Fig 1: The relative localisation of fibronectin and myosin10 is altered in the Golgi of FKRP-deficient cells.Fibronectin and myosin10 protein localisation within the trans-Golgi, as determined by STED microscopy and Gaussian distribution line profile analyses, images representative of a minimum of three repeats separated by a minimum of a week and three technical repeats. a STED images of myosin10: blue, fibronectin: red and Golgi 58 k protein: green. Merged image marked for Golgi with dashed lines and white solid lines marking the location of the line profiles quantitated in b. b Individual line profiles, normalised to maximum and using Gaussian distribution curve in Fiji image analysis software, fibronectin red, myosin10 blue, means marked with dotted lines. c Co-localisation of myosin10 and fibronectin from the Golgi identified by a 58 K antibody stain, Pearson correlation analysis (*P < 0.05), (***P < 0.0001) (n = 9), box-and-whisker plot, middle line = mean, box = 95% confidence interval, error bars = SEM, one-way ANOVA analysis. d Quantitation of the difference between individual line profile means, analysed from Gaussian fit curves in b (n = 9), (*P < 0.05), (***P < 0.0001). Box-and-whisker plot, middle line = mean, box = 95% confidence interval, error bars = SEM. e–h Analysis of Airyscan data in Imaris image analysis software. e Images from the Airyscan rendered in 3D, myosin10 in yellow, fibronectin in blue and Golgi: Golgi Reassembly Stacking Protein 2 (GORASP2) in green, scale bar = 2 µm. f Pearson’s correlation of fibronectin and myosin10 using Golgi marker: GORASP2 was used as a mask for analysis. Box-and-whisker plots, error bars represent a 95% confidence interval and middle box line represents mean (***P < 0.0001). One-way ANOVA analysis (n = 9 for each sample), one-way ANOVA analysis. g Sphericity of fibronectin within Golgi, Imaris generated surfaces and software analysis. Box-and-whisker plots, error bars represent a 95% confidence interval and middle box line represents mean (***P < 0.0001). One-way ANOVA analysis (n = 9 for each sample). h Airyscan data with cells rendered in 3D and statistically coded for sphericity in a heat map (with a value of one (red) indicating a perfect sphere) reveal the altered Golgi structure in patient cells, scale bar 2 µm. i Schematic of FKRP action. The schematic illustrates that in healthy control cells, FKRP is required to correctly localise myosin10 within the terminal Golgi, a process important for sialylation of fibronectin, that in turn regulates its binding to collagen. In FKRP deficiency, myosin10 is no longer anchored correctly and fibronectin is consequently not sialylated correctly. This lack of sialylation results in a failure of collagen–fibronectin binding at the MBM, which ultimately leads to a loss of MBM stability and an inability of individual muscle fibres to resist passive force.
Fig 2: Knockdown of GORASP2 decreased the contractility of primary human myometrial smooth muscle cells (hMSMCs). (A) hMSMCs were transfected with si-NC or si-GORASP2 for 72 h, treated with 3% O2 for 2 h and stained for GORASP2, N = 3. (B,C) Expression of GORASP2, OXTR and Connexin 43 in hMSMCs treated with normoxia or 3% O2 for 2 h after transfection with si-NC or si-GORASP2 for 72 h, N = 3. (D) Contractility assessment of hMSMCs transfected with si-NC or si-GORASP2 for 48 h, treated with 10 nM oxytocin and 3% O2 for 2 h, N = 3; (E): Gel area statistics of hMSMCs contraction. * p < 0.05, ** p < 0.01.
Fig 3: The effects of GORASP2 on energy production in hMSMCs. (A) Volcanic plots of RNA expression treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 48 h, N = 3. (B–F) KEGG-enriched pathways via Gene Set Enrichment Analysis reveal five significantly downregulated pathways related to metabolism. (G) Validation of genes related to the energy metabolism pathways using RT-qPCR, N = 3. (H) ATP levels in hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h. The ATP level (ng) was normalized with the protein amount (μg Prot) obtained from the groups, N = 3. (I,J) Oxygen consumption rate (OCR) of hMSMCs treated with 300 μM CoCl2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h. Subsequent additions of ATP synthase inhibitor oligomycin, uncoupler FCCP, ETC complex I inhibitor rotenone and respiratory complex III inhibitor antimycin A were performed. Basal respiration, ATP-production coupled respiration, maximal respiration and spare respiratory capacity were calculated from the OCR trace, N = 3. (K,L) Mitochondrial ROS detected in hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h, N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4: The effects of GORASP2 on the biological functions of hMSMCs. (A,B) Assessment of autophagy via the expression levels of LC3b II/I and Beclin 1 in hMSMCs treated with normoxia or 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h, N = 3. (C) Immunofluorescence of LC3b in hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 48 h, N = 3. (D,E) Intracellular reactive oxygen species (ROS) detected in hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h, N = 3. (F,G) Apoptosis ratios in hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h. The lower right quadrant (Q3, Annexin V+/PI-) represents early apoptotic cells, the upper right quadrant (Q2, Annexin V+/PI+) represents late apoptotic cells and necrotic cells, and the upper left quadrant (Q1, Annexin V-/PI+) is considered to be within the scope of the assay error. The total amount of Q2 and Q3 were analyzed between the groups. N = 3. (H,I) The cell cycle of hMSMCs treated with 3% O2 for 2 h after transfected with si-NC or si-GORASP2 for 72 h. The levels of G1, S and G2 phases were analyzed between the groups; N = 3. ns, no statistical difference, * p < 0.05, ** p < 0.01.
Fig 5: Golgi reassembly stacking protein 2 (GORASP2) is up-regulated in human in-labor myometrium. (A) Proteomics study comparing in-labor and non-labor myometrium. N = 10. (B,C) Expression of GORASP2 in in-labor and non-labor myometrium analyzed using Western blot. Non-Labor: N = 6, In-Labor: N = 7. (D) Electron microscopy images of human in-labor and non-labor myometrium. GO represents the Golgi apparatus; N indicates the nucleus; RER indicates the rough endoplasmic reticulum. (E) Visualization of GORASP2 in in-labor and non-labor myometrium via immunofluorescence, N = 3. * p < 0.05.
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