Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton
- Duke University School of Medicine, United States
- University of North Carolina, United States
Figures
Figure 1 with 2 supplements
Vimentin engages in O-GlcNAc-mediated protein-protein interactions within assembled IFs.
(A) O-GlcNAc transferase (OGT) uses the nucleotide-sugar donor UDP-GlcNAc to add O-GlcNAc to protein substrates. O-GlcNAcase (OGA) removes O-GlcNAc moieties. 5SGlcNAc and Thiamet-G are specific …
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Figure 1—source data 1
Proteomic analysis of vimentin GlcNDAz crosslinks.
Full proteomics results are available for download as a Scaffold (.sf3) file on the eLife web site. Free software to view Scaffold files is available at http://www.proteomesoftware.com/products/scaffold/download/.
- https://doi.org/10.7554/eLife.31807.006
Figure 1—figure supplement 1
Analysis of vimentin GlcNDAz crosslinks.
(A) 293T cells transfected with vimentin-myc-6xHis were treated with DMSO vehicle or GlcNDAz for 24 hr, treated with vehicle, 100 µM 5SGlcNAc, or 50 µM Thiamet-G for an additional 24 hr, and then …
Figure 1—figure supplement 2
Vimentin GlcNDAz crosslinks are not affected by phosphatase treatment.
293T cells were GlcNDAz-crosslinked as in Figure 1 and extracted in non-denaturing buffer. Cell lysates were normalized to 1 mg/ml, mock-treated or treated with lambda phosphatase for 1 hr at 30°C, …
Figure 2 with 2 supplements
Specific glycosylation sites in the vimentin head domain are required for homotypic, O-GlcNAc-mediated interactions.
293T cells were transfected with GFP only (control) or WT or mutant vimentin-myc-6xHis constructs as indicated for 24 hr, subjected to GlcNDAz crosslinking, and analyzed by IB. Tubulin is a loading …
Figure 2—figure supplement 1
Quantitative examination of S49 glycosylation.
293T cells were transfected with vimentin-myc-6xHis constructs as indicated for 24 hr and then treated with vehicle only or 100 µM GalNAz for an additional 24 hr. Lysates were subjected to click …
Figure 2—figure supplement 2
Mutations in putative extracellular GlcNAc-binding sites of vimentin do not reduce intracellular GlcNDAz crosslinking.
293T cells were transfected with vector or WT or mutant vimentin-myc-6xHis constructs as indicated for 24 hr, subjected to GlcNDAz crosslinking, and analyzed by IB (D21H3 antibody). Tubulin is a …
Figure 3 with 3 supplements
Head domain glycosylation sites are required for vimentin IF assembly and morphology in vivo.
(A) A vimentin−/− HeLa clone was stably transduced with empty vector (control) or expression constructs encoding WT or mutant vimentin-mEmerald and then imaged by laser scanning confocal …
Figure 3—figure supplement 1
Construction and validation of vimentin-reconstituted cells.
Successful CRISPR/Cas9-mediated deletion of vimentin was verified by actin-normalized qPCR (A) and IB (D21H3 antibody) (B). mRNA from WT and vimentin knockout mouse embryonic fibroblasts (MEFs) …
Figure 3—figure supplement 2
Head domain glycosylation sites are required for vimentin IF assembly and morphology in vivo.
(A) A vimentin−/−293T clone was stably transduced with empty vector (control) or expression constructs encoding WT or mutant vimentin-mEmerald and was imaged by laser scanning confocal fluorescence …
Figure 3—figure supplement 3
S49A mutation does not affect vimentin stability.
293T cells were transfected with vimentin-myc-6xHis constructs as indicated and treated with either vehicle (DMSO) or 12 µM MG132 for 13 hr. Lysates were analyzed by fluorescent IB (V9 antibody for …
Figure 4
Vimentin O-GlcNAcylation is required for cell migration.
Vimentin−/− HeLa cells reconstituted with empty vector or WT, S49A or Y117L vimentin-mEmerald expression constructs were serum-starved for 72 hr, treated with either vehicle control (DMSO), 100 µM …
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Figure 4—source data 1
Full statistical analyses of cell migration data.
(A) Migration data were first compared by analysis of variance (ANOVA) using JMP software. The results of the ANOVA, as well as the effect test, are displayed. (B) Pairwise t-tests for all combinations of data points were performed. Level and –Level indicate the sample and the sample it is being compared to, respectively. The calculated difference, standard error, and upper and lower confidence levels (CL) are shown, as well as the p value for each t-test. (C) Comprehensive comparison of all samples and conditions. Any two samples that do not share a letter in column two are statistically significantly different from each other.
- https://doi.org/10.7554/eLife.31807.015
Figure 5 with 2 supplements
Chlamydia trachomatis requires OGT activity and vimentin glycosylation sites to maintain inclusion integrity during infection.
(A) HeLa cells reconstituted with WT vimentin-mEmerald were infected with Chlamydia for ten hours and treated with DMSO vehicle or 50 µM 5SGlcNAc for an additional 20 hr. Cells were fixed and …
Figure 5—figure supplement 1
No effect of Thiamet-G treatment on Chlamydia inclusion size.
HeLa cells reconstituted with WT (n = 36), S49A (n = 40) or Y117L (n = 34) vimentin-mEmerald were infected with Chlamydia for 10 hr and treated with DMSO vehicle or 50 µM Thiamet-G for an additional …
Figure 5—figure supplement 2
The S49A mutation does not prevent Chlamydia-induced vimentin cleavage.
HeLa cells reconstituted with WT, S49A or Y117L vimentin-mEmerald were infected with Chlamydia (MOI = 0.5) for 30 hr, lysed, and analyzed by anti-GFP (mEmerald tag), tubulin, and MOMP IB, as …
Figure 6 with 1 supplement
Site-specific glycosylation regulates the form and function of vimentin IFs.
Our data suggest a model wherein glycosylation of the N-terminal vimentin head domain (red), particularly on S49, promotes homotypic vimentin-vimentin interactions, and assembly and/or maintenance …
Figure 6—figure supplement 1
Partial conservation of S49 among vimentin orthologs and human type III IF proteins.
(A) Human vimentin S49 is conserved across several vertebrate vimentin orthologs. (B) S49 is conserved in human desmin but not peripherin or glial fibrillary acidic protein (GFAP), the other type …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (HeLa) | vimentin -/- | this paper | ||
| Cell line (293T/17) | 293T vimentin -/- | this paper | ||
| Cell line (HeLa) | HeLa | |||
| Cell line (293T/17) | 293T/UAP1 | PMCID: PMC3323966 | Stably expressing AGX1(F383G) | |
| Antibody | Anti-O-GlcNAc RL2 | Santa Cruz Biotechnology | Santa Cruz: sc-59624 | 1:300 |
| Antibody | anti-myc 9E10 | Santa Cruz Biotechnology | Santa Cruz: sc-40 | 1:300 |
| Antibody | anti-tubulin | Sigma-Aldrich | Sigma: T6074 | 1:10,000 |
| Antibody | anti-nucleoporin-62 | BD Biosciences | BD Biosciences: 610498 | 1:1,000 |
| Antibody | anti-vimentin D21H3 | Cell Signaling | Cell Signaling: 5741 | 1:1,000; 1:100 |
| Antibody | anti-vimentin V9 | Sigma-Aldrich | Sigma-Aldrich: V6389 | 1:1,000 |
| Antibody | anti-GFP | Thermo Fisher Scientific | ThermoFisher: A11122 | |
| Antibody | Goat anti-rabbit IgG | Southern Biotechnology | Southern Biotech: 4030-05 | 1:5,000 |
| Antibody | goat anti-mouse IgG | Southern Biotechnology | Southern Biotech: 1030-05 | 1:5,000 |
| Antibody | goat anti-mouse k light chain | Southern Biotechnology | Southern Biotech: 1050-05 | 1:5,000 |
| Antibody | Goat anti-rabbit IgG (H+L) Alexa Fluor 594 conjugate | Thermo Fisher Scientific | Thermo Fisher Scientific: A-11012 | 1:5,000 |
| Antibody | goat anti-mouse IgG (H+L) Alexa Fluor 594 conjugate | Thermo Fisher Scientific | Thermo Fisher Scientific: A-11005 | 1:5,000 |
| Antibody | goat anti-rabbit IgG (H+L) IRDye 800CW conjugate | Li-Cor | Li-Cor: 925-32211 | 1:5,000 |
| Antibody | goat anti-mouse IgG (H+L) IRDye 800CW conjugate | Li-Cor | Li-Cor: 925-32210 | 1:5,000 |
| Recombinant DNA reagent | mEmerald-vimentin-N-18 | Addgene | Addgene: 54301 | |
| Recombinant DNA reagent | pLenti CMV/TO Hygro DEST | Addgene plasmid: 17291 | ||
| Recombinant DNA reagent | pLenti CMV Neo DEST (705-1) | Addgene plasmid: 17392 | ||
| Recombinant DNA reagent | mEmerald-vimentin-N-18/ pLenti6 CMV Neo | this paper | ||
| Recombinant DNA reagent | mEmerald-vimentin-N-18/ pLenti6 CMV Hygro | this paper | ||
| Chemical compound, drug | Thiamet-G | Duke Small Molecule Synthesis Facility | ||
| Chemical compound, drug | Ac45SGlcNAc | Benjamin Swarts, Central Michigan University | ||
| Chemical compound, drug | Advansta ECL | Advansta | ||
| Sequence-based reagent | vimentin sgRNA | Duke Functional Genomics Facility | ||
| Sequence-based reagent | vimentin sgRNA | Duke Functional Genomics Facility | ||
| Sequence-based reagent | vimentin sgRNA | Duke Functional Genomics Facility | ||
| Biological sample (virus) | Cas9 Lentivirus | Duke Functional Genomics Facility | ||
| Commercial assay or kit | pENTR Directional TOPO cloning kit | Thermo Fisher Scientific | Thermo Fisher Scientific: K240020 | |
| Commercial assay or kit | Gateway LR Clonase II Enzyme | Thermo Fisher Scientific | Thermo Fisher Scientific:11791100 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.31807.021
