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Syntenin-knock out reduces exosome turnover and viral transduction - PubMed

  • ️Fri Jan 01 2021

Syntenin-knock out reduces exosome turnover and viral transduction

Rudra Kashyap et al. Sci Rep. 2021.

Abstract

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Syntenin-knock out reduces the AAV-mediated expression of tau-P301L in mouse brain. Micrographs of brain sections illustrating the expression of human tau protein after intracerebral injection of AAV-6-tau4R-P301L viral particles in the left hemisphere of wild-type mice (WT, top) or syntenin-knock out mice (KO, bottom). Note the low expression of tau in KO brain, both at caudal (−) and rostral (+) positions relative to the site of injection. Histogram on the right indicates the number of ‘dark’ pixels (with density above an arbitrarily set threshold) per complete brain section (left + right hemispheres). Data were collected using 4 mice per group and 4 sections per mice. Values are expressed as mean ± SEM. Scale bar is 1 mm.

Figure 2
Figure 2

Syntenin-knock out reduces the exosomal secretions of syntenin cargo by primary MEFs in culture. (a) Exosome characterization by nanoparticle-tracking. Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. Particle numbers and sizes were analyzed by Nanosight. (b) Left: Western blot analysis of proteins present in the lysates and exosome preparations of WT and Synt KO MEFs in primary culture. Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. Right: Bar graphs showing the levels of exosomal marker proteins in Synt-KO MEFs, relative to the levels measured in WT cells (taken as 100%). Note the concurrent decreases of ALIX, CD63 and syndecan 1-CTF (SDC1-CTF), and potential syndecan cargo such as B1-integrin, fibronectin, and EGFR, but not CD81 and TSG101, in the exosomes of Synt KO MEFs.

Figure 3
Figure 3

Loss of syntenin expression in primary MEFs leads to a decrease in HSPG expression. (a) Cell surface heparan sulfate abundance. Staining of total native HS, with mAb 10E4, in untreated cells (left panels), and of the residual delta-HS, with mAb 3G10, in cells treated with both heparitinase and chondroitinase ABC (right panels), in WT (top panels) and Synt-KO (bottom panels) MEFs. Compared to WT MEFs, Synt-KO MEFs show less intense fluorescence, for both 3G10 and 10E4, suggesting a reduction in both the number of HS chains and total mass of HS present at cell surfaces. Bar graphs on the right correspond to relative 10E4 fluorescence intensity per cell (taking WT cells as 100%). (b) Heparan sulfate proteoglycan abundance. (Left) Western blot of cell lysates, either left undigested (−) or digested (+) with heparitinase, chondroitinase ABC or a combination of both enzymes, and stained for delta-HS, using mAb 3G10, revealing the expression of HSPGs in WT and Synt-KO MEFs. Note that, compared to WT cells, Synt-KO MEFs show lesser amounts of delta-HS tagged PG core proteins (tentatively identified as the syndecans 1–4). (Right) Bar graphs represent the quantification of three independent experiments, taking band intensities in WT cells as 100%.

Figure 4
Figure 4

Loss of syntenin expression in primary MEFs limits retroviral transduction and exosome uptake. (a) Retroviral transduction. Retrovirus encoding LUC IRES eGFP, produced using phoenix packaging cells, was incubated with MEFs (WT and with Synt KO, shown on the left) and with MCF-7 cells (Ctrl and Synt-CRISPR, shown on the right), for 48 h. Cells expressing eGFP were quantified by flow cytometry. Bar graphs represent the percentage of cells transduced by retrovirus (i.e. expressing eGFP), relative to the percentage of WT cells that was transduced (taken as 100%). (b) Exosome uptake. MEFs, either WT or Synt-KO, were incubated with exosomes derived from MCF-7 cells expressing eGFP-syntenin (eGFP-Synt), at 37 °C for 8 h, to allow for exosome uptake, and then subjected to confocal laser scanning microscopy. Left: Representative confocal micrographs of MEFs, showing the accumulation of eGFP-Synt (green) and DAPI (blue) staining of the nuclei. Exosomes loaded with eGFP-Synt yield more puncta in WT cells than in Synt-KO cells. Right: quantification of mean eGFP fluorescence per cell.

Figure 5
Figure 5

ALIX-binding syntenin rescues retroviral transduction in syntenin 1-negative MCF-7 cells. (a) Representative confocal micrographs of MCF-7 cells showing the distribution of heparan sulfate (as detected by mAb 10E4, green) and the DAPI (blue) staining of the nuclei, upon syndecan over expression (syndecans 1–4, individually and all four together in co-transfection) in control (Ctrl) and in syntenin 1-negative (Synt-CRISPR) MCF-7 cells. (b) Retroviral transfection was analyzed using flow cytometry. Retrovirus encoding LUC IRES eGFP, produced using phoenix packaging cells, was incubated for 48 h with Ctrl and with Synt-CRISPR MCF-7 cells, all or not over-expressing syndecans. Cells expressing eGFP were quantified by flow cytometry. (c) Wild-type MCF-7 cells and Synt-CRISPR MCF-7 cells were transfected with expression plasmid vector encoding mCherry (empty vector), mCherry-syntenin (wild-type syntenin), mCherry-syntenin ∆ALIX (syntenin defective in ALIX-binding) or mCherry-syntenin K215A (syntenin defective in cargo recycling), replated and then incubated for 48 h with retrovirus encoding LUC IRES eGFP. Fluorescent protein expressions were quantified by flow cytometry. eGFP expression in mCherry-expressing cells was taken as a measure of syntenin effects on retroviral transduction. In every experiment, the percentage of wild type cells transfected with empty vector and expressing mCherry that were expressing eGFP was taken as 100 percent. n = 6, bars represent mean values ± SD; n.s., non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

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