Journal: Human Molecular Genetics

Article Title: Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease

doi: 10.1093/hmg/ddv220

Figure Lengend Snippet: Identification of endolysosomes in AP-5 patient lines. ( A ) Cryo-immunoelectron microscopy of patient-derived fibroblasts (p.Q578*) labelled with antibodies against the late endosomal/lysosomal markers, LAMP1, LBPA and CD63. Note that the enlarged endocytic structures label positive for all markers, with LAMP1 labelling restricted to the limiting membrane, and LBPA and CD63 largely restricted to intraluminal whorls and striations. Scale bar = 200 nm. ( B ) Live imaging of control and patient-derived fibroblasts following incubation with Magic Red Cathepsin B reagent for 45 min at 37°C. Note that the enlarged structures in the patient lines are positive for cathepsin B activity and are therefore hydrolytically active. Scale bar = 20 µm. ( C ) Live imaging of control and patient-derived fibroblasts incubated with Lysotracker Red at 37°C. Note that the enlarged structures in the patient lines are positive for Lysotracker Red and are therefore acidic. Scale bar = 20 µm.

Article Snippet: Antibodies used in this study include in-house antibodies against clathrin, AP-5 ζ (monoclonal antibody used for IF), µ5 and SPG11 monoclonal ( ) and spastizin [PER antibody ( )] and commercial antibodies against spastizin (Atlas HPA035693), EEA1 (BD Transduction Labs E41120), LAMP1 (Abcam ab24170 and H4A3), CIMPR (2G11; Calbiochem 444105), AP-2 µ2 (AP50; BD Transduction Labs 611351), LC3 (4E12; MBL M152-3B), LBPA (Jean Gruenberg), CD63 (H5C6; BD Biosciences), tubulin (DM1A, Abcam ab7291) and actin (C4, Abcam ab3280).

Techniques: Immuno-Electron Microscopy, Derivative Assay, Imaging, Incubation, Activity Assay

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: Molecular Nutrition & Food Research

Article Title: Bovine Milk‐Derived Extracellular Vesicles Inhibit Catabolic and Inflammatory Processes in Cartilage from Osteoarthritis Patients

doi: 10.1002/mnfr.202100764

Figure Lengend Snippet: Characterization of commercial milk‐derived extracellular vesicles. Within 2 h, EVs were isolated using ultracentrifugation. A) Particle size distribution of isolated vesicles was determined using a NS300. Data presented is a combination of eight separate isolations, error bars represent mean ± SEM. B) Electron microscopy confirmed spherical morphology and biolayer membrane structure. C) Sucrose density gradient following standard ultracentrifugation‐based isolation shows particles in the range of 1.16–1.20 g mL –1 , which is the described range for exosome‐like vesicles. D) Western blotting confirmed the presence of EV‐markers ALIX, HSP70, CD63, and CD81.

Article Snippet: Antibodies used; CD81 (B‐11), Santa Cruz, sc‐166029; HSP70 (3A3), Santa Cruz, sc‐32239: CD63 (H5C6), BD Pharmingen, 556019; ALIX (1A12), Santa Cruz, sc‐53540.

Techniques: Derivative Assay, Isolation, Electron Microscopy, Western Blot

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: CD63-pHluorin is sorted into acidic MVBs and released via exosomes. (a) Proposed model for the visualization of MVB–PM fusion: a pH-sensitive optical reporter (CD63-pHluorin) is quenched when facing the acidic lumen of the MVB. Upon fusion, low luminal pH is immediately neutralized, resulting in a sudden increase in fluorescent intensity. EC, extracellular. (b) Immunofluorescent colabeling of total CD63 (red) and CD63-pHluorin (green) in HeLa cells. PCC, Pearson’s correlation coefficient. (c) TIRF images of a CD63-pHluorin–expressing HeLa cell at normal and elevated intracellular pH (NH 4 Cl superfusion). On the right, a heat map revealing acidic vesicles close to the PM was obtained by subtracting the fluorescent intensity values of the normal pH from the high-pH condition. (d) EM images of an MVB close to the PM (left) and EVs aligning the PM (right) labeled with gold particles directed to GFP (10 nm) in CD63-pHluorin–expressing HeLa cells. (e) Imaging flow cytometry of the number of late endosomes per cell in a 2.5-µm optical section in CD63-pHluorin–expressing cells (left) or immunostaining against LAMP1 in nontransfected cells (right; n > 2,000 cells). (f) Volume distribution of endosomes based on analysis of whole-cell confocal scans (error bars represent SD; n = 3). The blue area accounts for 75% of the total number of endosomes and covers the 400–600-nm-diameter range. (g) Immunogold labeling on purified exosomes with gold particles (10 nm) coupled to anti-GFP antibody. (h) Western blotting analysis on untransfected (−) and CD63-pHluorin–transfected (+) cells and purified exosomes for total CD63 and GFP. (i) Example of a localized sudden increase in fluorescence at the PM before the event (1), during the event (2), and right before disappearance of the signal (3). (j) Left: total projection of fusion events (bright spots) over a time course of 3 min onto two cells (blue). Right: representative example of CD63-pHluorin–expressing HeLa cell. N, nucleus. Bars: (b, c, and j) 10 µm; (i) 2.5 µm. (k) Effect of incubation with GW4896 (5 µM; n ≥ 8 cells per condition) and nSMase-2 knockdown ( n ≥ 22 cells per condition) on fusion activity in HeLa cells. *, P < 0.05; ***, P < 0.001 using Student’s two-tailed two-sample t test. Whiskers in the box plots represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter. (l) Western blotting analysis on purified exosomes from GW4896- and control-treated HeLa cells for CD63 and CD81.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Expressing, Labeling, Imaging, Flow Cytometry, Immunostaining, Purification, Western Blot, Transfection, Fluorescence, Incubation, Activity Assay, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: CD63-pHluorin fusion events are derived from MVBs. (a) Left three panels: live imaging of fusion events (indicated by white arrows) over a time course of 12 s onto one cell before the event (left), at the start of the event (middle), and right before fixation of the cell (3). Right: inset showing a magnification of the localized sudden increase in fluorescence at the PM (highlighted by a dashed line square) right before fixation. (b) Left: correlation of light microscopy signal of a fusion event observed by live imaging with EM pictures of the first section of the cell facing the coverslip (low magnification). Right: correlation of light microscopy signal with the first slice of the electron tomographic reconstruction of the first section of the cell facing the coverslip. The orange circle indicates the error range (167 nm) of the correlation performed by eC-CLEM. (c) 3D model of the electron tomographic reconstruction. The ER is depicted in light violet. Dense compartments are depicted in brown. The structure of interest is depicted in red and orange. (d) Bottom side view of the 3D model of the compartment of interest in its surroundings. The white arrow indicates the opening of the MVB where ILVs are released. (e) 3D model showing the MVB isolated from its environment. ILVs secreted through the opening of the MVB are depicted in white. (f) Top view of the secretory profile of the MVB that correlates with the fluorescence burst of the CD63-pHluorin fusion event.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Derivative Assay, Imaging, Fluorescence, Light Microscopy, Isolation

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: MVB–PM fusion is distinct from other forms of vesicle-mediated exocytosis. (a) Schematic model showing the markers used in this study for the different types of cargo delivery of vesicles fusing with the PM. (b) Time-lapse imaging (heat maps) of a fusion event of the exosomal protein CD63-pHluorin. (c) Time-lapse images of soluble (NPY-pHluorin) and membrane protein (VAMP2-pHluorin) fusion events. (d) Fluorescent signal duration of NPY (mean = 0.85 s), VAMP2 (mean = 2.12 s), and CD63 (mean = 106.55 s) fusion events. n ≥ 13 events per reporter. (e) 3D heat maps of three consecutive CD63-pHluorin fusion event frames. (f) Western blot for exosomal markers (CD63 and Alix) on EVs purified from the supernatant (soluble) and EVs attached to the cell surface (PM attached) isolated after short trypsinization of the cells. (g) Direct comparison between signal duration of fusion events of CD81- and CD9-pHluorin relative to CD63-pHluorin. n ≥ 20 events per reporter. ***, P < 0.001; ****, P < 0.0001 using Student’s two-tailed two-sample t test.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Imaging, Western Blot, Purification, Isolation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: GPCR activation triggers MVB–PM fusion in single cells in a calcium-independent manner. (a) Schematic model of imaging setup. (b) Fusion activity of HeLa cells stimulated with KCl (70 mM), caffeine (20 mM), or histamine (100 µM). n ≥ 8 cells per condition. (c) Total projection of fusion events over a 60-s time course onto cells before (top) and after (bottom) stimulation with histamine (100 µM). Pseudocolored as in . (d) Measurement of individual HeLa cells ( n = 14) before and during stimulation with histamine (100 µM). (e) Mean fusion kinetics of CD63-pHluorin HeLa cells ( n = 6) showing the distribution of fusion events over time (dark blue line; SD is in light blue) and the calcium levels (red) during histamine stimulation (gray-shaded block). (f) Heat maps revealing calcium responses (measured by Fluo-4) upon histamine stimulation obtained by subtracting the fluorescent intensity values before stimulation from those after 8-s stimulation. Cells were nontreated or incubated with a buffer with fast (BAPTA) or slow (EGTA) calcium-binding kinetics. Bars, 10 µm. (g) Quantification of fusion activity of histamine-stimulated HeLa cells in the presence of EGTA (top) or BAPTA (bottom) buffers. n ≥ 10 cells per condition. (h) Measurement of individual HUVEC cells ( n = 30) before and after stimulation with histamine (100 µM). *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. All t tests were unpaired except for d and h. Whiskers in the box plots (b and g) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Activation Assay, Imaging, Activity Assay, Blocking Assay, Incubation, Binding Assay, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: The GPCR downstream effector SNAP23 regulates MVB–PM fusion. (a) Network of proteins of interest together with direct interactors with altered phosphorylation levels upon histamine (100 µM) stimulation as identified by phosphoproteomics in HeLa and HUVEC cells. Proteins of interest are depicted with a blue rim. FC, fold change. (b) Graph showing the signal intensity values of phosphorylated peptides from proteins of interest before and after stimulation with 100 µM histamine. Data represent means ± SD of two technical replicates per condition. *, P < 0.05 (P = 0.048) using Student’s two-tailed two-sample t test. (c) Western blotting analysis on SNAP23 protein expression in six different cell lines. (d) Confocal analysis of FL (GFP-SNAP23-FL) and truncated (GFP-SNAP23-CΔ9) GFP-SNAP23 (in gray)–transfected SiHa cells labeled for CD63 (red). (e) Total projection of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9 over 3 min. Pseudocolored as in . Bars, 10 µm. (f) Quantification of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9. n ≥ 10 cells per condition. (g) Confirmation of SNAP23 knockdown (KD) at the protein level in HeLa cells. (h) Effect of SNAP23 knockdown on MVB–PM fusion in HeLa cells. n ≥ 17 cells per condition. (i) Confirmation of SNAP23 and syntaxin-4 knockdown in HeLa cells at the mRNA level. Data represent means ± SD. (j) Effect of the knockdown of SNAP23 or syntaxin-4 on the fusion activity of HeLa cells. n ≥ 11 cells per condition. ctrl, nontransfected; siCTRL, control siRNA. *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. Whiskers in the box plots (f, h, and j) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Two Tailed Test, Western Blot, Expressing, Transfection, Labeling, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca 2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P < 0.01; ****, P < 0.0001 using Student’s two-tailed two-sample t test. All t tests were paired except for b and f. Whiskers in the box plots in b and f represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Activation Assay, Activity Assay, Transfection, Western Blot, Isolation, Labeling, Two Tailed Test

Journal: Journal of Extracellular Biology

Article Title: Analysis of extracellular vesicle microRNA profiles reveals distinct blood and lymphatic endothelial cell origins

doi: 10.1002/jex2.134

Figure Lengend Snippet: Characterization of enriched extracellular vesicles. (a) Concentration and (b) size of the P10 (microvesicle fraction), P100 (small EV fraction) and S100 (supernatant) fraction after the differential ultracentrifugation enrichment protocol. Particles per mL or nm derived from NTA measurements (11 positions, three technical replicates) are plotted for LEC (blue, n = 5) and BEC (red, n = 2). (c) Representative size distribution histograms for the P10 and P100 fraction from one LEC and BEC donor. (d) Concentration of lipid membrane dye (CMG) stained EVs determined by FITC‐triggered flowcytometry. EVs/mL are plotted for 14 technical replicates for LEC (blue, n = 5) and BEC (red, n = 2). (e) Box plots of the mean fluorescence intensity (MFI) of EV markers (tetraspanins CD81, CD63), endothelial cell surface marker (CD31) and the isotype control detected for CMG‐positive events for the P10 and P100 fraction. Dashed lines are the average MFI of the isotype controls for LEC ( n = 5) and BEC ( n = 2). (f) Percentage of events detected in the respective size range gate of small EVs (grey, <200 nm), intermediate EVs (middle grey, 200–500 nm) and large EVs (dark grey, >500 nm). Data is derived from 14 technical replicates of CMG‐stained P10 and P100 fraction derived from LEC ( n = 5) and BEC ( n = 2). (g) Representative scatter plots for P10 and P100 fraction derived from one LEC and BEC donor. (h) Representative cryo‐TEM images of EV enriched fractions P10 and P100 from LEC and BEC (Scale Bar = 100 nm).

Article Snippet: Staining of respective antigens was performed by addition 1 μL directly PE‐conjugated antibodies for CD63, CD81 (Clones REA1055 and REA513, Miltenyi Biotec, Bergisch Gladbach, Germany) and CD31 (Clone WM59, BD Pharmingen).

Techniques: Concentration Assay, Derivative Assay, Membrane, Staining, Fluorescence, Marker, Control