Synphilin-1 regulates mechanotransduction in rigidity sensing through interaction with zyxin

Abstract

Background: Synphilin-1 has been studied extensively in the context of Parkinson's disease pathology. However, the biophysical functions of synphilin-1 remain unexplored. To investigate its novel functionalities herein, cellular traction force and rigidity sensing ability are analyzed based on synphilin-1 overexpression using elastomeric pillar arrays and substrates of varying stiffness. Molecular changes are analyzed using RNA sequencing-based transcriptomic and liquid chromatography-tandem mass spectrometry-based proteomic analyses.

Results: Synphilin-1 overexpression reduces cell area, with a decline of local contraction on elastomeric pillar arrays. Cells overexpressing synphilin-1 exhibit an impaired ability to respond to substrate rigidity; however, synphilin-1 knockdown restores rigidity sensing abilities. Integrated omics analysis and in silico prediction corroborate the phenotypic alterations induced by synphilin-1 overexpression at a biophysical level. Zyxin emerges as a novel synphilin-1 binding protein, and synphilin-1 overexpression reduces the nuclear translocation of yes-associated protein.

Conclusion: These findings provide novel insights into the biophysical functions of synphilin-1, suggesting a potential protective role to the altered extracellular matrix, which may be relevant to neurodegenerative conditions such as Parkinson's disease.

Keywords: Mechanobiology; Multi-omics; Rigidity sensing; Synphilin-1; Zyxin.

Fig. 1

Cells overexpressing synphilin-1 exhibited reduced spreading area with decreased lamellipodia formation and focal adhesion. A Cell morphology of human embryonic kidney 293 (HEK293) and synphilin-1-overexpressing HEK293 (Synph-293) cells from bright-field, F-actin staining, and scanning electron microscopy analyses (scale bars: 20 μm). B Cell spreading area of HEK293 and Synph-293 cells (n = 50). Data represent the mean + SD. C Proportion of lamellipodia structures at the edge of HEK293 and Synph-293 cells (n = 13). Data represent the mean + SD. Individual value is represented as a hollow circle. D Focal adhesion analysis of cells using paxillin staining images (scale bar: 10 μm). E, F Number (E) and total area (F) of focal adhesions of cells (n = 24/18/31/47). The inner solid line and cross sign indicate the median and mean values, respectively. Statistical significance was determined using an independent sample t-test with Levene’s test. ns: no significance

Fig. 2

Synphilin-1 overexpression induced cellular contraction toward the center, with diminished local contraction. A Schematic representation of elastomeric pillar arrays and pillar force formula. B Schematic representation of the directionality (γ) of pillars. C Pillar deflection map of HEK293, Synph-293, and synphilin-1-knockdown Synph-293 (Synph-293-KD) cells (scale bar: 4 μm) (scale arrow: 3 nN). The yellow line indicates the cell boundary. The length and direction of the cyan arrows indicate the magnitude and direction of traction force vectors, respectively. D Number of pillars covered by cells (n = 50). E Average traction force of pillars beneath cells (n = 50). F Total traction force of pillars beneath cells (n = 50). G Spatial traction force distribution at the edge of cells (scale bar: 1 μm) (scale arrow: 1 nN). Red arrows indicate contractile units. Hγ analysis of the pillars of the edge of cells (n = 50). The inner solid line and cross sign indicate the median and mean values, respectively. Statistical significance was determined using a one-way ANOVA, followed by Tukey’s HSD comparison test

Fig. 3

Synphilin-1 overexpression induced the loss of cellular rigidity sensing on substrates with different stiffness. A Actin staining images of cells on substrates with different stiffness (scale bar: 20 μm). B–D Spreading area of cells on substrates with different stiffness in HEK293 (B), Synph-293 (C), and Synph-293-KD cells (D) (n = 50). E Focal adhesion of cells on substrates with different stiffness (scale bar: 20 μm). F–H Total area of focal adhesions of cells on substrates with different stiffness in HEK293 (F), Synph-293 (G), and Synph-293-KD cells (H) (n = 50). The inner solid line and cross sign indicate the median and mean values, respectively. Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD (B, F, G) and Games-Howell (C, D, H) comparison test

Fig. 4

Integrated omics analyses encompassing transcriptome and proteome datasets predicted the phenotype of synphilin-1 overexpression. A Volcano plot of gene expression. Points satisfying the threshold p < 0.05 and fold change ± 1.5 are denoted by green (down-regulated in Synph-293 cells) and red (up-regulated in Synph-293 cells). B Volcano plot of protein expression. Points satisfying the threshold p < 0.05 and fold change ± 1.2 are denoted by green (down-regulated) and red (up-regulated). C Comparison analysis of cellular functions between transcriptome and proteome datasets using Ingenuity Pathway Analysis (IPA) software. Numbers in the table indicate “activation z-score (− Log₁₀(p-value))”. D Transcriptome network with an in silico prediction of Synph-293 cell phenotype using IPA software. E Proteome network with Synph-293 phenotype prediction. F Gene expression level of transcriptome network-related genes in Synph-293 cells. GAPDH was used as an internal control for gene expression normalization. Data represent the mean + SD of three independent experiments. G Relative protein abundance levels of proteome network-related proteins in Synph-293 cells. Data represent the mean + SD. H Integrated omics network with the prediction of cellular functions. Molecules with opposite expression patterns are presented in yellow. Statistical significance was determined using an independent sample t-test with Levene’s test

Fig. 5

Synphilin-1 interacted with zyxin in the cytosol and affected the subcellular location of YAP. A Expression patterns of synphilin-1 and zyxin in Synph-293 cells (scale bar: 10 μm). Images represent one z-position of confocal z-stack images. B Scatter plot of the pixel intensity of synphilin-1 and zyxin channels of cytosol area. C, D Co-immunoprecipitation (Co-IP) assay between synphilin-1 and zyxin. Cells were subjected to cross-linking using 1% formaldehyde before lysis. Cell lysates were immunoprecipitated with synphilin-1 (C) and zyxin antibodies (D). N.C.: negative control. E–G Prediction of single protein and protein-protein structures using AlphaFold 3. Protein structures indicate zyxin with six Zn²⁺ ions (E), FLAG-synphilin-1 (F), and FLAG-synphilin-1-zyxin with six Zn²⁺ ions (G). H Subcellular expression pattern of YAP in HEK293 and Synph-293 cells (scale bar: 10 μm). I Quantification of nuclear YAP expression intensity ratio in synphilin-1-overexpressing HEK293 cells (n = 27/27/16/16). Lines represent the mean ± SD. Individual value is represented as a hollow circle. J Protein expression of Ser127 phosphorylated YAP (pYAP (S127)) and total YAP in synphilin-1-overexpressing HEK293 cells. K Relative expression level of pYAP (S127). L Relative expression level of total YAP. Data represent the mean + SD. Individual value is represented as a red circle. Statistical significance was determined using an independent sample t-test with Levene’s test

Fig. 6

Synphilin-1 regulated substrate rigidity sensing by interacting with zyxin and affecting YAP localization. Under conditions of synphilin-1 overexpression, the following observations were made. Synphilin-1 interacts with endogenous zyxin in the cytosol. This interaction reduces the transportation of zyxin into complexes containing focal adhesion and causes interference in mechanotransduction signaling, especially signaling through the nuclear translocation of YAP. Therefore, synphilin-1-overexpressing cells on the pillar array are unable to spread to further pillars, and they retract their protrusions. In addition, the synphilin-1-overexpressing cells do not respond to substrates of various stiffness through changes in morphology, including spreading area and focal adhesion. These phenomena can be reversed by synphilin-1 knockdown in cells overexpressing synphilin-1