of CtBP1/BARS had no apparent effects on actin ruffling (Supplementary Figure 1D). We thus sought to distinguish whether CtBP1/BARS acts by controlling the formation or the fission of the macropinocytic cup, by using multiple approaches. In the first approach, we modified the macropinocytosis assay as follows: we fixed the cells without prior washing (instead of washing them extensively, as for the previous experiments in Figure 1C–F), so as not to remove the dextran present in the invaginated cups; moreover, we used fluorescein-conjugated ‘fixable’ lysine-containing dextran (FITC-dextran). While under the ‘extensive washing’ conditions used in previous experiments, the number of macropinosomes was strongly reduced in CtBP1/BARS-inhibited cells (e.g., by injection of the DN NBD mutant) (Figure 3B), under these ‘no wash’ conditions, the CtBP1/BARS-inhibited cells showed a number of dextran-positive round macropinosome-like structures that were comparable to those seen in control cells (Figure 3A, upper panels, and Figure 3B). This suggests that in these inhibited cells, macropinocytic cups can form but cannot develop into complete macropinosomes. Figure 3.Characterization of the role of CtBP1/BARS in macropinosome formation in A431 cells. (A) Cells were microinjected with GST or NBD–GST, as indicated, and incubated for 1 h in serum-free Ringer's buffer and for 8 min in Ringer's buffer containing 50 ng/ml EGF and FITC-conjugated dextran. The cells were then fixed without the pre-fixing wash, to detect both fully incorporated macropinosomes and formed macropinocytic cups still connected with the plasma membrane. To distinguish between these, the fixed cells were incubated in PBS at either neutral pH (PBS) or pH 5 (acidic medium), to quench fluorescence of the macropinosomes connected with the plasma membrane. The time frame of our confocal analysis is less than 10 min. (B, C) Quantification of macropinosomes in cells without or with the pre-fixing wash (B) and of closed macropinosomes (C), in cells treated as in (A), as indicated (see Materials and methods). (D) Cells were transfected with NBD–YFP (6 h overexpression) and incubated for 1 h in serum-free Ringer's buffer and for 8 min in Ringer's buffer containing 50 ng/ml EGF and TRITC- and biotin-conjugated dextran. The cells were then fixed without the pre-fixing wash, as indicated in (A). To distinguish between fully incorporated and still connected macropinosomes, the fixed cells were stained (without permeabilization) with streptavidin-633 (to reveal the latter). Arrow, NBD-YFP at the macropinocytic cup. (E) Quantification of closed macropinosomes in cells transfected with YFP or NBD–YFP and treated as in (D). (F) Cells were transfected with NBD–YFP (6 h of overexpression), incubated for 1 h in serum-free Ringer's buffer and then with 50 ng/ml EGF and TRITC-labelled dextran. Representative frames of time-lapse imaging for NBD–YFP (green) and TRITC-dextran (red) and merged signals are shown, as indicated. Upper panels, 90–320 s: arrows, macropinosomes not yet sealed. More than 50 cells were analysed under each experimental condition and the data are means±s.d. from three independent experiments. Scale bars: (A, D) 10 μm; (F) 1 μm. Download figure Download PowerPoint To test this conclusion more directly, we exposed the fixed cells to a pH 5.0 medium, exploiting the pH sensitivity of the fluorescence of FITC-dextran, which is quenched below pH 5.5. The rationale here is that under these conditions, the macropinocytic cups that were still connected with the plasma membrane should lose their fluorescence, whereas the sealed macropinosomes should not. Strikingly, the vast majority of the dextran-positive macropinosome-like structures in these CtBP1/BARS-inhibited cells indeed showed a consistent bleaching of fluorescence after acidification, indicating that these structures were still connected with the extracellular space (Figure 3A and C). In contrast, in control (GST-injected) cells, a much smaller fraction of the dextran-positive structures showed this loss of fluorescence (Figure 3A and C). Similar results were obtained using TRITC-dextran conjugated with biotin as the fluid-phase marker and Alexa633-conjugated streptavidin as a probe to test the accessibility of extracellular molecules to the macropinosomal lumen after fixation. Under these conditions, the macropinosomes that were formed in the presence of the CtBP1/BARS DN mutant NBD again remained accessible to the external medium (Figure 3D and E). Thus, these data indicate that in cells where CtBP1/BARS function is impaired, the macropinocytic cup is formed normally whereas fission is inhibited. As a third approach, A431 cells were transfected with the CtBP1/BARS DN mutant NBD–YFP, to follow its living dynamics after EGF stimulation. The rationale here was that if this inhibitory construct localizes to the macropinocytic cups like CtBP1/BARS, it should allow visualization of cups that are unable to close. Indeed, upon EGF stimulation, NBD–YFP was seen on plasma membrane ruffles and cup-like structures (see Supplementary Movies 3 and 4). However, these cups did not develop into macropinosomes and NBD–YFP remained on their surface often for long times (up to 200 s), until the cup ‘aborted’ into a normal plasma membrane ruffle (Figure 3F, 90–320 s, arrow). These results confirm that CtBP1/BARS is required for mac
Referência(s)