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Ceramide-rich microdomains facilitate nuclear envelope budding for non-conventional exosome formation

Abstract

Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B4 (LTB4) refs. 1,2. We previously demonstrated that LTB4 and its synthesizing enzymes, 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and leukotriene A4 hydrolase, are packaged and released in exosomes3. Here we report that the biogenesis of the LTB4-containing exosomes originates at the nuclear envelope (NE) of activated neutrophils. We show that the neutral sphingomyelinase 1 (nSMase1)-mediated generation of ceramide-enriched lipid-ordered microdomains initiates the clustering of the LTB4-synthesizing enzymes on the NE. We isolated and analysed exosomes from activated neutrophils and established that the FLAP/5-LO-positive exosome population is distinct from that of the CD63-positive exosome population. Furthermore, we observed a strong co-localization between ALIX and FLAP at the periphery of nuclei and within cytosolic vesicles. We propose that the initiation of NE curvature and bud formation is mediated by nSMase1-dependent ceramide generation, which leads to FLAP and ALIX recruitment. Together, these observations elucidate the mechanism for LTB4 secretion and identify a non-conventional pathway for exosome generation.

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Fig. 1: The LTB4-synthesizing machinery is packaged in NE-derived buds and cytosolic vesicles in activated neutrophils.
Fig. 2: nSMase1 facilitates the recruitment of the LTB4-synthesizing machinery on lipid-ordered NE microdomains.
Fig. 3: nSMase-dependent enrichment and co-localization of FLAP with ceramide-positive structures on the NE of activated neutrophils.
Fig. 4: nSMase1 and ceramide are present within and are required for the generation of NE-derived 5-LO/LBR positive NE buds and cytosolic vesicles.
Fig. 5: 5-LO-positive and CD63-negative punctae are present within LBR-positive vesicles.

Data availability

All the raw data and associated statistic calculations presented have been provided as ‘source data’ for the respective figures. Owing to the large size of high-resolution z-stack microscopy images, the raw microscopy images are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Platelet Pharmacology and Physiology Core at the University of Michigan for providing human blood from healthy volunteers. We are grateful to D. Cai and Y. Li (University of Michigan) for their assistance in the expansion microscopy experiments. We also acknowledge R. Majumdar and C. Saunders as well as J. Y. Park for their intellectual contributions. Finally, many thanks to members of the Parent laboratory, and P. Hanson (University of Michigan) and P. Coulombe (University of Michigan) for their valuable suggestions and help during the progress of this work. This work was supported by funding from the University of Michigan School of Medicine (C.A.P.), by the Lucchesi Predoctoral Fellowship Award (S.C.), by an American Heart Association Predoctoral Award (F.J.-J.) and by R01 AI152517 (C.A.P.).

Author information

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Contributions

S.B.A., S.C., F.J.-J. and C.A.P. designed the experiments. S.B.A., S.C. and F.J.-J. performed the experiments and analysed the results. S.B.A., S.C. and C.A.P. wrote and edited the manuscript. F.J.-J. edited the manuscript.

Corresponding author

Correspondence to Carole A. Parent.

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Extended data

Extended Data Fig. 1 Characterization of LBR-positive NE buds and cytosolic vesicles.

A Representative Airyscan microscopy image of fixed PMNs uniformly stimulated with either DMSO or 20 nM fMLF showing the distribution of FLAP (green) and 5-LO (red) (n = 3). The yellow arrowheads point to nuclear buds. Scale bar is 5 µm, in the inset scale bar is 2 µm. B Box-whisker plots showing the size distribution of LBR-positive NE buds and cytosolic vesicles in PMNs chemotaxing towards fMLF (n = 5), where each red dot represents the value from the individual bud (68) and cytosolic vesicle (99), plotted as box-whisker plot with whiskers indicating the range. The boundary of the box closest to zero indicates 25th percentile, while the farthest one indicates 75th percentile, where the line within the box represents median. The indicated P value was determined using the two-tailed Mann-Whitney test. C Examples of fixed PMNs chemotaxing towards 100 nM fMLF and stained for LBR (green), Lamin B1 (red), and Hoechst (blue) acquired using Airyscan microscopy (n = 3). Scale bar is 5 µm, in the inset it is 1 µm. NE buds are shown in yellow boxes and cytosolic vesicles are shown in blue boxes. The yellow arrowheads point to nuclear buds and the blue arrowheads point to cytosolic vesicles. Source numerical data are available in the source data file.

Source data

Extended Data Fig. 2 nSMase1 regulates fMLF-induced perinuclear lipid order.

A Representative western blot image showing the levels of nSMase1 in Scr or nSMase1 CRISPR KO HL-60 cell lysates. GAPDH was used as a loading control (n = 2). * Denotes non-specific band detected by the nSMase1 antibody. B Representative fluorescence microscopy images, showing the lipid-ordered (Lo) domains, lipid-disordered (Ld) domains, RGB images of Ld, Lo, and Hoechst merged, and GP images of either Scr or nSMase 1 KO dHL-60 cells chemotaxing towards 100 nM fMLF stained with di-4ANEPPDHQ (n = 3). Pseudo-colored GP images are based on the colormap, with white/red shades depicting lipid ordered regions and blue/darker shades representing lipid disordered regions. Scale bar is 5 µm. C Histogram depicting the gaussian distribution of the GP pixel intensity obtained from the cytosol and perinuclear ROI (as depicted in Fig. 2b). D Graph showing the median value of the perinuclear GP intensity distribution obtained from the GP images of Scr and nSMase1 KO dHL-60 cells. Data is quantified from 9 cells out of three independent experiments and is presented as box-whisker plot where whiskers indicate the range, and the boundary of the box closest to x-axis indicates 25th percentile, while the farthest one indicates 75th percentile and the line within the box represents the median. Each red dot represents the value from one cell. Indicated P value is determined using two-tailed Mann-Whitney test. Source numerical data and unprocessed blots are available in the source data file.

Source data

Extended Data Fig. 3 Characterization of NE membranes from WT dHL-60 cells.

A Schematic illustration of the methodology used to isolate DRM and DSM fractions from the isolated NE. B Representative western blot showing the efficiency of the fractionation protocol using resting WT dHL60 cells (n = 2). Scanned unprocessed blots are available in the source data file.

Extended Data Fig. 4 nSMase1-GFP is enriched at sites of nuclear budding.

Representative time-lapse images of dHL-60 cells expressing nSMase1-GFP chemotaxing towards 100 nM fMLF. The zoomed section of the images shows the nSMase1-GFP signal as fluorescence intensity spectrum (scale on right) and Hoechst in grayscale. Scale bar is 2 µm, in the zoomed images it is 1 µm. N = 6 cells acquired from two independent experiments. Also, see Supplementary Movie 4.

Extended Data Fig. 5 Characterization of CD63 positive vesicles in activated neutrophils.

A Representative four-fold expansion microscopy image of fixed human PMNs chemotaxing towards 100 nM fMLF, captured using Airyscan microscopy, stained for CD63 (magenta), LBR (yellow) and ceramide (cyan) (n = 3). Scale bar 5 µm. In the inset, the scale bar is 400 nm. B Line profiles showing the presence of 50–100 nm ceramide (cyan) positive punctae within the CD63 (magenta) positive MVBs from panel A. C Graph showing the distribution of the median diameter of CD63-positive MVBs. Data are plotted as box-whisker plot where whiskers indicate the range, and the boundary of the box closest to 200 nm indicates 25th percentile, while the farthest one indicates 75th percentile and the line within the box represents the median. The red dots represent data of 52 CD63-positive MVBs (red dots) from 8 cells pooled from two independent experiments. D Representative Airyscan microscopy images of fixed PMNs uniformly stimulated with 100 nM fMLF for 30 min and stained with LBR (green) and CD63 (red) (n = 3). Enlarged images are depicted in inverted grayscale. Scale bar is 5 µm. E Box-whisker plots showing the number (top) and median diameter (bottom) of CD63-positive vesicles per cell, as shown in panel C, where whiskers indicate the range, and the boundary of the box closest to zero or 200 nm indicates 25th percentile, while the farthest one indicates 75th percentile and the line within the box represents the median. P value calculated using two-tailed Mann-Whitney test yield non-significant values (n = 3). Source numerical data are available in the source data file.

Source data

Extended Data Fig. 6 Characterization of exosomes isolated from activated Scr and nSMase1 KO dHL-60 cells.

A Histogram showing the particle count and size of exosomes purified from either Scr or nSMase1 KO dHL60 cells stimulated with 100 nM fMLF for 15 min. Data were obtained from nanoparticle tracking analysis (NTA) of the isolated exosomes and is plotted from three independent experiments as mean ± SEM. The dotted line parallel to the y-axis, at 180 nm, indicate the segregation of two exosome populations, small (0–180 nm) and large (181–360 nm). B Quantification of the area under the curve from the NTA data. Data from three independent experiments are presented as paired experiments. P-value was obtained using two-way RM ANOVA. C Representative western blot images showing the levels of FLAP, 5-LO, Flotillin 2, and CD63 in pooled fractions 4–9 of density-gradient purified exosomes isolated from either Scr or nSMase1 KO dHL-60 cells stimulated with 100 nM fMLF for 15 min. Scr cell lysate represents the amount of protein from 1/100th the number of cells used for exosome isolation (n = 4). D Bar graph showing the quantifications of the band intensity of FLAP, 5-LO, Flotillin 2, and CD63 in Scr and nSMase1 KO exosomes. Four data points are plotted as mean ± SEM where each red dot represents the value from one experiment. P values determined using two-tailed paired multiple t-test are reported. E Bar graph showing exosomal LTB4 levels from the Scr or nSMase1 KO dHL60 cells stimulated with 100 nM fMLF for 15 min. Data from three independent experiments are plotted as mean ± SEM. P value was obtained using two-tailed ratio paired t-test. Source numerical data and unprocessed blots are available in the source data file.

Source data

Extended Data Fig. 7 Characterization of exosomes isolated form activated PMNs.

A Representative western blot showing the distribution of FLAP, CD63, Flotillin 2, TSG101, and ALIX in various fractions of density-gradient purified exosomes isolated from the supernatant of PMNs stimulated with 100 nM fMLF for 15 min (n = 4–5). B Bar graph showing the arbitrary band intensity of the indicated proteins. Data are plotted as mean ± SEM of at least three independent experiments. Source numerical data and unprocessed blots are available in the source data file.

Source data

Extended Data Fig. 8 ALIX and LBR distribution in activated PMNs.

Representative Airyscan microscopy images of fixed PMNs chemotaxing towards 100 nM fMLF and stained for LBR (green) and ALIX (red) (n = 3). Scale bar is 5 µm. In the inset, the scale bar is 1 µm. NE buds are shown in yellow boxes and cytosolic vesicles are shown in blue boxes.

Supplementary information

Supplementary Information

Supplementary Table 1

Reporting Summary

Supplementary Video 1

Stitched time-lapse images of di-4ANEPPDHQ-stained PMNs chemotaxing towards 100 nM fMLF, showing Lo regions (green, left), Ld regions (red, centre) and the corresponding GP image. Pseudo-coloured GP images are based on the colour map, with white/red shades depicting lipid-ordered regions and blue/darker shades representing lipid-disordered regions. For colour map, see Fig. 2a. Images were captured at 15 s intervals and stitched together to create a movie at 20 frames per second.

Supplementary Video 2

Stereoscopic rendering of olive-coloured Hoechst-stained isolated nuclei, showing the intensity distribution of FLAP staining, as presented in Fig. 3a.

Supplementary Video 3

Stereoscopic rendering of FLAP, ceramide and Hoechst-stained isolated nuclei, showing the differential localization of FLAP and ceramide, as presented in Fig. 3c.

Supplementary Video 4

Time-lapse movie of dHL60 cells expressing nSMase1-eGFP (green) and stained with Hoechst (blue) chemotaxing towards 100 nM fMLF. Images were captured at 15 s intervals and stitched together to create a movie at twp frames per second.

Source data

Source Data Fig. 1

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Source Data Fig. 2

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Unprocessed western blots or dot blots.

Source Data Fig. 3

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Source Data Extended Data Fig. 1

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Source Data Unprocessed Blots_ED Fig. 7

Unprocessed western blots.

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Arya, S.B., Chen, S., Jordan-Javed, F. et al. Ceramide-rich microdomains facilitate nuclear envelope budding for non-conventional exosome formation. Nat Cell Biol (2022). https://doi.org/10.1038/s41556-022-00934-8

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