Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway
- Molecular Cell Biology Division, Department of Biology and Center of Cellular Nanoanalytics, Osnabrück University, Germany
- Cell Death and Metabolism Group, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, Denmark
- Laboratoire de Bioimagerie et Pathologies, Université de Strasbourg, France
- Biophysics Division, Department of Biology and Center of Cellular Nanoanalytics, Osnabrück University, Germany
- Division of Medical Genetics, Department of Pediatrics, University of Utah, United States
- Department of Genetics, University Medical Center Utrecht, Netherlands
- Children’s Hospital, University of Helsinki and Helsinki University Hospital, Finland
- Department of Cell Biology, Yale School of Medicine, United States
Figures
Figure 1
SMS2 contains an autonomous ER export signal.
(A) Membrane topology of SMS family members and chimeric protein SMSr/211-77. Active site residues are marked in red. The position of a conserved IXMP sequence motif is marked by an arrow. (B) …
Figure 2
Pathogenic SMS2 variants support bulk production of SM in the ER.
(A) HeLa SMS1/2 double KO (ΔSMS1/2) cells transduced with doxycycline-inducible constructs encoding FLAG-tagged SMS2, SMS2I62S, SMS2M64R or their enzyme-dead isoforms (D276A) were grown for 16 hr in …
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Figure 2—source data 1
Unprocessed and uncropped image files of the immunoblots.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig2-data1-v2.pdf
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Figure 2—source data 2
Raw data of the quantitative analysis of SM species.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig2-data2-v2.xlsx
Figure 3
Cells expressing pathogenic variant SMS2M64R accumulate SM in the ER.
(A) Workflow for affinity purification of the ER from HeLa cells expressing wildtype or pathogenic SMS2 variants. (B) HeLa wildtype (WT) or ΔSMS1/2 cells transduced with empty vector (EV) or …
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Figure 3—source data 1
Unprocessed and uncropped image files of the immunoblots.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig3-data1-v2.pdf
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Figure 3—source data 2
Raw data of the quantitative analysis of lipid species in whole cells or isolated ER.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig3-data2-v2.xlsx
Figure 4
Lipid composition of the PM of cells expressing wildtype or pathogenic SMS2 variants.
(A) Workflow for affinity purification of the PM from HeLa cells expressing wildtype or pathogenic SMS2 variants. (B) HeLa wildtype (WT) or ΔSMS1/2 cells transduced with empty vector (EV) or …
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Figure 4—source data 1
Unprocessed and uncropped image files of the immunoblots.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig4-data1-v2.pdf
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Figure 4—source data 2
Raw data of the quantitative analysis of lipid species in whole cells or isolated PM.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig4-data2-v2.xlsx
Figure 5
Luminal SM reporter EqtSMSS enables visualization of an ER-resident SM pool in SMS2M64R-expressing cells.
(A) Human osteosarcoma U2OS cells co-transfected with mCherry-tagged VAPA (ER, magenta) and empty vector (control), SMS2M64R or SMS2M64R/D276A and luminal GFP-tagged SM reporter EqtSMSS or its SM …
Figure 6
Pathogenic SMS2 variants disrupt transbilayer SM asymmetry.
(A) HeLa ΔSMS1/2 cells transduced with doxycycline-inducible SMS2, SMS2M64R, SMS2I62S or their enzyme-dead isoforms (D276A) were transfected with cytosolic GFP-tagged SM reporter EqtSMcyto or its SM …
Figure 7
SMS2M64R-expressing cells fail to concentrate SM on their surface and exhibit imbalances in lipid order.
(A) HeLa wildtype (WT) or ΔSMS1/2 cells transduced with doxycycline-inducible SMS2, SMS2M64R or their enzyme-dead isoforms (D276A) were treated with doxycycline (1 μg/ml, 16 h), incubated with …
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Figure 7—source data 1
Raw data of the quantitative analysis of ratiometric fluorescence microscopy images captured under the conditions indicated.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig7-data1-v2.xlsx
Figure 8
Pathogenic SMS2 variants perturb subcellular cholesterol pools.
(A) HeLa ΔSMS1/2 cells transduced with doxycycline-inducible SMS2, SMS2M64R or SMS2I62S were co-transfected with GFP-tagged EqtSMcyto (green) and mCherry-tagged cytosolic sterol reporter D4H (red). …
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Figure 8—source data 1
Raw data of the quantitative analysis of cell viability under the conditions indicated.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig8-data1-v2.xlsx
Figure 9
Patient-derived fibroblasts display perturbations in SM distribution and lipid order.
(A) Control (WT) or patient-derived human skin fibroblasts carrying heterozygous missense variants c.185T>G (p.I62S) or c.191T>G (p.M64R) in SGMS2 were co-transfected with mCherry-tagged VAPA (ER, ma…
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Figure 9—source data 1
Raw data of the quantitative analysis of ratiometric fluorescence microscopy images captured under the conditions indicated.
- https://cdn.elifesciences.org/articles/79278/elife-79278-fig9-data1-v2.xlsx
Appendix 1—figure 1
SMS2 contains an autonomous ER export signal.
HeLa cells transfected with empty vector (EV) or FLAG-tagged SMS2, SMS2I62S, SMS2M64R, SMSr, SMSr/211-77, SMSr/211-77I62S or SMSr/211-77M64R were fixed, immunostained with a-FLAG (green) …
Appendix 1—figure 2
Doxycycline-induced expression of SMS2 variants in stably transduced HeLa cells.
HeLa ΔSMS1/2 cells transduced with doxycycline-inducible Flag-tagged SMS2, SMS2I62S, SMS2M64R or their enzyme-dead isoforms (D276A) were grown for 16 h in the absence or presence of 1 μg/ml …
Appendix 1—figure 3
Pathogenic SMS2 variants support bulk production of SM in the ER.
(A) HeLa ΔSMS1/2 cells transduced with doxycycline-inducible FLAG-tagged SMS2, SMS2I62S, SMS2M64R or their enzyme-dead isoforms (D276A) were grown for 16 h in the absence or presence of 1 μg/ml …
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Appendix 1—figure 3—source data 1
Raw data of the quantitative analysis of lipid species under the conditions indicated.
- https://cdn.elifesciences.org/articles/79278/elife-79278-app1-fig3-data1-v2.xlsx
Appendix 1—figure 4
Mobilization of EqtSMcyto and LysSMcyto by pathogenic SMS2 variants.
(A) HeLa ΔSMS1/2 cells transduced with FLAG-tagged SMS2, SMS2I62S, SMS2M64R or their enzyme-dead isoforms (D276A) were transfected with GFP-tagged EqtSMcyto (green) and grown for 16 h in the absence …
Appendix 1—figure 5
EqtSMcyto-positive puncta formed in cell expressing pathogenic SMS2 variants do not co-localize with a wide range of organellar markers.
HeLa ΔSMS1/2 cells transduced with doxycycline-inducible SMS2M64R were transfected with EqtSMcyto (green) and treated with 1 μg/ml doxycycline for 16 h. Next, cells were fixed, immunostained with …
Appendix 1—figure 6
Pathogenic SMS2 variants perturb subcellular cholesterol pools.
(A) HeLa wildtype orΔSMS1/2 cells were co-transfected with GFP-tagged EqtSMcyto (green) and mCherry-tagged D4H (red). After 16 h, cells were fixed, counterstained with DAPI (blue) and imaged by …
Appendix 1—figure 7
Patient-derived fibroblasts display perturbations in subcellular cholesterol pools.
Control (WT) or patient-derived human skin fibroblasts carrying heterozygous missense variants c.185T>G (p.I62S) or c.191T>G (p.M64R) in SGMS2 were transfected with mCherry-tagged D4H (magenta), …
Author response image 1
Author response image 2
Tables
Appendix 1—table 1
Primers used for site-directed mutagenesis of SMS2.
| Primer name | Sequence |
|---|---|
| SMS2(I62S)-F | 5’-CGGACTATATCCAAAGTGCTATGCCCACTGAATC-3’ |
| SMS2(I62S)-R | 5’-GATTCAGTGGGCATAGCACTTTGGATATAGTCCG-3’ |
| SMS2(M64R)-F | 5’-CTATATCCAAATTGCTAGGCCCACTGAATCAAGG-3’ |
| SMS2(M64R)-R | 5’-CCTTGATTCAGTGGGCCTAGCAATTTGGATATAG-3’ |
| SMS2(D276A)-F | 5’-CGAACACTACACTATCGCTGTGATCATTGC-3’ |
| SMS2(D276A)-R | 5’-GCAATGATCACAGCGATAGTGTAGTGTTCG-3’ |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/79278/elife-79278-transrepform1-v2.pdf
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Appendix 1—figure 3—source data 1
Raw data of the quantitative analysis of lipid species under the conditions indicated.
- https://cdn.elifesciences.org/articles/79278/elife-79278-app1-fig3-data1-v2.xlsx
