Polarized sorting of Patched enables cytoneme‐mediated Hedgehog reception in the Drosophila wing disc
2020; Springer Nature; Volume: 39; Issue: 11 Linguagem: Inglês
10.15252/embj.2019103629
ISSN1460-2075
AutoresLaura González‐Méndez, Ana‐Citlali Gradilla, D. Sánchez‐Hernández, Esperanza González, Adrián Aguirre‐Tamaral, Carlos Jiménez‐Jiménez, Milagros Guerra, Gustavo Aguilar, Germán Andrés, Juan Manuel Falcón‐Pérez, Isabel Guerrero,
Tópico(s)Plant Reproductive Biology
ResumoArticle20 April 2020free access Source DataTransparent process Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc Laura González-Méndez Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ana-Citlali Gradilla Corresponding Author [email protected] orcid.org/0000-0003-0992-2557 Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author David Sánchez-Hernández Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Esperanza González Exosomes Lab. Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain Search for more papers by this author Adrián Aguirre-Tamaral Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Carlos Jiménez-Jiménez Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Milagros Guerra Electron Microscopy Unit, Centro de Biología Molecular Severo Ochoa, (CSIC-UAM), Nicolás Cabrera 1, Universidad Autonoma de Madrid, Madrid, Spain Search for more papers by this author Gustavo Aguilar Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Growth and Development, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Germán Andrés orcid.org/0000-0003-0265-5409 Electron Microscopy Unit, Centro de Biología Molecular Severo Ochoa, (CSIC-UAM), Nicolás Cabrera 1, Universidad Autonoma de Madrid, Madrid, Spain Search for more papers by this author Juan M Falcón-Pérez Exosomes Lab. Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Derio, Spain Search for more papers by this author Isabel Guerrero Corresponding Author [email protected] orcid.org/0000-0001-6761-1218 Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Laura González-Méndez Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ana-Citlali Gradilla Corresponding Author [email protected] orcid.org/0000-0003-0992-2557 Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author David Sánchez-Hernández Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Esperanza González Exosomes Lab. Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain Search for more papers by this author Adrián Aguirre-Tamaral Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Carlos Jiménez-Jiménez Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Milagros Guerra Electron Microscopy Unit, Centro de Biología Molecular Severo Ochoa, (CSIC-UAM), Nicolás Cabrera 1, Universidad Autonoma de Madrid, Madrid, Spain Search for more papers by this author Gustavo Aguilar Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Growth and Development, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Germán Andrés orcid.org/0000-0003-0265-5409 Electron Microscopy Unit, Centro de Biología Molecular Severo Ochoa, (CSIC-UAM), Nicolás Cabrera 1, Universidad Autonoma de Madrid, Madrid, Spain Search for more papers by this author Juan M Falcón-Pérez Exosomes Lab. Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Derio, Spain Search for more papers by this author Isabel Guerrero Corresponding Author [email protected] orcid.org/0000-0001-6761-1218 Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Laura González-Méndez1,‡, Ana-Citlali Gradilla *,1,‡, David Sánchez-Hernández1, Esperanza González2, Adrián Aguirre-Tamaral1, Carlos Jiménez-Jiménez1, Milagros Guerra3, Gustavo Aguilar1,4, Germán Andrés3, Juan M Falcón-Pérez2,5,6 and Isabel Guerrero *,1 1Tissue and Organ Homeostasis, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Madrid, Spain 2Exosomes Lab. Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain 3Electron Microscopy Unit, Centro de Biología Molecular Severo Ochoa, (CSIC-UAM), Nicolás Cabrera 1, Universidad Autonoma de Madrid, Madrid, Spain 4Growth and Development, Biozentrum, University of Basel, Basel, Switzerland 5IKERBASQUE, Basque Foundation for Science, Bilbao, Spain 6Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Derio, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +34 911964465; E-mail: [email protected] *Corresponding author. Tel: +34 911964465; E-mail: [email protected] EMBO J (2020)39:e103629https://doi.org/10.15252/embj.2019103629 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient. Synopsis Hedgehog (Hh) gradient formation in the Drosophila wing disc is enabled by Hh exosomal transport along filopodia-like structures called cytonemes at the epithelial cell basolateral surface. Here, Hh receptor Patched (Ptc) is shown to undergo similar trafficking process to interact with Hh at discrete, synapse-like cytoneme contact sites. Ptc internalization from apical surfaces is required for its ESCRT-mediated sorting into multi-vesicular bodies. Tetraspanin Tsp96F directly interacts with Ptc and regulates its exosomal sorting. SNARE complex components are required for Ptc-containing exosome fusion at cytonemes. Introduction The Hedgehog (Hh) family of proteins plays a central role in development in a wide range of organisms; it is needed for early patterning, cell migration, axon guidance and adult stem cell maintenance, and it is also implicated in cancer. As a morphogen, Hh can act at a distance to pattern tissues through concentration-dependent differential activation of target genes (reviewed in Briscoe & Therond, 2013). The graded distribution of Hh and the ability of receiving cells to respond specifically to different Hh concentrations are tightly regulated processes. Hh signalling mechanisms have been extensively studied in the Drosophila wing imaginal disc. The disc is a pseudo-stratified epithelium divided into a posterior (P) and an anterior (A) compartment, with different cell adhesion affinities. The P compartment cells produce the lipid-modified Hh molecule, which disperses across the receiving A compartment cells, decreasing in concentration as it spreads away from the A/P compartment border. We have previously demonstrated in the wing disc and abdominal histoblast nest epithelia that cytonemes act as transporters for membrane-associated Hh to achieve the restricted spatial distribution essential for tissue patterning (Bischoff et al, 2013). The A compartment cells also extend basolateral cytonemes oriented towards the P compartment for Hh reception, contributing as well to the formation of the Hh signal gradient (Gonzalez-Mendez et al, 2017). Hh interacts with its receptor complex at cytoneme-mediated cell–cell contacts (Chen et al, 2017; Gonzalez-Mendez et al, 2017). The complex includes the Hh canonical receptor Patched (Ptc), a 12-transmembrane-domain protein, as well as the adhesion molecules and Hh co-receptors Interference of Hedgehog (Ihog) and Brother of Ihog (Boi), all localizing to cytonemes (Bilioni et al, 2013; Chen et al, 2017; Gonzalez-Mendez et al, 2017). In the absence of the ligand Hh, the main receptor Ptc acts as a signal repressor by inhibiting the transducing molecule, the GPCR transmembrane protein Smoothened (Smo), such that the pathway can only be activated after Hh reception (Alcedo et al, 1996; van den Heuvel & Ingham, 1996; Denef et al, 2000). In turn, Ptc is also up-regulated in a graded manner, being itself a transcriptional target of the Hh pathway (Chen & Struhl, 1996; Briscoe et al, 2001; Torroja et al, 2005). As for the co-receptors Ihog and Boi (Bilioni et al, 2013), they are found in both Hh-producing and Hh-receiving cytonemes, maintaining Hh levels for correct reception (Zheng et al, 2010; Hsia et al, 2017). Other components necessary for Hh reception, including Dispatched (Disp) (Burke et al, 1999; Ma et al, 2002), the Wif ortholog Shifted (Shf) (Glise et al, 2005; Gorfinkiel et al, 2005), and the glypicans Dally-like (Dlp) (Desbordes & Sanson, 2003; Lum et al, 2003a) and Dally (Takeo et al, 2005), are all localized in cytonemes (Callejo et al, 2011; Bilioni et al, 2013; Gonzalez-Mendez et al, 2017). The finding of discrete contact places where components needed for Hh signalling colocalize suggests a contact-dependent mechanism for Hh reception. In this mechanism, receptor, co-receptors and ligand are present in close proximity in a dynamic manner, as previously described for synaptic-like processes (Huang et al, 2019). A key feature of the synaptic signal transfer is the discrete localization of both presenting and receiving elements at either side of the contacting cytonemes. Vesicle trafficking is crucial for this focal localization, and it includes the machineries needed for vesicle formation, translocation and fusion with target membranes. In fact, a multivesicular body (MVB)-mediated recycling process to direct the ligand Hh towards its basolateral secretion has been described in the Drosophila wing disc (Callejo et al, 2011; Bilioni et al, 2013; Gradilla et al, 2014). In addition, biochemical and functional characterization of Hh and Sonic Hh (Shh) vesicles has identified them as extracellular (EVs) (Fleury et al, 2014; Gradilla et al, 2014; Matusek et al, 2014; Vyas et al, 2014; Parchure et al, 2015; Coulter et al, 2018). Indeed, basolateral EVs containing both Hh and co-receptor Ihog are transported via cytonemes in an anterograde direction towards Hh-receiving cells in Drosophila (Gradilla et al, 2014); similarly, vesicles containing the vertebrate Shh are also transported along filopodia-like structures in the chicken limb bud (Sanders et al, 2013). To date, the mechanisms driving the polarized transport and location at cytonemes of the Hh-receptor Ptc remain unidentified. Here, we find that several members of the SNARE protein complex mediate Ptc final presentation at basolateral membranes and cytoneme contacts in the wing disc. In addition, we describe a preceding vesicular sorting to polarize Ptc to reach cytonemes that requires the ESCRT machinery. We found that this complex, known for its function in multivesicular bodies (MVBs) generation (reviewed in Babst, 2011), transports Ptc to cytonemes in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. In vivo imaging of these Ptc-loaded dynamic vesicles in the abdominal histoblasts confirmed the inclusion of MVB markers and revealed an extracellular vesicle (EVs) signature, coinciding with our findings of Ptc presence in EVs secreted by wing disc cells in culture. Loss of function of several ESCRT and SNARE proteins results in Ptc retention in vesicular sorting compartments as well as Hh reception impairment. These data uncover the role of these proteins in Ptc inclusion in MVBs for polarized sorting of Ptc and its final extracellular exposure at cytoneme membranes. Results Hh reception at cytoneme contacts resembles a synaptic process Direct contact between Hh-presenting and Hh-receiving cytonemes at specific sites along their membranes has been revealed using the GRASP technique (Chen et al, 2017; Gonzalez-Mendez et al, 2017), initially developed to study neuronal synapses. It labels sites of close cell–cell contacts (20–40 nm, a distance comparable to a synaptic cleft) through complementation of two GFP barrels tagged to membrane proteins (Feinberg et al, 2008). Both Ptc and Hh colocalize at fluorescent GRASP sites along cytonemes, indicating potential specific cell–cell membrane contact sites between producer and receptor cells for Hh reception (Gonzalez-Mendez et al, 2017). Thus, Hh reception can be envisioned as a synaptic-like process reviewed in Gonzalez-Mendez et al (2019) and we set out to further investigate the mechanisms for Ptc localization and Hh reception at these cytoneme contacts. Rapid signal transfer at neuronal synapses is partly mediated by the vesicle fusion proteins SNARE, which form different complexes together with diverse attachment and targeting proteins (reviewed in Wang et al, 2017). Two functionally interchangeable Syb homologs are encoded in Drosophila (Bhattacharya et al, 2002), one neuron specific (nSyb) (DiAntonio et al, 1993; Bhattacharya et al, 2002) and another one, epithelial Syb, more broadly expressed. In active synaptic contacts, nSyb is a major component of synaptic vesicles, facilitating membrane fusion for the release of proteins in a zipper manner with t-SNARE proteins at target membranes (reviewed in Sudhof, 2013). Interestingly, colocalization of Ptc with a tagged version of nSyb (nSyb-GFP) is observed in discrete punctae distributed along the apico-basal axis of the wing disc epithelium and at cytonemes emanating from Hh-receiving cells (Fig 1A). Similarly, the calcium sensor Synaptotagmin 1 (Syt1-GFP) colocalizes with Ptc in lateral punctae and can be visualized at Hh-receiving cytonemes (Fig 1B), indicating a synaptic-like process for Hh reception. Figure 1. The SNARE proteins Synaptobrevin (Syb) and Synaptotagmin (Syt1) are located at receiving cytonemes GFP-tagged Syb protein expressed in Hh-receiving cells colocalizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal confocal sections (arrows) and can be detected along cytonemes marked with Syb-GFP (arrowhead). Expression of the GFP-tagged Syt1 also colocalizes with immuno-labelled endogenous Ptc at vesicles (lateral confocal section, arrows point at vesicles with colocalization) and decorates the cytoneme membranes (basal confocal section, arrowheads indicate cytonemes). GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1–10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. C′ Inset from C, note the fluorescent discrete punctae (arrows) along Hh-producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area. Scheme to the right depicts GRASP experiment. Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal confocal sections, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytonemes). Data information: Scale bars 10 μm. Download figure Download PowerPoint Thus, to confirm that exocytosis occurs in close contact to the basal membranes of Hh-receiving cells, as it should in a synaptic scenario, we performed the GRASP experiment using the neuronal Synaptobrevin (nSyb). Through the combined use of the Gal4/UAS and LexA/LexAop systems, we co-expressed the UAS-nSyb construct fused to one of the GFP barrels (nSyb-GFP1–10) (Macpherson et al, 2015) in Hh-receiving cells and a plasma membrane-anchored protein fused to the complementary GFP barrel (CD4-GFP11) in Hh-producing cells. We observed discrete GFP-positive dots along Hh-sending cytonemes stabilized by Ihog-RFP expression (Fig 1C), as well as colocalizing with endogenous Ptc (Appendix Fig S1). This result indicates that exocytosis of Ptc-including nSyb vesicles occurs at discrete contact sites between receiving and sending cytonemes. In accordance, expression of a tagged version of the Syb functional direct partner, the t-SNARE Syx1A, also localizes to discrete dots at Hh-receiving basal plasma membranes and along receiving cytonemes and shows a basally polarized complex for vesicle fusion (Fig 1D). Altogether, these data suggest a synaptic-like process for Hh reception at basal receiving cytonemes. SNARE complex function in Ptc vesicle fusion Since the SNARE proteins Syb, Syt1 and Syx1A colocalize with Ptc and this complex can function on recycling as well as positioning of receptors at plasma membrane (reviewed in Bin et al, 2018), we explored the SNARE complex involvement in Ptc presentation in the wing disc epithelium by knocking down SNARE components and analysing the effect in the localization of endogenous Ptc. Ptc sorting for a basal presentation was suggested after blocking vesicle fusion by epithelial Syb down-regulation in the dorsal compartment of the wing disc; as it resulted in acute basal accumulation of endogenous Ptc (Fig 2A top panel-dorsal and 2B′) compared to the very low basal levels in the ventral wild-type cells (Fig 2A control and 2B) which in turn are due to Ptc rapid endocytosis after normal Hh reception (Lu et al, 2006). Similar, although more discrete, basal accumulation of Ptc was also observed after down-regulation of the calcium sensor Syt1 (Fig 2A lower panel). Figure 2. Polarized Ptc vesicle fusion to basolateral membranes is SNARE complex dependent Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc when knocking down Syb or Syt1. Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh-producing cells in wild-type receiving cells (left panel), or B′) when either blocking exocytosis by down-regulating Syb (middle panel) or B″) blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild-type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrows), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows). Confocal apical (left) and basal (right) images of a shits mutant wing disc-expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note, the dorsal reduction of Ptc at plasma membrane after blocking exocytosis. Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild-type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). Note, a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (−1 = basal, +1 = apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N = 7–16, box shows lower and upper quartiles, and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis. Data information: Scale bars 10 μm. Source data are available online for this figure. Source Data for Figure 2 [embj2019103629-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint This basal accumulation appears in large vesicle-like punctae in a fairly restricted area (Fig 2B′). Syb inhibition might be blocking the exocytic process for Ptc membrane deposition and exposure for Hh reception as Syb mutant phenotype is different to the one observed when blocking endocytosis. After inhibiting the rapid endocytosis of Ptc by expressing a dominant negative form of dynamin (UAS-shiK44A), Ptc accumulates in apical and basolateral plasma membranes including cytonemes through the whole receiving territory (Fig 2B″, yellow arrows). We next tested Ptc localization after dorsal compartment expression of the Syb RNAi in a shi−/− (shits1) mutant background, where endocytosis is also being blocked. In this experiment, the ventral side of the wing disc is kept as control for Syb RNAi expression. Under Syb and shi down-regulation conditions, Ptc accumulation at membranes was reduced despite blocking internalization (Fig 2C). Furthermore, this reduction is much greater at the basal membrane (Fig 2C right panel), strongly suggesting that Syb mediates Ptc presentation at basolateral plasma membranes and that in its absence Ptc appears to aberrantly accumulate intracellularly. To further test the putative SNARE-dependent vesicle fusion for Ptc exocytosis/deposition at the plasma membrane, we quantified Ptc levels along the apical/basal axis using a GFP-tagged Yac insertion and an Integrated Density parameter. This parameter allows numerical translation and localization of the Ptc buildup phenotype in a systematic manner (see Materials and Methods). This confirmed again a basolateral shift of the highest levels of Ptc when Syb function is inhibited (Fig 2D). In agreement with our hypothesis for Syb function, induction of the RNAi for α-Snap (AKA SnapII), which controls disassembly of the SNARE complex after fusion to plasma membranes (revised in Sudhof, 2004), showed high levels of Ptc being present at the basal side of the epithelium (Fig 2D). However, inhibition of other SNAREs (Sec22, Snap24 and Snap29), reported as vesicle fusion regulators in different organelles such as the endoplasmic reticulum or Golgi (revised in Littleton, 2000; Wang et al, 2017), showed more variability within each experiment; maximum levels of Ptc were distributed all along the apico/basal axis, probably as a result of vesicle fusion blockage at different stages of Ptc vesicle trafficking (Fig 2D, Appendix Fig S2). ESCRT machinery requirement for MVB-mediated Ptc trafficking To characterize the Ptc punctate structures that traffic towards the basal side of the wing disc, we performed immuno-electron microscopy imaging of anterior cells expressing Ptc-GFP with an α-GFP antibody. Interestingly, we observed Ptc signal at the apical membrane (Fig 3A,a), while intracellular Ptc was found in MVBs, subapically but also close to the basal membrane (Fig 3A,a,b,c). Furthermore, analysis of EM images suggests the presence of two polarized MVB types that show differences in density and structure. Denser MVBs are mainly located basally and include a greater number of Ptc positive intraluminal vesicles (ILV) (Fig 3A,c1), while subapical MVBs tend to be less dense with more Ptc on the outer MVB membrane (Fig 3A,b1); these denser endosomes might be the ones involved in the polarization of Ptc. Figure 3. Patched traffics from the apical to the basal side of the wing disc through Multivesicular Bodies Electron microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 h in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium (a) anti-GFP gold labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. (b) Ptc immunogold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) Note that subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. (c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), (c1) Note that basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV). Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. Note that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild-type control on the ventral side. Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire (Shi DN). Note the same apical and basal accumulation as after expression of Rab5DN, and comparing to the ventral wild-type half. Data information: Scale bars (B, C), 10 μm. Download figure Download PowerPoint We have previously described that after inhibition of endocytosis through expression of the dominant negative form of Shi (ShiK44A) there was a basolateral as well as an apical retention of Ptc (Fig 3C; Callejo et al, 2011), which also happened after expression of the dominant negative form of Rab5 (Fig 3B). The apical internalization of Ptc seems to be independent from reception because it has been described that Hh reception occurs at the basolateral membrane (Callejo et al, 2011; Chen et al, 2017; Gonzalez-Mendez et al, 2017). Thus, this apical endocytosis could initiate the charging of Ptc in MVB to later redirect Ptc towards the basolateral side of the epithelium. The ESCRT machinery is largely involved in the formation of MVBs for extracellular vesicle biogenesis, polarized cell localization and degradation by lysosomes (reviewed in Babst, 2011). Knocking down the ESCRT components Hrs, Tsg101, Vps22 and Shrub gave rise to abnormal accumulation of Ptc in punctate structures. These aberrant structures are positive for endosomal markers such as Rab4, Rab5 and Rab7 (Fig EV1), indicating that the treatment results in a general blockage of the vesicle progression. Thus, when inhibiting ESCRT-0 Hrs, the first protein to initiate MVB formation at the endocytic compartment, Ptc accumulates mainly at this subapical compartment, probably due to a buildup of aberrant initiating MVBs. In the case of subsequent ESCRT-I TSG101, ESCRT-II Vps22 and ESCRT-III Shrub, needed later in MVB formation, the Ptc accumulation tends to shift towards the basal side of the epithelium, suggest
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