Circadian regulation of protein cargo in extracellular vesicles

Abstract

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography-mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Fig. 1.. Primary tendon cells in culture produce small EVs and maintain circadian rhythms.

(A) Western blot analysis of whole-cell lysate and EVs purified from conditioned medium. Small EV extracts (EV) were positive for exosome markers, flotillin-1, CD9, and plasma membrane markers, including MMP14, and were negative for markers of other membrane compartments. (B) TEM images of purified small EVs showed membrane-bound vesicles. Scale bars, 50 nm. (C) Measurements from TEM images showed that the median diameter of small EVs released in media was 37.7 ± 13.8 nm (SD) (338 EVs measured from two independent conditioned medium purifications). (D) Representative TEM images showing features of the endosomal pathway [endocytic vesicles (Ves), multivesicular bodies (MVB), and lysosomes (Lyso)] and matrix assembly features [endoplasmic reticulum (ER) and collagen]. Scale bars, 500 nm. (E) Representative real-time bioluminescence recording of PER2::Luciferase activity in synchronized cells showed a circadian rhythm maintained for ~6 days with a mean period of 25.1 ± 0.1 hours (SD) (n = 6). (F) Quantification of MMP14 protein during 24 hours normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control (n = 3 technical replicates except n = 2 for 24 hours; bars show SEMs; **P < 0.0086, Kruskal-Wallis test). (G) Quantification of gelatinase activity of MMP2 (62-kDa band) relative to 0 hour (n = 3 from three independent assays; bars show SEMs; *P = 0.0479 Kruskal-Wallis test). See data file S1 for original scans of Western blots and gels and uncropped TEM images. AU, arbitrary units.

Fig. 2.. Time series fibroblast small EV isolation for proteomics analysis.

(A) Schematic diagrams the experimental setup for time series isolation of EVs from the first and (B) second 24 hours after synchronization with dexamethasone (Dex) and (C) from control (no synchronization) cells. Arrows above the bars indicate fresh medium changes. Arrows below the bars indicate media collected for sample isolations. (D) Representative NTA plot showing hydrodynamic diameters of small EVs released overnight into conditioned medium. The histograms are for each of the five technical replicates, each outlined in a different color, with a median diameter of 174.3 ± 77 nm (SD; n = 5 technical replicates from one experiment). (E) Box-and-whisker plot showing that the median hydrodynamic diameter of small EVs for 24 hours was unchanged (~170 nm). Data from one experiment are shown. (F) Box plot showing the distribution of protein abundances detected by LC-MS in triplicate samples of small EVs isolated during 48 hours after synchronization. (G) Principal components analysis (PCA) showing the first two principal components of variances between the 48-hour time series samples. (H) Top 10 significantly enriched terms, ranked by their enrichment scores that are overrepresented in the 1015 proteins detected in the small EV samples by LC-MS. Values in bars indicate the number of proteins in the functional term. (I) Venn diagram showing the comparison of all proteins detected in the EV samples by LC-MS with proteins from the Vesiclepedia database.

Fig. 3.. Identification and phase characteristics of circadian small EV proteins.

(A) Heatmap showing the mean expression of the 176 circadian rhythmic proteins identified by MetaCycle analysis (q < 0.2) in the second 24-hour dataset. (B) Phase distribution of peak abundances of the MetaCycle-identified rhythmic proteins. (C) Heatmap showing the mean expression of the 88 circadian rhythmic proteins identified by the Gaussian process (S median > 0.3, SE <0.2) in the 48-hour time series. (D) Phase distribution of peak abundances of the Gaussian process–identified rhythmic proteins. (E) Venn diagram showing the comparison of circadian small EV proteins identified by the Gaussian process and MetaCycle analysis with circadian proteins identified in the soluble fraction of mouse tendon tissue. (F) Top 10 significant enriched terms in circadian proteins identified by both Gaussian process and MetaCycle analyses, ranked by their enrichment scores. Values in bars indicate the number of proteins in the functional term. (G to J) Comparison of the phase distribution of peak abundances of Gaussian process–identified (GP) and MetaCycle-identified (MC) proteins belonging to ribosomal/ribonucleoprotein/RNA binding (G), cytoskeletal (H), and ECM (I) and related to vesicles/endocytosis (J) functional terms. Values in brackets indicate the number of proteins.

Fig. 4.. Circadian periodicity analysis provides a proteomic time signature for fibroblast small EVs.

(A) Correlation between the MetaCycle-derived peak phase and the mean peak phase derived from the Gaussian process for the 30 common proteins (R² = 0.8954). Proteins are expressed using gene names. (B) Pattern of peak abundance of circadian small EV proteins validated by at least two analyses with less than 6-hour peak phase time. (C) Schematic of how the protein composition of small EVs from healthy fibroblast changes for 24 hours.

Fig. 5.. EV MMP14 level is regulated via the endosomal pathway.

(A) Representative images of immuno-EM analysis of small EVs purified from primary mouse tendon fibroblast cultures labeled with antibodies specific to the catalytic domain of MMP14 and visualized with 10-nm gold-gold particles (black dots). Scale bars, 50 nm. (B) Representative Western blot analysis of lysates from tenocytes treated with small interfering RNAs (siRNAs) targeting Flot1. (C) Quantification of protein levels from Western blot analysis of cellular flotillin-1 and MMP14 from control cells and cells treated with siRNAs targeting Flot1 (n = 3 independent experiments, normalized to GAPDH loading control; ****P < 0.0001 and *P = 0.0371, unpaired t tests). (D) Representative Western blot analysis of small EVs released by tenocytes with depleted flotillin-1. (E) Quantification of protein levels from Western blot analysis of EV flotillin-1 and MMP14 from control cells and cells treated with siRNAs targeting Flot1 (n = 4 independent experiments, 5 μg of protein loaded; **P < 0.0032 for flotillin-1 and **P < 0.0034 for MMP14, unpaired t tests). (F) Representative confocal fluorescence microscopy images showing increased fluorescence intensity for MMP14 in flotillin-1–depleted cells. Scale bars, 20 μm. (G) Representative confocal fluorescence microscopy images showing increased colocalization of MMP14 with early endosomes, localized via early endosome antigen-1 (EEA-1), in cells treated with siRNAs targeting Flot1. Scale bars, 10 μm. (H) Line scan analysis of confocal images shows reduced MMP14 staining outside early endosomes. See data file S1 for original scans of Western blots and gels and uncropped TEM images.