Synphilin-1 regulates mechanotransduction in rigidity sensing
- 주제(키워드) mechanobiology , multi-omics , rigidity sensing , synphilin-1 , zyxin
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Gwang Lee
- 발행년도 2025
- 학위수여년월 2025. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034909
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Synphilin-1 is a cytoplasmic protein identified initially through interaction with α-synuclein and has been primarily studied in the context of the pathogenesis of Parkinson’s disease. However, the physiological and pathological roles of synphilin-1 remain incompletely understood, and in particular, the biophysical functions of synphilin-1 have yet to be explored. In this study, the mechanobiological role of synphilin-1 was investigated through analyses of cellular traction forces and rigidity sensing abilities using elastomeric pillar arrays and substrates of varying stiffness. To elucidate the underlying molecular mechanisms of the phenotypes induced by synphilin-1 overexpression, comprehensive transcriptomic and proteomic analyses were performed using RNA sequencing and liquid chromatography-tandem mass spectrometry. Bioinformatic analyses of the omics data were conducted using Ingenuity Pathway Analysis to predict relevant molecular pathways. Synphilin-1 overexpression results in reduced cell spreading area and declined cellular local contraction on elastomeric pillar arrays. Synphilin-1-overexpressing cells exhibit an impaired mechanosensitivity to substrate stiffness, while synphilin-1 knockdown restores rigidity sensing abilities. Integrated transcriptomic and proteomic omics analysis, supported by in silico prediction, corroborates the phenotypic changes induced by synphilin-1 overexpression at a molecular level. Zyxin was identified as a novel binding partner of synphilin-1, and synphilin-1 overexpression correlates with reduced nuclear translocation of yes-associated protein (YAP), indicating attenuation of mechanotransduction signaling pathways. These findings provide novel insights into the biophysical functions of synphilin- 1, suggesting a potential protective function in response to the extracellular matrix alterations, which may be relevant to neurodegenerative conditions such as Parkinson’s disease.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1. Cell culture and transfection 5
2.2. Scanning electron microscopy (SEM) 5
2.3. Immunocytochemistry 6
2.4. Polydimethylsiloxane (PDMS) substrate preparation 7
2.5. Elastomeric pillar array fabrication 7
2.6. Pillar displacement measurement 8
2.7. RNA sequencing 10
2.8. RNA extraction and RT-qPCR 10
2.9. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 12
2.10. Identification and quantification for proteome data 12
2.11. Formaldehyde cross-linking and co-immunoprecipitation 13
2.12. Co-immunoprecipitation 14
2.13. Single protein and protein-protein structure prediction 14
2.14. Polyacrylamide (PA) gel fabrication 14
2.15. Cell viability measurement 15
2.16. Statistical analysis 15
3. Results 16
3.1. Synphilin-1-overexpressing cells exhibited reduced spreading area with decreased lamellipodia formation and focal adhesion 16
3.2. Synphilin-1 overexpression induced cellular contraction towards the central region with diminished local contractions 19
3.3. Synphilin-1 overexpression attenuated cellular rigidity sensing across substrates with different stiffness 21
3.4. Integrated transcriptomics and proteomics analyses predicted the phenotype of synphilin-1 overexpression 24
3.5. Synphilin-1 interacted with zyxin in the cytosol 30
3.6. Synphilin-1 overexpression affected the subcellular localization of YAP 34
3.7. Elevated collagen I expression in patients with PD and the protective effects of synphilin-1 overexpression against excessive collagen I 38
4. Discussion 40
CONCLUSION 45
REFERENCES 46
Appendix Figures 50
Appendix Tables 62
