Abstract
Human embryonic stem cells (hESCs) can differentiate to three germ layers within biochemical and biomechanical niches. The complicated mechanical environments in vivo could have diverse effects on the fate decision and biological functions of hESCs. To globally screen mechanosensitive molecules, three typical types of mechanical stimuli, i.e., tensile stretch, shear flow, and mechanical compression, were applied in respective parameter sets of loading pattern, amplitude, frequency, and/or duration, and then, iTRAQ proteomics test was used for identifying and quantifying differentially expressed proteins in hESCs. Bioinformatics analysis identified 37, 41, and 23 proteins under stretch pattern, frequency, and duration, 13, 18, and 41 proteins under shear pattern, amplitude, and duration, and 4, 0, and 183 proteins under compression amplitude, frequency, and duration, respectively, where distinct parameters yielded the differentially weighted preferences under each stimulus. Ten mechanosensitive proteins were commonly shared between two of three mechanical stimuli, together with numerous proteins identified under single stimulus. More importantly, functional GSEA and WGCNA analyses elaborated the variations of the screened proteins with loading parameters. Common functions in protein synthesis and modification were identified among three stimuli, and specific functions were observed in skin development under stretch alone. In conclusion, mechanomics analysis is indispensable to map actual mechanosensitive proteins under physiologically mimicking mechanical environment, and sheds light on understanding the core hub proteins in mechanobiology.
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Abbreviations
- ACY1:
-
Aminoacylase-1
- ANOVA:
-
Analysis of variance
- ANXA4:
-
Annexin A4
- BASP1:
-
Brain abundant membrane attached signal protein 1
- BP:
-
Biological process
- CC:
-
Cellular component
- CFL2:
-
Cofilin 2
- ES:
-
Enrichment score
- ESCs:
-
Embryonic stem cells
- EXOSC5:
-
Exosome component 5
- FC:
-
Fold change
- FDR:
-
False discovery rate
- GART:
-
Phosphoribosylglycinamide formyltransferase
- GO:
-
Gene ontology
- GSEA:
-
Gene set enrichment analysis
- hESCs:
-
Human embryonic stem cells
- HIST1H1B:
-
Histone cluster 1 H1 family member b
- HSP70:
-
Heat shock protein family A
- ITGB1:
-
Integrin beta1
- iTRAQ:
-
Isobaric tags for relative and absolute quantitation
- LAMB1/C1:
-
Lamin B1/C1
- MAPK:
-
Mitogen-activated kinase-like protein
- mESCs:
-
Mouse embryonic stem cells
- MDC1:
-
Mediator of DNA damage checkpoint 1
- MF:
-
Molecular function
- MFI:
-
Mean fluorescence intensity
- MTHFD1:
-
Methylenetetrahydrofolate dehydrogenase 1
- NES:
-
Normalized enrichment score
- PCA:
-
Principal component analysis
- PHD:
-
Plant homeodomain
- PSMs:
-
Protein spectrum matches
- Q-Q plot:
-
Quantile-sample quantile plot
- RhoA:
-
Ras homolog family member A
- RPL35A:
-
Ribosomal protein L35a
- RT:
-
Room temperature
- STON2:
-
Stonin 2
- TFI:
-
Total fluorescence intensity
- UHRF1:
-
Ubiquitin like with PHD and ring finger domains 1
- WGCNA:
-
Weighted gene co-expression network analysis
- YAP:
-
Yes associated protein 1
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Acknowledgements
The authors thank to Drs. Yan Zhang and Xiaohua Lei for technical assistance. LC-MS/MS analysis was conducted in Beijing Institute of Genomics, Chinese Academy of Sciences. FX-4000TM tension unit was used in Beijing Anzhen Hospital, Capital Medical University and FX-5000TM compression unit was used in Shandong Key Laboratory of Biophysics, Dezhou University. This work was supported by National Natural Science Foundation of China Grants 31661143044, 31627804, 31870931, 31470907, and Frontier Science Key Project of Chinese Science Academy grant QYZDJ-SSW-JSC018.
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ML, JW, FZ and DL conceived the project; FZ, JW and DL performed the experiments and analyzed the data; DL and LZ prepared the hESCs and analyzed the data; BS compiled the pump control software; YG designed the flow chamber; YW analyzed the data and prepared the figures; ML, ZF, JW, and DL wrote the paper.
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10237_2020_1378_MOESM1_ESM.tif
Fig. S1. Integrality test of collected protein samples under distinct mechanical stimuli. Entire set of proteins used for iTRAQ analysis was defined using SDS-PAGE assay under tensile stretch (a-b), shear flow (c-d), or mechanical compression (e-f) with v (TIF 3026 kb)
10237_2020_1378_MOESM2_ESM.tif
Fig. S2. Reliability test of iTRAQ data sets under distinct mechanical stimuli. Plotted were linear regression analyses of PSMs (a, d, g), Unique Peptides (b, e, h), and Coverage (c, f, i) in intermittent-continuous stretch (a-c), pulsatile-steady shear (TIF 4794 kb)
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Fig. S3. Normality test of fold change (FC) for all the identified proteins under distinct mechanical stimuli. Plotted were probability density functions (a-c) and Q-Q plots (d-f) under tensile stretch (a, d), shear flow (b, e), or mechanical compression (TIF 1734 kb)
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Fig. S4. Circos plot for differential protein distribution on chromatins under tensile stretch (a), shear flow (b), or mechanical compression (c). Short black lines in the innermost circles denote genes encoding differential proteins (TIF 1293 kb)
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Fig. S5. Enrichment analysis of GSEA presented in single mechanical stimuli. Sign (NES) in x-axis in B-D defines the direction of enrichment upon the definitions of FC values in (a). Plotted were the enriched terms for cellular component (CC) (b), biologi (TIF 3100 kb)
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Fig. S6. GO analysis of correlated expression modules obtained from WGCNA analysis under distinct mechanical stimuli. (a-c) Enrichment of green module genes in CC (a), BP (b), and MF (c), respectively. (d-f) Enrichment of blue module genes in CC (d), BP (TIF 1941 kb)
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Fig. S7. Ribosome-related GSEA enrichment under typical stretch (a-c), shear (d-f) and compression (g-l). Enriched at 0.1 Hz stretch were translational initiation (a), large ribosomal subunit (b) and ribosomal subunit (c), at 1.1 Pa shear were translation (TIF 6172 kb)
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Fig. S8. Morphology analysis and DNA expression in nuclei under typical stretch, shear and compression. Cellular nuclei were stained by Hoechst 33342. Nuclear area (a, f, k), circularity (b, g, l), aspect ratio (c, h, m), and mean (d, i, n) or total (e, j (TIF 1229 kb)
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Zhang, F., Wang, J., Lü, D. et al. Mechanomics analysis of hESCs under combined mechanical shear, stretch, and compression. Biomech Model Mechanobiol 20, 205–222 (2021). https://doi.org/10.1007/s10237-020-01378-5
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DOI: https://doi.org/10.1007/s10237-020-01378-5