ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells

Report16 March 2021free access Transparent process ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells Takahide Matsui Corresponding Author Takahide Matsui [email protected] Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Futaba Osaki Futaba Osaki Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Shu Hiragi Shu Hiragi Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Yuriko Sakamaki Yuriko Sakamaki Microscopy Research Support Unit Research Core, Tokyo Medical and Dental University, Tokyo, Japan Search for more papers by this author Mitsunori Fukuda Corresponding Author Mitsunori Fukuda [email protected] orcid.org/0000-0002-8620-5853 Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Takahide Matsui Corresponding Author Takahide Matsui [email protected] Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Futaba Osaki Futaba Osaki Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Shu Hiragi Shu Hiragi Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Yuriko Sakamaki Yuriko Sakamaki Microscopy Research Support Unit Research Core, Tokyo Medical and Dental University, Tokyo, Japan Search for more papers by this author Mitsunori Fukuda Corresponding Author Mitsunori Fukuda [email protected] orcid.org/0000-0002-8620-5853 Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Author Information Takahide Matsui *,1, Futaba Osaki1, Shu Hiragi1, Yuriko Sakamaki2 and Mitsunori Fukuda *,1 1Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan 2Microscopy Research Support Unit Research Core, Tokyo Medical and Dental University, Tokyo, Japan *Corresponding author. Tel: +81 22 795 3641; E-mail: [email protected] *Corresponding author. Tel: +81 22 795 7731; E-mail: [email protected] EMBO Reports (2021)22:e51475https://doi.org/10.15252/embr.202051475 PDFDownload PDF of article text and main figures.AM PDF 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 Exosomes, important players in cell–cell communication, are small extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods of studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of small extracellular vesicles (more specifically CD9- and CD63-positive, Annexin I-negative small extracellular vesicles, which we refer to as exosomes herein) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. We also identify GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical exosome-specific protein. We further demonstrate that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome release. Thus, two independent machineries, the ALIX–Syntenin1–Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells. SYNOPSIS Distinct types of exosomes (or small extracellular vesicles) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. Two independent machineries, the ALIX–Syntenin1–Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), mediate the polarized exosome release from epithelial cells. Polarized epithelial cells asymmetrically release two distinct types of exosomes (apical and basolateral exosomes) with distinct protein compositions. ALIX and ceramide independently mediate apical and basolateral exosome release, respectively. Introduction Cells release extracellular vesicles (EVs) of different sizes and intracellular origin. EVs can be largely divided into two categories, exosomes and microvesicles (also known as ectosomes) (Cocucci & Meldolesi, 2015). Microvesicles (~ 50 nm to 1 μm in diameter) are formed from the plasma membrane by budding and/or shedding mechanisms (Cocucci et al, 2009; Cocucci & Meldolesi, 2015), whereas exosomes are small EVs (sEVs; around 100 nm in diameter) that are derived from multivesicular bodies (MVBs) and released by well-organized systems. Exosomal cargos, such as proteins, lipids, and nucleic acids, are selectively incorporated into intraluminal vesicles (ILVs), i.e., precursors of exosomes, in MVBs. The MVBs are then transported to the plasma membrane, and after fusing with it, the ILVs are released into the extracellular space as exosomes (Pegtel & Gould, 2019; Kalluri & LeBleu, 2020). It has recently been reported that a single cell releases various types (e.g., sizes and contents) of EVs or exosomes (i.e., exosomal heterogeneity) (Colombo et al, 2013; Kowal et al, 2016; Zhang et al, 2018). Although several distinct mechanisms of exosome biogenesis have been reported (Mathieu et al, 2019; Kalluri & LeBleu, 2020), how these mechanisms are differently used or regulated within a single cell remains completely unknown. This is mainly because the results of the studies varied with the techniques and devices used to conduct them. Hence, the mechanisms by which heterogeneous exosomes are produced within cells are poorly understood. The Madin–Darby canine kidney (MDCK) cell line is a well-known epithelial cell line, which has clearly defined apical–basolateral asymmetry (i.e., apical and basolateral domains), and for that reason, MDCK cells are often used as an in vitro model for studying the mechanism of polarization (Simmons, 1982). Once non-polarized cells release heterogeneous exosomes into the extracellular space, it is extremely difficult to distinguish and collect them separately. However, if heterogeneous exosomes are asymmetrically released from polarized MDCK cells, it would be possible to easily collect apical and basolateral exosomes separately. Thus, we assumed that MDCK cells would become a good model for studying exosomal heterogeneity without using special techniques and devices. Here, we established a method of purifying exosomes (sEVs that are positive for representative exosome markers but negative for the known microvesicle marker Annexin I) released from polarized MDCK cells and found that the polarized cells release distinct types of exosomes (apical and basolateral exosomes) having different protein compositions. Moreover, we showed that the endosomal sorting complexes required for transport (ESCRT) machinery is not unexpectedly required for exosome release from polarized MDCK cells, and instead, ALIX and ceramide independently regulate apical and basolateral exosome release, respectively. Results and Discussion Polarized MDCK cells release distinct types of exosomes from apical and basolateral side To investigate differences between apical and basolateral exosomes, we first purified all sEVs (≤~ 200 nm in diameter) from apical and basolateral MDCK culture media by polyethylene glycol (PEG) precipitation (Rider et al, 2016; Cocozza et al, 2020) (Fig EV1A) and found that five well-known EV marker proteins (exosome markers: Flotillin-1, CD63, CD9, and CD81; and a microvesicle marker: Annexin I) (Jeppesen et al, 2019) were asymmetrically recovered in the apical and basolateral PEG pellets (Fig 1A), consistent with the previous reports (Chen et al, 2016; Banfer et al, 2018). Moreover, by using a density gradient floatation assay, each EV marker in the PEG pellets was floated into the same fraction (Fr. 5 in Fig 1B), suggesting that the PEG pellets contained membranous organelles having the same density. Furthermore, an immunofluorescence analysis showed that CD63 (enriched in apical sEVs) was distributed in MVB-like intracellular punctate structures, whereas CD9 (enriched in basolateral sEVs) was mainly localized at the basolateral membrane and partially distributed in intracellular punctate structures, some of which overlapped CD63-positive dots in polarized MDCK cells (Fig EV1B). The CD9 enrichment in basolateral sEVs may be related to its basolateral membrane localization; however, CD63, which is enriched in apical sEVs, did not show apical membrane localization. Taken together, these results allowed us to conclude that a single MDCK cell secretes at least two types of sEVs presumably from different origins that have different EV markers: Flotillin-1-, CD63-, and Annexin I-enriched vesicles from the apical side, and CD9- and CD81-enriched vesicles from the basolateral side. Click here to expand this figure. Figure EV1. Structure of MVBs and sEVs in polarized MDCK cells Scheme of sEV and exosome isolation methods used in this study. Polarized MDCK cells were immunostained with anti-CD63 and anti-CD9 antibodies. Arrowheads, CD63- and CD9-double-positive dots; arrows, CD63-positive dots; and double-headed arrows, CD9-positive dots. Scale bar, 20 μm. Polarized MDCK cells were analyzed by conventional electron microscopy. Small ILVs and a large ILV were indicated by arrowheads and an arrow, respectively. It is noteworthy that almost all of the ILVs measured < 100 nm in diameter. Scale bar, 100 nm. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral side of the MDCK cells were purified by PEG precipitation and analyzed by NTA. Representative NTA traces were shown. Note that both PEG pellets showed a broad particle size distribution, suggesting that PEG pellets contain various types of sEVs. Quantification of the NTA data obtained in five independent experiments of (D). Mean ± s.e.m. was shown. PEG pellets prepared as in (D) were immunonegative stained with anti-CD9 antibody and analyzed by electron microscopy. Scale bar, 100 nm. The percentages of CD9-positive sEVs in 10 images were calculated. Mean ± s.e.m. was shown. Download figure Download PowerPoint Figure 1. Heterogeneous exosome release from polarized MDCK cells MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles. PEG pellets prepared as in (A) were subjected to OptiPrep flotation analysis. MDCK cells stably expressing human CD63 were cultured as in (A). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody. sEVs prepared as in (C) were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. Quantification of the NTA data obtained in five independent experiments. MDCK cells were cultured as in (A). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. sEVs prepared as in (F) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. Quantification of the NTA data obtained in five independent experiments. Data information: (A and B) Annexin I blots were separately obtained on different days using the same samples. (E and H) *P < 0.01 (two-sided Student's unpaired t-test). Mean ± s.e.m. was shown. Download figure Download PowerPoint Next, we turned our attention to the size difference between apical and basolateral sEVs. An electron-microscopic analysis of the MDCK cells revealed that most of the ILVs in their MVBs were less than 100 nm in diameter (Fig EV1C). A nanoparticle tracking assay (NTA) showed that both the apical and basolateral PEG pellets mainly contained 50–100 nm particles together with a smaller number of larger particles and that their size distributions and concentrations were similar (Fig EV1D and E). Moreover, immunonegative staining of the PEG pellets with anti-CD9 antibody showed that CD9-positive sEVs (approximately 10–20% of PEG pellets) were also less than 100 nm in diameter (Fig EV1F and G). These observations indicated that the size of apical and basolateral sEVs is not much different. Because the PEG pellets contained Annexin I-positive sEVs (Fig 1A) and/or CD9-negative sEVs (Fig EV1D–G), we decided to use purified sEV samples (CD63- and CD9-positive sEVs) rather than all sEV samples (PEG pellet and P100) to study the mechanisms of exosomal heterogeneity. To do so, we performed direct immunoaffinity capture of CD63- and CD9-positive sEVs by using anti-CD63- and anti-CD9-specific antibodies, respectively (see Jeppesen et al, 2019 for details) (Fig EV1A). As shown in Fig 1C, both apical and basolateral CD63-positive sEVs contained CD9 and CD81 but not Annexin I, and CD63 was more abundant in the apical sample than in the basolateral sample, the same as in the PEG pellets (Fig 1A). Moreover, NTA showed that most of the apical and basolateral CD63-positive sEVs were < 100 nm in diameter (Fig 1D), consistent with the results of the electron-microscopic analyses (Fig EV1C), and the concentration of the apical CD63-positive sEVs was higher than that of the basolateral sEVs (Fig 1E). Since CD63 was not localized at the plasma membrane (Fig EV1B) and the CD63-positive sEVs were negative for Annexin I (Fig 1C), we concluded that CD63-positive sEVs are most likely to mainly consist of exosomes. In contrast to the CD63-positive EVs, the CD9-positive sEVs were more abundant in the basolateral sample than in the apical sample (Fig 1F and H), but they were similar in size to the CD63-positive sEVs (Fig 1G). Consistent with a previous report (Jeppesen et al, 2019), no Annexin I was detected in the CD9-positive sEV samples, the same as in the CD63-positive sEVs (Fig 1C and F), even though CD9 was predominantly localized at the plasma membrane (Fig EV1B). Since the CD9-positive sEVs contained CD63 and CD81, the majority of CD9-positive sEVs are likely to be exosomes, although we could not completely rule out the possibility that CD9-positive, Annexin I-negative microvesicles were included in the basolateral sEV sample. Taken together, these results suggested that polarized MDCK cells differentially release CD63- and CD9-positive exosomes from their apical and basolateral sides, respectively. GPRC5C is a novel apical exosome-specific protein To further clarify the difference between the apical and basolateral exosomes, we searched for apical exosome- or basolateral exosome-specific proteins by liquid chromatography–tandem mass spectrometry (LC-MS/MS; Tables EV1–EV3). We used all sEVs collected by ultracentrifugation (Fig EV1A) to perform LC-MS/MS, because there were too little CD63- or CD9-positive sEVs to analyze. Silver staining of the proteins from apical and basolateral sEVs yielded similar band patterns (Fig 2A), and 84% of the proteins detected by LC-MS/MS in the two types of sEVs were identical (Fig 2B). One of the proteins detected, GPRC5C (G protein-coupled receptor class C group 5 member C), was detected only in the apical sample. GPRC5C is an orphan receptor that belongs to the GPRC5 family (Robbins et al, 2000) and is involved in renal acid–base homeostasis (Rajkumar et al, 2018), but it had never been reported as an exosome protein. GPRC5C was detected in the apical PEG pellet alone (Fig 2C) and floated into Fr. 5, the same as other EV marker proteins (Fig 2D). Moreover, GPRC5C was co-immunopurified only with apical CD9- and CD63-positive sEVs (Fig 2E and F), strongly suggesting that GPRC5C is a novel exosome protein, and that MDCK cells release at least two types of CD9- and CD63-positive exosomes (a GPRC5C-positive type from the apical side and a GPRC5C-negative type from the basolateral side). Figure 2. GPRC5C is an apical exosome-specific protein MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining. P100 prepared as in (A) were analyzed by LC-MS/MS. Venn diagrams represent the number of proteins detected in each sample with minimum three independent peptides. See also Tables EV1–EV3. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. PEG pellets were subjected to OptiPrep flotation analysis. MDCK cells were cultured as in (C). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. MDCK cells stably expressing human CD63 were cultured as in (C). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody. Note that GPRC5C was detected only in the apical sEV samples in all experiments performed. Data information: (C and D) Annexin I blots were separately obtained on different days using the same samples. Download figure Download PowerPoint Inhibition of the ESCRT machinery promotes exosome release The release of heterogeneous exosomes from a single cell requires the production of ILVs or MVBs having different properties. The ESCRT machinery is known to regulate EV formation from the plasma membrane and cargo sorting (Nabhan et al, 2012; Matusek et al, 2014; Hurley, 2015; Christ et al, 2017; Vietri et al, 2020). However, involvement of ESCRT in exosome biogenesis has been controversial, because some groups have reported that MVBs can be generated and exosomes are released in an ESCRT-independent manner (Trajkovic et al, 2008; Colombo et al, 2013). We therefore attempted to determine whether the ESCRT machinery is involved in the polarized exosome release from MDCK cells. The ESCRT machinery and its associated proteins can be divided into six functionally distinct subcomplexes: ESCRT-0/I/II/III, VPS4, and other ESCRT-associated proteins. We first knocked down the component(s) (HRS [ESCRT-0], TSG101 [ESCRT-I], EAP20 and 30 [ESCRT-II], CHMP6 [ESCRT-III], VPS4A/B, and ALIX [other ESCRT-associated proteins]) of each subcomplex and examined the amounts of exosome markers released (Fig 3A and B). Knockdown (KD) of most of the ESCRT proteins (HRS, TSG101, EAP20/30, CHMP6, and VPS4A/B) promoted CD9-positive sEV secretion from both sides. Moreover, the numbers of sEVs released from these cells increased without any change in their size (Fig 3C and D). It is generally thought that MVBs fuse with lysosomes for their degradation rather than with the plasma membrane to release exosomes and that they are essential for the lysosomal function via the endocytic pathway (Raiborg & Stenmark, 2009; Henne et al, 2011; Huotari & Helenius, 2011). Since lysosomal dysfunction has been shown to result in larger MVBs and to promote exosome release from breast cancer cells (Latifkar et al, 2019), we hypothesized that exosome release is accelerated by the lysosomal dysfunction caused by ESCRT-KD. To test this hypothesis, we exposed MDCK cells to the vacuolar ATPase inhibitor bafilomycin A1 to abolish the lysosomal function and confirmed that CD9-positive sEV release from both sides was upregulated in an exposure-time-dependent manner (Fig EV2A–D). Moreover, enlarged MVBs were observed in the bafilomycin A1-exposed cells, the same as in the HRS-KD and VPS4-KD cells (Fig EV2E and F) (Stuffers et al, 2009), indicating that most of the ESCRT-KDs affect the lysosomal function, thereby promoting exosome release. Figure 3. ALIX, but not the ESCRT machinery, is required for apical exosome release MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. The intensity of the bands shown in (A) was measured in three independent experiments. sEVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. Quantification of the NTA data obtained in five independent experiments. Data information: (A) CD63 blots were separately obtained on different days using the same samples. (B and D) *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Exosome release is upregulated by inhibition of lysosomal function MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the cells were cultured in EV-depleted medium with or without 100 nM bafilomycin A1 (Baf A1) for the times indicated until the medium was harvested. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. The intensity of the bands shown in (A) was measured in three independent experiments. sEVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. Quantification of the NTA data obtained in five independent experiments of (C). Polarized MDCK cells were cultured in EV-depleted medium with or without 100 nM Baf A1 for 6 h and cells were immunostained with anti-CD63 antibody. Scale bars, 20 μm (1 μm, insets). MDCK cells were transfected with siControl or the siRNAs indicated. After 3 days, the culture medium was replaced with EV-depleted medium. One day later, the cells were immunostained with anti-CD63 antibody. Scale bars, 20 μm (1 μm, insets). MDCK cells were transfected with the siRNAs indicated, cultured as in (E), and analyzed by conventional electron microscopy. Scale bars, 400 nm. Note that in addition to normal MVBs, enlarged and ILV-less MVB-like structures were often observed in HRS-KD cells. MDCK cells were transfected with siControl or two independent siALIX. The cells were then transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. The intensity of the bands shown in (H) was measured in three independent experiments. Data information: (A and H) CD63 blots were separately obtained on different days using the same samples. (B, D and I) *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown. Download figure Download PowerPoint Depletion of ALIX specifically reduces apical exosome release Unlike these ESCRT-KDs, ALIX-KD specifically decreased apical CD9- and CD63-positive sEV release and did not affect basolateral sEV release (Figs 3, EV2H and I, and EV3). Because the size of the MVBs in the ALIX-KD cells seemed to be unaffected, unlike the HRS-KD or VPS4-KD cells (Fig EV2F and G), ALIX is likely to be involved in apical exosome release independently of the ESCRT machinery. Actually, ALIX is also known to form a ternary complex with Syntenin1 and Syndecan1 and to regulate exosome biogenesis (Baietti et al, 2012; Ghossoub et al, 2014). As shown in Fig EV4, Syntenin1-KD and Syndecan1-KD phenocopied ALIX-KD (Figs 3 and EV2H and I), strongly suggesting that ALIX regulates apical exosome release together with Syntenin1 and Syndecan1, and independently of the ESCRT machinery. Since ALIX has been shown to mediate the sorting of exosome cargo proteins (Dores et al, 2012; Dores et al, 2016; Larios et al, 2020) and Syntenin1 can bind CD63 (Latysheva et al, 2006), an abundant protein in apical exosomes (Fig 1), the ALIX–Syntenin1–Syndecan1 complex presumably regulates the cargo protein sorting to apical exosomes and may also be involved in the efficient exosome formation in MDCK cells. Click here to expand this figure. Figure EV3. The effect of ALIX-KD and GW4869 treatment on release of CD63-positive sEVs and all sEVs MDCK cells stably expressing human CD63 were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody or ultracentrifugation. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. The intensity of the bands shown in (A) was measured in three independent experiments. sEVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. Quantification of the NTA data obtained in five independent experiments of (C). Data information: (B and D) *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Syntenin1 and Syndecan1 are specifically involved in the apical exosome release MDCK cells were transfected with siControl or the siRNAs indicated. The cells were then transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated. The intensity of the bands shown in (A) was measured in three independent experiments. EVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. Quantification of the NTA of data obtained in five independent experiments of (C). Data information: (A) CD63 blots were separately obtained on different days using the same samples. (B and D) *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown. Download figure Download PowerPoint Inhibition of ceramide synthesis specifically inhibits basolateral exosome release To identify the mechanism of the ALIX/ESCRT-independent basolateral exosome release, we turned our attention to the sphingolipid ceramide, because it is enriched in exosomes and regulates formation and release of EVs independently of the ESCRT machinery (Trajkovic et al, 2008; Menck et al, 2017). Ceramide is formed as a result of the hydrolytic removal of the phosphocholine moiety of sphingomyelin by sphingomyelinases (SMases), and the neutral SMase inhibitor GW4869 is often used as an effective drug to suppress EV release (Trajkovic et al, 2008; Menck et al, 2017; Verweij et al, 2018; Catalano & O'Driscoll, 2020). When MDCK cells were treated with GW4869, basolateral CD9- and CD63-positive sEV release was specifically reduced without affecting apical sEV release (Figs EV3 and EV5A–D). Essentially, the same results were obtained by nSMase2-KD (Fig EV5E–H), indicating that ceramide is involved only in basolateral CD9- and CD63-positive exosome release. Click here to expand this figure. Figure EV5. Ceramide is specifically involved in the basolateral exosome release MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the cells were cultured in EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples w