Secreted exosomes induce filopodia formation
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, United States
- Center for Extracellular Vesicle Research, Vanderbilt University School of Medicine, United States
- Department of Biological Sciences, Vanderbilt University, United States
- Kanagawa Cancer Center, Japan
- Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, United States
- Department of Biomedical Informatics, Vanderbilt University Medical Center, United States
- Cell Imaging Shared Resource EM Facility, Vanderbilt University, United States
Figures
Figure 1 with 1 supplement
Exosome markers localize to the base and tips of filopodia in cancer cells and cortical neurons.
(A) Representative confocal image of HT1080 cells stained with phalloidin-Alexa fluor 488 and CD63 shown in red. The red channel has been edited using brightness and contrast tools for ease of visibility. Note the localization of the exosome marker CD63 in extracellular deposits and at or near the tips of filopodia (arrowheads). Representative of 20 images. Scale bar is 10 mm in each panel. (B) Time series of pHluorin-M153R-CD63-mScarlet movie in HT1080 cells. Yellow arrowheads indicate fusion sites and yellow arrows indicate filopodia. Note a filopodium forming shortly after MVE fusion. (C) Representative kymographs showing MVE docking (red), fusion (yellow), and filopodia formation in HT1080 cells. Yellow arrowheads denote MVE fusion events, and black arrowheads denote the formation of a filopodium. Each pixel is 10 s x 0.2857 mm. (D) Quantification of the time elapsed between MVE fusion and filopodia formation. n=420 kymographs from 46 cells from three independent experiments (biological replicates). (E) Primary cortical neurons were co-transfected with GFP-Rab27b (green) and mCherry as a filler to visualize filopodia (red) on DIV5 and fixed for imaging on DIV6. SV2 negative staining (no signal) identifies these structures as filopodia instead of dendritic spines. Arrows in merged images indicate localization of GFP-Rab27b to tips and bases of filopodia. Scale bars = 5 µm. (F) Percent GFP-Rab27b localization to tips and bases of filopodia in 70 individual cortical neurons from three independent experiments (biological replicates). Red line indicates the median. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
Figure 1—video 1
Visualization of exosome secretion and filopodia formation dynamics.
HT1080 cells stably expressing pHLuorin_M153R-CD63-mScarlet were seeded onto glass bottom MatTek plates and imaged live. Images were taken every 10 s.
Figure 2 with 3 supplements
Exosomes promote filopodia formation and stability in cancer cells.
(A) EVs secreted from equal numbers of control (shLacZ), Rab27a-KD, and Hrs-KD B16F1 cells over 48 hr were quantified using NanoSight particle tracking analysis (N=3 biological replicates). (B) Representative images of control (shLacZ), Hrs-KD, and Rab27a-KD B16F1 cells stained with rhodamine-phalloidin. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. (C) Quantification of filopodia from images as in B (≥27 total cells per condition from three biological replicates). Filopodia number per 500 mm2 cell area. (D) Representative images of filopodia in B16F1 control (shLacZ) and exosome-depleted (shHrs) cells treated for 18 hr with LEVs or SEVs isolated from control cells. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. (E) Quantification of filopodia from images as in D (≥20 total cells per condition from three biological replicates). (F) Filopodia number in B16F1 shScr cells treated with indicated numbers of purified LEVs or SEVs, for 18 hr (≥25 total cells per condition from three biological replicates). (G) Control (shLacZ) and exosome-depleted (shHrs) B16F1 cells were transiently transfected with tdTomato-F-tractin to visualize filopodia formation. Live images were taken every 30 s for 15 min and newly formed filopodia were counted at each time point. Only filopodia that form and fully retract during the duration of the video were quantified. (≥20 total cells per type per biological replicate, from three biological replicates). (H) Lifetime of newly formed filopodia from G. Lifetime is defined as the time from first formation of the filopodia to full retraction. Bars represent mean and error bars are SEM. Scale bars in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
Figure 2—figure supplement 1
Characterization of B16F1 cells and EV preparations.
(A) Western blot of Hrs-KD in B16F1 cells. (B) Western blot of Rab27a-KD in B16F1 cells. (C) Nanoparticle tracking analysis representative traces showing particle size distributions that correspond to the data for control SEVs and LEVs in Figure 2A. (D) Western blot of gradient fractions for SEVs prepared by the cushion density gradient (DG) method. TCL, total cell lysate. LEVs also run on gel. (E) TEM of negatively stained LEVs and SEVs prepared by the cushion-DG method. Scale bar = 200 nm in each image.
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Figure 2—figure supplement 1—source data 1
PDF file containing the original western blots from Figure 2—figure supplement 1A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
PDF file containing the original western blots from Figure 2—figure supplement 1B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data3-v1.zip
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Figure 2—figure supplement 1—source data 4
Original files for western blot analysis displayed in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data4-v1.zip
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Figure 2—figure supplement 1—source data 5
PDF file containing the original western blots from Figure 2—figure supplement 1D, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data5-v1.zip
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Figure 2—figure supplement 1—source data 6
Original files for western blot analysis displayed in Figure 2—figure supplement 1D.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp1-data6-v1.zip
Figure 2—figure supplement 2
Characterization of HT1080 cells and EV preparations and comparison of graphing methods.
(A) WB of Rab27a KD in HT1080 cell lysates. (B) TEM of SEVs (purified by cushion DG) and LEVs from HT1080 cells. Scale bar = 200 nm in each image. (C) Secretion rates of SEVs from HT1080 cell lines (N=3). Nanoparticle tracking analysis traces of SEVs from shScr and shRab27a HT1080 cells showing size (diameter) distribution of SEVs and particles/mL/cell. (D) Representative images showing filopodia in control and Rab27a-KD H1080 cells. Images have been edited with brightness and contrast tools for ease of visibility. Scale bars in wide field and zoom insets = 10 mm. (E) Quantification of filopodia in control and Rab27a-KD HT1080 cell lines. ≥20 cells per condition per biological replicate, from three biological replicates. (F) Data from graph in E displayed as filopodia per cell. (G) Data in Figure 2C displayed as filopodia per cell. (H). Data from Figure 2E displayed as filopodia per cell. (I) Data from Figure 2F displayed as filopodia per cell. (J) Data from Figure 2G displayed as filopodia per cell. (K) Cell areas of cells used for quantification in Figure 2G. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 2—figure supplement 2—source data 1
PDF file containing the original western blots from Figure 2—figure supplement 2A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig2-figsupp2-data2-v1.zip
Figure 2—video 1
Live imaging of filopodia in B16F1 control and shHrs cells.
B16F1 cells stably expressing shHrs or control plasmid (shLacZ) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek Plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted, de-noised, and brightness/contrast was adjusted to facilitate visualization of filopodia.
Figure 3 with 2 supplements
Exosomes promote filopodia, spine, and synapse formation in cortical neurons.
Primary cortical neurons were co-transfected with plasmids for expression or inhibition of exosome regulatory molecules and mCherry (red) as a cytoplasmic filler to image neuronal protrusions, then fixed and immunostained with SV2 (pseudo-colored in cyan) to visualize synapses. Filopodia were identified as thin SV2-negative, mCherry-positive protrusions. Spines were identified as dendritic protrusions that co-localize with SV2. Synapses were identified as SV2-positive puncta present on both dendritic protrusions and dendritic shafts. (A, C) Representative images of primary rat cortical neurons co-transfected at DIV6 when filopodia typically form (A) or DIV12 when synapses typically form (C) with GFP or GFP-Rab27b (green, left images) and mCherry (red) and co-stained with SV2 (blue, right images). (B, D) Quantification of filopodia (DIV6), spine and synapse density (DIV12) from images as in A and C. (E–G) Images and analysis from neurons transfected with control shRNA (NTshRNA) or shRNAs against Rab27b (E) or Hrs (G) and immunostained for SV2. Quantification of filopodia (DIV6), spine and synapse density (DIV12) for Rab27b-KD (F) or Hrs-KD (H) neurons. (I, J) Rescue experiments. Control and KD neurons (as indicated) expressing shRNAs and mCherry were treated with purified SEVs on DIV5 for 24 hr at a dose of 200 EVs per neuron, then fixed and stained for SV2 at DIV6. Filopodia, spine, and synapse density were quantified from more than 30 primary or secondary dendritic shafts from three independent experiments (biological replicates) for each condition. Scale bars = 5 mm. Error bars, SEM. *p<0.05, **p<0.01, ***p<0.001.
Figure 3—figure supplement 1
Exosome secretion enhances filopodia, spine, and synapse numbers in primary hippocampal neurons.
(A, B) Primary rat hippocampal neurons were co-transfected with GFP-Rab27b or GFP, and mCherry filler (red) on DIV5 and immunostained for SV2 (cyan) on DIV6. Overlay images showing localization of GFP-Rab27b at the base and tips of filopodia and quantification of percent GFP-Rab27b localization at tips and bases of filopodia in hippocampal neurons are shown in Figure 1E and F. (A) Representative mCherry and SV2 overlay images. (B) Quantification of filopodia, spine, and synapse density from images as in A. (C–F) Hippocampal neurons were transfected with NTshRNA or shRNAs against Rab27b (C, D) or Hrs (E, F) and immunostained for SV2 at DIV6 for filopodia analysis or DIV12 for spine and synapse analysis. Quantification of the number of filopodia, spines, and synapses for Rab27b-KD (D) or Hrs-KD (F) neurons. Filopodia, spine, and synapse density quantified from ≥30 primary or secondary dendritic shafts from three independent experiments (biological replicates) for each condition. Scale bar = 5 µm. Error bars, SEM. *p<0.05, **p<0.01, ***p<0.001.
Figure 3—figure supplement 2
Analysis of exosome gene knockdown and SEV rescue in cortical neurons.
(A, B) Representative images of neurons co-transfected with NTshRNA, Hrs shRNA, or Rab27b shRNA, and mCerulean, as indicated, and immunostained for Hrs or Rab27b. Blue arrows indicate mCerulean-expressing neurons, which are the ones assumed to be cotransfected with shRNA and are quantitated for filopodia, spine, and synapse density. White arrows indicate non-mCerulean-expressing neurons. (C, D) Quantitation of Hrs (C) or Rab27b (D) immunofluorescence levels in mCerulean-expressing neurons for the indicated transfection conditions. (E–G) Analysis of SEVs purified from cortical neurons (CN). (E) Nanoparticle tracking analysis trace showing size distribution. (F) Western blot analysis of positive (TSG101, Flotillin-1, Alix) and negative (GM130) markers. (G) Negative stain TEM image. (H, I) Representative images of control and exosome-KD cortical neurons +/-addback of purified SEVs (+SEV). Arrows indicate filopodia. Scale bar = 5 mm. Quantitation is in Figure 3I and J. Error bars, SEM. *p<0.05, **p<0.01, ***p<0.001.
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Figure 3—figure supplement 2—source data 1
PDF file containing the original western blots from Figure 3—figure supplement 2F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 2F.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig3-figsupp2-data2-v1.zip
Figure 4 with 3 supplements
Endoglin is an SEV-enriched cargo that promotes filopodia formation.
(A) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see Supplementary file 1 for the full proteomics results). (B) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for endoglin, and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. (C) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. (D) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. (E) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). (F) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in Figure 5E. (G, H) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. (G) De novo filopodia formation. (H) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 4—source data 1
PDF file containing original blot for Figure 4A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for Coomassie blue gel displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data2-v1.zip
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Figure 4—source data 3
PDF file containing the original western blots from Figure 4B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data3-v1.zip
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Figure 4—source data 4
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data4-v1.zip
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Figure 4—source data 5
PDF file containing the original western blots from Figure 4C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data5-v1.zip
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Figure 4—source data 6
Original files for western blot analysis displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-data6-v1.zip
Figure 4—figure supplement 1
KD of endoglin affects filopodia numbers but not EV numbers in B16F1 melanoma cells.
(A) Nanoparticle tracking analysis traces for B16F1 control (shScr) and endoglin-KD (shEng) SEVs. (B) SEV secretion rates from B16F1 shEng stable lines. N=5 biological replicates. (C) Representative western blot of Endoglin-KD in transient siRNA-transfected B16F1 cells. (D) Filopodia numbers in siRNA-transfected B16F1 cells (≥23 cells per condition per biological replicate, from three biological replicates). (E) Images from control and shHrs cells incubated with purified EV, corresponding to graph in Figure 4F. Scale bar in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 4—figure supplement 1—source data 1
PDF file containing the original western blots from Figure 4—figure supplement 1C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1C.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
HT1080 Endoglin-KD cells have reduced filopodia.
(A) Western blot of Endoglin KD in HT1080 cells. (B) Nanoparticle tracking analysis traces of SEVs purified from shScr and shEng HT1080 cells showing size distribution (diameter) of SEVs and particles/mL/cell (N=3 biological replicates). (C) SEV secretion rates of HT1080 shScr and shEng HT1080 cells. (D) Representative images of HT1080 shScr and shEng cells. Images have been edited with brightness and contrast for ease of visibility. Scale bar in wide field and zoom insets = 10 mm. (E) Quantitation of filopodia density for control and shEng HT1080 cells.≥20 cells per condition per biological replicate, from four biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 4—figure supplement 2—source data 1
PDF file containing the original western blots from Figure 4—figure supplement 2A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 2A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig4-figsupp2-data2-v1.zip
Figure 4—video 1
Live imaging of filopodia in B16F1 control and shEng cells.
B16F1 cells stably expressing shEng or control plasmid (shScr) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted and brightness/contrast was adjusted to facilitate visualization of filopodia.
Figure 5 with 1 supplement
Exosomal endoglin controls motility and metastatic colony formation.
(A) Cartoon diagram of metastatic colony assay in avian embryos. On day 0, fluorescent HT1080 cells were injected (100,000 cells per egg) into the vein of the chicken embryo. On day 4, the egg was opened, the embryo was sacrificed, and a circular tool was used to punch holes through the shell. The chorioallantoic membrane (CAM) was peeled away from the shell, placed on a glass slide with a coverslip, and immediately imaged. The cartoon was created using BioRender.com. (B) Representative low power wide field images of colony formation in the CAM. Scale bar = 200 mm. (C) Representative high-power wide field images of colony formation in the CAM. Scale bar = 100 mm. (D, E) Quantification of CAM colony number (D) and size (E) from high-power images as in C. 4–7 eggs were harvested per replicate for each condition for three biological replicates. (D) Colony number is graphed per field of view using 25–30 fields of view per egg. (E) Quantification of the percent of large (≥5000 mm2) colonies formed by control and shEng HT1080 cells. (F) 3D invasion in collagen. HT1080 cell spheroids were seeded in collagen gels and imaged for 8 hr. Invasion is quantified as fold area increase in the size of each spheroid over 8 hr. Scale bar = 100 mm. Error bars, SEM. ns, not significant; *p<0.05; ** p<0.01; *** p<0.001.
Figure 5—video 1
Endoglin controls 3D migration in type I collagen.
The indicated HT1080 cell types were induced to form spheroids and then mixed into 3D type I collagen. Spheroids were imaged every 30 min for 8 hr. Scale bar = 100 mm.
Figure 6 with 1 supplement
Exosomal endoglin promotes filopodia formation through THSD7A.
(A) Native gel Western blot of B16F1 SEVs. (B) Standard western blot of HT1080 SEVs. (C) Western blot of cortical neuron total cell lysate (TCL) and SEVs. (D) Representative images and quantitation of filopodia number in control (lipofectamine) and THSD7A-mScarlet-transfected HT1080 cells. Arrowheads indicate THSD7A at the ends of filopodia (white arrowheads) or in extracellular deposits (red arrowheads). Scale bars in wide field and zoom insets = 10 mm. (E) (Left) Western blot of control shRNA (NTC) and shTHSD7A (C-04, C05, C-06) - expressing HT1080 cell lines. Vinculin is used as a loading control and numbers below the blot indicate normalized THSD7A levels. (Right) Filopodia counts in control and shTHSD7A HT1080 cells. ≥20 cells per condition per biological replicate, from three biological replicates. (F) THSD7A coated coverslips rescue filopodia defect in shEng B16F1 and HT1080 cells.≥20 cells per condition per biological replicate, from three biological replicates. (G, H) Cortical neurons were transfected with a FLAG-THSD7A expression vector (Kuo et al., 2011) or vector control, fixed, and stained with an antibody against THSD7A, and imaged by confocal microscopy. (G) Representative images. Arrows indicate THSD7A localization to the tips of filopodia. Scale bar = 5 mm. (H) Quantification of filopodia in neurons expressing FLAG-THSD7A or control vector. n=42 neurons from three separate experiments (biological replicates). (I) Rescue of filopodia numbers in shHrs neurons plated on dishes coated with various concentrations of recombinant human THSD7A, as indicated. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 6—source data 1
PDF file containing the original western blots and Ponceau stain from Figure 6A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot and Ponceau analysis displayed in Figure 6A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data2-v1.zip
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Figure 6—source data 3
PDF file containing the original western blots from Figure 6B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data3-v1.zip
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Figure 6—source data 4
Original files for western blot analysis displayed in Figure 6B.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data4-v1.zip
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Figure 6—source data 5
PDF file containing the original western blots from Figure 6C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data5-v1.zip
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Figure 6—source data 6
Original files for western blot analysis displayed in Figure 6C.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data6-v1.zip
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Figure 6—source data 7
PDF file containing the original western blots from Figure 6E, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data7-v1.zip
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Figure 6—source data 8
Original files for western blot analysis displayed in Figure 6E.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-data8-v1.zip
Figure 6—figure supplement 1
Analysis of candidate EV cargoes for rescue of filopodia defects in shEng cells.
(A) Western blot showing b1-integrin, TGFb1, ALK1 levels in control (shScr) and endoglin-KD (shEng) B16F1 SEVs. (B) Filopodia density analysis of B16F1 shScr and shEng cells treated with BMP-9.≥20 cells per condition per biological replicate, from three biological replicates. (C) Filopodia density analysis of B16F1 shScr and shEng cells treated with TGFb1.≥20 cells per condition per biological replicate, from three biological replicates. (D) Filopodia density analysis of B16F1 shScr and shEng cells plated on 20 µg/ml fibronectin (FN) or 100 µg/mL poly-D-lysine (PDL). ≥20 cells per condition per biological replicate, from three biological replicates. (E) Filopodia density analysis of B16F1 shScr and shEng cells plated on PDL or 2 µg/mL rhTHSD7A for the indicated time points, then fixed and stained for filopodia. ≥20 cells per condition per biological replicate, from biological replicates.
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Figure 6—figure supplement 1—source data 1
PDF file containing the original western blots from Figure 6—figure supplement 1A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig6-figsupp1-data2-v1.zip
Figure 7
Endoglin controls THSD7A trafficking to exosomes.
(A) Western blot analysis of total cell lysates (TCL) and SEVs from HT1080 control and shEng cells +/-rescue with WT endoglin or control expression vectors. The figure was made from cropped images of membranes to remove irrelevant lanes. (B) Quantification of endoglin expression (normalized to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. (C) Quantification of THSD7A expression (relative to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. (D) Quantification of filopodia in HT1080 control cells and shEng cells rescued with WT endoglin expression. N=3, at least 30 total cells per condition. (E) Representative confocal images of THSD7A-mScarlet-expressing control and shEng HT1080 cells immunostained for CD63. Box 1 shows extracellular THSD7A and CD63 deposits. Box 2 shows intracellular CD63-positive MVEs. For both boxes, the zoomed images have been adjusted for brightness and contrast (to equivalent levels for control and shEng cells) for easy visualization. Note that the overlap of THSD7A (magenta) and CD63 (green) gives a white signal, pointed out with white arrowheads in the shEng merged image in Zoom 2. Scale bar is 10 mm in wider field view and 5 mm in zoom insets. (F) Quantification of colocalization of internal CD63 and mScarlet signals in HT1080 cells from nonadjusted images.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 7—source data 1
PDF file containing the original western blots from Figure 7A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig7-data1-v1.zip
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig7-data2-v1.zip
Figure 8 with 1 supplement
Filopodia formation induced by THSD7A depends on Cdc42 activity.
Control and endoglin-KD HT1080 cells were plated on coverslips coated with poly-D-lysine (PDL) or THSD7A. In some cases, cells were treated with the Cdc42 inhibitor ML141 (10 µM) or transfected with the dominant active Cdc42 mutant Q61L, as indicated.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
Figure 8—figure supplement 1
Total cell lysates of B16F1 cells were seeded on PDL +/-THSD7 A and treated with or without TGF-b1 and inhibitor.
Cell lysates were probed for phospho-Smad and total Smad levels. Representative of three blots. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.
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Figure 8—figure supplement 1—source data 1
PDF file containing the original western blots from Figure 8—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig8-figsupp1-data1-v1.zip
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Figure 8—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/101673/elife-101673-fig8-figsupp1-data2-v1.zip
Figure 9
Model of exosome-induced filopodia formation.
(A) In tumor cells, endoglin and THSD7A are trafficked into intralumenal vesicles (ILV) in multivesicular endosomes (MVEs) for secretion. Inhibiting the exosome biogenesis pathway by blocking Hrs or inhibiting MVE docking by blocking Rab27a subsequently reduces exosome secretion and filopodia formation. SEVs carrying THSD7A can induce filopodia on target cells via Cdc42, leading to increased cell motility and metastasis. When endoglin levels are lowered (such as by KD), THSD7A is retained inside cells in CD63-positive endosomes, and its levels in SEVs are greatly decreased. The drop in THSD7A levels in endoglin-KD EVs could be due either to a lack of trafficking into ILVs or, alternatively, enhanced lysosomal degradation of THSD7A-containing MVEs. Given that THSD7A accumulates in CD63-positive endolysosomal compartments in endoglin-KD cells (Figure 7E and F), the latter possibility seems more likely. The cartoon was created using BioRender.com. (B) In primary neurons, exosome biogenesis is controlled by the formation of ILVs by Hrs and MVE docking is controlled by Rab27b. Knockdown of either of these proteins results in reduced formation of filopodia, dendritic spines, and synapses in both cortical and hippocampal neurons. Similar to cancer cells, THSD7A is carried in neuronal SEVs and induces filopodia. The cartoon was created using BioRender.com.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-CD63 (Mouse monoclonal) | Abcam | ab8219 | IF 1:500 |
| Antibody | anti-mouse Endoglin (Rabbit polyclonal) | Cell Signaling | 3290 | WB 1:1000 |
| Antibody | anti-human Endoglin (Rabbit monoclonal) | Cell Signaling | 4335 | WB 1:1000 |
| Antibody | anti-CD63 (Rabbit polyclonal) | Abcam | ab68418 | WB 1:1000 |
| Antibody | anti-Beta actin (Rabbit monoclonal) | Cell Signaling | 4970 | WB 1:5000 |
| Antibody | anti-TSG101 (Rabbit polyclonal) | Abcam | ab30871 | WB 1:1000 |
| Antibody | anti-Flotillin-1 (Rabbit polyclonal) | Cell Signaling | 3253 | WB 1:1000 |
| Antibody | anti-TGFbeta1 (Rabbit polyclonal) | Abcam | ab92486 | WB 1:1000 |
| Antibody | anti-HSP70 (Mouse monoclonal) | Santa Cruz | sc-24 | WB 1:1000 |
| Antibody | anti-CD29 (beta1-integrin; Mouse monoclonal) | BD Biosciences | 610467 | WB 1:1000 |
| Antibody | anti-ALK1/ACVRL1 (Rabbit polyclonal) | Abcepta | AP7807a | WB 1:1000 |
| Antibody | anti-Rab27a (Rabbit monoclonal) | Cell Signaling | 69295 | WB 1:1000 |
| Antibody | anti-Hrs (M-79) (Rabbit polyclonal) | Santa Cruz | sc-30221 | WB 1:1000 |
| Antibody | rabbit anti-GM130 (Rabbit monoclonal) | ThermoFisher | MA5-35107 | WB 1:1000 |
| Antibody | anti-THSD7A (Rabbit polyclonal) | Sigma | HPA000923 | WB 1:1000 |
| Antibody | anti-Vinculin (Mouse monoclonal) | Sigma-Aldrich | V9131 | WB 1:1000 |
| Antibody | anti-THSD7A (Rabbit polyclonal) | Nicola Tomas, University Medical Center Hamburg-Eppendorf | WB 1:1000 | |
| Antibody | anti-pSmad1/5/9 (Rabbit monoclonal) | Cell Signaling | 13820 | WB 1:1000 |
| Antibody | anti-Smad1 (Rabbit monoclonal) | Cell Signaling | 6944 | WB 1:1000 |
| Antibody | anti-pSmad2 (Rabbit monoclonal) | Cell Signaling | 3108 | WB 1:1000 |
| Antibody | anti-Smad2 (Rabbit monoclonal) | Cell Signaling | 5339 | WB 1:1000 |
| Antibody | Anti- SV2 (Mouse monoclonal) | Developmental Studies Hybridoma Bank | IF 1:1000 | |
| Antibody | anti-Flotillin-1 (Mouse monoclonal) | BD Biosciences | 610820 | WB 1:1000 |
| Antibody | anti-Alix (Mouse monoclonal) | Cell Signaling | 2171 | WB 1:1000 |
| Antibody | anti-GM130 (Mouse monoclonal) | BD Biosciences | 610822 | WB 1:1000 |
| Cell line (Mus musculus) | B16F1 | ATCC | mouse melanoma | |
| Cell line (Homo sapiens) | HT1080 | ATCC | human fibrosarcoma | |
| Recombinant DNA reagent | mCherry-pTαS2 (plasmid) | Donna J. Webb, Vanderbilt University | Expression of mCherry in neurons | |
| Recombinant DNA reagent | GFP-pTαS2 (plasmid) | Donna J. Webb, Vanderbilt University | Expression of GFP in neurons | |
| Recombinant DNA reagent | mScarlet-pTαS2 (plasmid) | This paper | Expression of mScarlet in neurons, described in Neuron methods: Reagents and constructs | |
| Recombinant DNA reagent | GFP-Rab27b (plasmid) | Addgene | 89447 | Expression of GFP-tagged human Rab27B; a gift from Wendy Westbroek to Addgene |
| Recombinant DNA reagent | pCMV-Tag4-THSD7A-FLAG (plasmid) | Yung-Jen Chuang, National Tsing Hua University, Taiwan | Expression of THSD7A-FLAG | |
| Recombinant DNA reagent | pCMV-Tag4 (plasmid) | Yung-Jen Chuang, National Tsing Hua University, Taiwan | Empty vector for C-terminal FLAG-tagged expression | |
| Recombinant DNA reagent | pCytERM_mScarlet_N1 (plasmid) | Addgene | 85066 | Expression of mScarlet-tagged cytochrome p450 aa 1–29; A gift from Dorus Gadella to Addgene |
| Recombinant DNA reagent | pCMV-Tag4-THSD7A-mScarlet (plasmid) | This paper | Expression of mScarlet-tagged THSD7A, described in Cancer cell methods: Plasmids and reagents | |
| Recombinant DNA reagent | pLenti6/V5-DEST (plasmid) | Thermo Fisher | V49610 | Empty vector for V5-tagged expression |
| Recombinant DNA reagent | pLenti6-WT-Eng-V5 (plasmid) | This paper | Expression of shRNA-resistant WT endoglin-V5, described in Cancer cell methods: Plasmids and reagents | |
| Recombinant DNA reagent | pRK5-myc-Cdc42-Q61L (plasmid) | Addgene | 12974 | Expression of myc-tagged Cdc42 constitutively active; a gift from Gary Bokoch to Addgene |
| Recombinant DNA reagent | pHluorin_M153R-CD63-mScarlet (plasmid) | BH Sung et al., Nat Commun., 2020 | Expression of dual color CD63 reporter of exosome secretion and uptake | |
| Recombinant DNA reagent | tdTomato-fTractin (plasmid) | Robert S. Fischer, NIH | Expression of tdTomato-tagged F-actin. Provided by Robert Fischer | |
| Transfected construct (M. musculus) | shHrs-1 | Dharmacon p-Blockit | 5’-GGAACGAACCCAAGTACAAGG-3’ | |
| Transfected construct (M. musculus) | shHrs-2 | Dharmacon p-Blockit | 5’-GCATGAAGACGAACCACATGC-3’ | |
| Transfected construct (M. musculus) | shRab27a-1 | Dharmacon p-Blockit | 5’-GTGCGATCAAATGGTCATGCC-3’ | |
| Transfected construct (M. musculus) | shRab27a-2 | Dharmacon p-Blockit | 5’-CGTTCTTCAGAGATGCTATGC-3’ | |
| Transfected construct (M. musculus) | pBlockit-shLacZ | Dharmacon p-Blockit | control shRNA | |
| Transfected construct (M. musculus) | shEng-1 | Dharmacon TRC lentiviral shRNA (pLKO.1) | TRCN0000094354 | 5’-TTAGGCTTCTAAGCAGCATGG-3’ |
| Transfected construct (M. musculus) | shEng-2 | Dharmacon TRC lentiviral shRNA (pLKO.1) | TRCN0000094355 | 5’-TATAGATGACAAACAGCAGGG-3’ |
| Transfected construct (M. musculus) | shScr | Dharmacon TRC lentiviral shRNA (pLKO.1) | control shRNA | |
| Transfected construct (H. sapiens) | shEng-1 | Dharmacon TRC lentiviral shRNA (pLKO.1) | TRCN0000083138 | 5’-ATCCAGGTTCAAATGACAGGG-3’ |
| Transfected construct (H. sapiens) | shEng-2 | Dharmacon TRC lentiviral shRNA (pLKO.1) | TRCN0000083141 | 5’-ATCATACTTGCTGACACCTGC-3’ |
| Transfected construct (H. sapiens) | shEng | Dharmacon TRC lentiviral shRNA (pLKO.1) | TRCN0000083139 | 5’-TAGTGGTATATGTCACCTCGC-3’ |
| Transfected construct (H. sapiens) | shTHSD7A | Dharmacon SMARTvector Lentiviral shRNAs | V3SH11240-226004853 | N/A, proprietary to Dharmacon |
| Transfected construct (H. sapiens) | shTHSD7A | Dharmacon SMARTvector Lentiviral shRNAs | V3SH11240-226644723 | N/A, proprietary to Dharmacon |
| Transfected construct (H. sapiens) | shTHSD7A | Dharmacon SMARTvector Lentiviral shRNAs | V3SH11240-227287134 | N/A, proprietary to Dharmacon |
| Transfected construct (H. sapiens) | NTC (non-targeting control) | Dharmacon SMARTvector Lentiviral shRNAs | N/A, proprietary to Dharmacon | |
| Transfected construct (Rattus norvegicus) | Hrs shRNA | pSUPER, doi: 10.1126/science.1068999 | 5’-AATGAACCCAAGTACAAGG-3’ For transfection of neurons | |
| Transfected construct (R. norvegicus) | Hrs shRNA | pSUPER Vector, doi: 10.1126/science.1068999 | 5’-ATGAAGAGCAACCACATGA-3’ For transfection of neurons | |
| Transfected construct (R. norvegicus) | Rab27b shRNA | pSUPER Vector, doi: 10.1126/science.1068999 | 5’-CCGATACACAGACAATAAA-3’ For transfection of neurons | |
| Transfected construct (R. norvegicus) | Rab27b shRNA | pSUPER Vector, doi: 10.1126/science.1068999 | 5’-TGAGTCAACTGCAGGCAAA-3’ For transfection of neurons | |
| Transfected construct (M. musculus) | Endoglin siRNA | Dharmacon ON-TARGETplus siRNA SMARTpool | L-045109-00-0005 | N/A, proprietary to Dharmacon |
| Transfected construct (M. musculus) | NTC (non-targeting control) | Dharmacon | D-001810-01-05 | N/A, proprietary to Dharmacon |
| Peptide, recombinant protein | human BMP-9 | R&D Systems | 3209 BP | recombinant protein |
| Peptide, recombinant protein | human TGF-beta1 | R&D Systems | 240-B-002 | recombinant protein |
| Peptide, recombinant protein | human Fibronectin | Cultrex | 3420-001-01 | recombinant protein |
| Peptide, recombinant protein | human THSD7A | R&D Systems | 9524-TH | recombinant protein |
| Other | EZSHPERE 96-well plate | SGC Techno Glass | AG4860900SP | spheroid collagen invasion assay plate |
| Other | type 1 A collagen solution | Nitta gelatin | Type-1-A | spheroid collagen invasion culture medium |
| Chemical compound, drug | ML141 | Millipore | 217708 | Cdc42 inhibitor |
| Other | OptiPrep Density Gradient Medium | Sigma | D1556 | iodixanol-based solution for density gradient |
| Commercial assay, kit | Coomassie Brilliant Blue R-250 Staining Solutions Kit | BioRad | 1610435 | protein stain |
| Chemical compound, drug | Phenylmethylsulfonyl fluoride (PMSF) | Research Products International | P20270 | Serine protease inhibitor |
| Chemical compound, drug | cOmplete Protease Inhibitor Cocktail | Roche | 4693116001 | protease inhibitor cocktail |
| Chemical compound, drug | TGF-beta1 inhibitor SB 431542 | Sigma-Aldrich | S4317 | |
| Chemical compound, drug | PhosSTOP | Roche | 4906845001 | Phosphatase inhibitor cocktail |
| Commercial assay or kit | BCA Protein Assay Kit | Pierce | 23225 | Protein quantification assay |
| Commercial assay or kit | Micro BCA Protein Assay Kit | Pierce | 23235 | Protein quantification assay |
| Chemical compound, drug | Lipofectamine RNAiMAX | Life Technologies | 13778150 | transfection reagent |
| Chemical compound, drug | Lipofectamine 2000 | Invitrogen | 11668–027 | transfection reagent |
Additional files
-
Supplementary file 1
Proteomics results from bands cut from colloidal blue gel in Figure 4A.
- https://cdn.elifesciences.org/articles/101673/elife-101673-supp1-v1.xlsx
-
Supplementary file 2
iTRAQ proteomics results from control and endoglin-KD B16F1 SEVs.
- https://cdn.elifesciences.org/articles/101673/elife-101673-supp2-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/101673/elife-101673-mdarchecklist1-v1.docx
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Secreted exosomes induce filopodia formation
eLife 13:RP101673.
https://doi.org/10.7554/eLife.101673.3
