Research ArticleCELL BIOLOGY

Actomyosin contractility confers mechanoprotection against TNFα-induced disruption of the intervertebral disc

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Science Advances  19 Aug 2020:
Vol. 6, no. 34, eaba2368
DOI: 10.1126/sciadv.aba2368
  • Fig. 1 Stimulation of NP cells with TNFα alters cellular morphological and biophysical properties.

    (A) NP cells in beads maintained round morphology. TNFα increased F-actin–rich processes. Scale bars, 20 μm. (B) Untreated and TNFα-treated NP cells from imaging flow cytometry [brightfield (BF), pMLC (green), nucleus (red)]. Scale bars, 10 μm. (C) Violin plots of circularity and (D) pMLC intensity demonstrate that TNFα decreased both (*P < 10−7 versus untreated). Solid lines, median; dotted lines, quartiles. (E) Heatmap (log2 fold change) of significantly changed myosin, Rho GTPases, cytoskeleton, and ARP (Actin-Related Proteins) genes in TNFα versus untreated groups. (F) TNFα-treated NP cells grown in 2D have more peripheral F-actin (red). No differences in microtubules were observed (i) (tubulin, green). Vimentin (ii) (green) appeared more peripherally spread in TNFα (white arrow. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (G) NP cells in alginate exhibited similar microtubules in untreated versus TNFα. Peripheral vimentin intensity increased in TNFα, with small vimentin-rich processes observed (white arrow). Scale bars, 10 μm. TNFα treatment decreased (H) cell radius (*P < 10−7 versus untreated, n = 180 to 193) and increased (I) hyperosmotic Lp (*P < 10−4 versus untreated, n = 5 to 12) and (J) hypo-osmotic Lp (*P = 0.024 versus untreated, n = 7 to 11). (K) Cell modulus decreased with TNFα treatment (*P < 10−7 versus untreated, n = 45 to 50). (H) to (K) represent means ± SD.

  • Fig. 2 Inhibiting myosin-II mimics the effects of TNFα on NP cell morphology and single-cell biophysical properties.

    (A) F-actin–rich processes were observed in NP cells treated with TNFα (10 ng/ml), 10 μM blebbistatin, and 10 μM Y27632 for 24 hours compared to untreated cells. Scale bars, 20 μm. (B) Circularity significantly decreased in all treatments (*P < 10−7 versus untreated). (C) Cell radius increased in blebbistatin and Y27632 groups, similar to TNFα (*P < 0.02 versus untreated, n = 133 to 193) but not in ML7. δ indicates P < 0.05 compared to TNFα. (D) Lp during hyperosmotic loading significantly increased in blebbistatin and TNFα groups but not in Y27632 or ML7 (*P < 0.01 versus untreated, n = 5 to 13). δ indicates P < 0.05 compared to TNFα. (E) Lp during hypo-osmotic loading was significantly higher in the TNFα group but not in blebbistatin, Y27632, or ML7 (*P < 0.02 versus untreated, n = 7 to 13). (F) Single-cell elastic modulus significantly decreased in TNFα, Y27632, and blebbistatin but not in ML7 (*P < 10−6 versus untreated, n = 40 to 50). δ indicates P < 0.05 compared to TNFα. Scatter plot lines in (C) to (F) represent means ± SD.

  • Fig. 3 Increasing actomyosin contractility with the RhoA activator CN03 protects against TNFα-induced morphological and biophysical changes.

    (A) NP cells treated with CN03 or CN03 + TNFα have similar morphology to untreated cells (F-actin, red). Scale bars, 20 μm. (B) CN03 + TNFα increased circularity versus TNFα to a level beyond that of untreated cells (*P < 10−7 versus untreated, §P < 10−7 versus TNFα, n = 13,000 to 20,000). (C) CN03 + TNFα protected against TNFα-induced decrease in pMLC levels [n and P values are the same as in (B)]. (D) Heatmap (log2 fold change) of significantly changed myosin, Rho GTPases, cytoskeleton, and ARP genes in TNFα versus untreated and CN03 + TNFα versus TNFα groups. (E) CN03 + TNFα cell radius was significantly decreased versus TNFα (*P < 10−7 versus untreated, §P < 10−7 versus TNFα, n = 180 to 239). (F) Hyperosmotic Lp in CN03 + TNFα significantly decreased versus TNFα or untreated (*P < 0.05 versus untreated, §P < 0.001 versus TNFα, n = 5 to 12). (G) Hypo-osmotic Lp significantly decreased in CN03 + TNFα versus TNFα (*P < 0.03 versus untreated, §P < 10−6 versus TNFα, n = 6 to 11). (H) Cell modulus in CN03 + TNFα increased versus TNFα, approaching untreated levels (*P < 10−6 versus untreated, §P < 0.001 versus TNFα, n = 45 to 50). (E) to (H) are means ± SD.

  • Fig. 4 Increasing actomyosin contractility decreases NF-κB nuclear localization and alters expression levels of TNFα-responsive genes.

    (A) Representative images of NP cells from untreated and TNFα-treated groups using imaging flow cytometry, showing brightfield, NF-κB (green), nucleus (Draq5, red), and merged NF-κB + Draq5 images. Scale bars, 10 μm. (B) Histograms of NF-κB nuclear colocalization (similarity score calculated by the Amnis IDEAS software) and normalized to untreated group showed an increase with TNFα stimulation that was mitigated with CN03 cotreatment (*P < 10−5 versus untreated, §P < 10−7 versus TNFα). (C) Western blot of nuclear fraction of NP cells from TNFα ± CN03 treatment groups. Cell lysates were isolated after 10, 30, or 60 min of treatment, showing NF-κB (p65) and loading control for nuclear fraction (lamin). Inflammatory stimulation led to increased nuclear NF-κB at all time points. Cotreatment with CN03 + TNFα decreased p65 at 30 and 60 min. (D) Volcano plot of transcriptomics data obtained for TNFα versus untreated. (E) Volcano plot of transcriptomics for CN03 + TNFα versus TNFα. Dotted line indicates threshold of significance P = 0.05. In red are genes with nonsignificant changes, and in black are genes with significant changes. (F) Heatmap (log2 fold change) of TNFα-responsive genes and NF-κB signaling genes that were found to have significant change [Padj (adjusted P value) < 0.05] in TNFα versus untreated and CN03 + TNFα versus TNFα groups (color bar, log2 fold change).

  • Fig. 5 Increasing actomyosin contractility protects tissue from TNFα-induced GAG loss.

    (A) Schematic of the explant-in-hydrogel culture system and respective treatment groups. NP explants were cast and cultured within 2% agarose hydrogel to prevent swelling and treated with culture media only (untreated), TNFα, CN03, or CN03 + TNFα for 14 days. (B) Confocal fluorescence of NP explant sections stained with phalloidin for F-actin visualization. Cells within tissue show that the morphological changes induced by TNFα treatment are prevented by coincubation with CN03. Scale bars, 100 μm. (C) Violin plot for circularity of NP cells within explants indicates that the decrease induced by TNFα is mitigated in CN03 + TNFα. Solid lines represent median, and dotted lines represent quartiles. *P < 0.05 compared to untreated, §P < 0.05 compared to TNFα. (D) Histological sections of NP explants after 14 days in the swelling-restricted organ culture system, stained with Alcian blue to show GAG content. Sections were generated from across the explant cross section, showing effects near the central (inner) and periphery (outer) of the explant. There was a reduction in GAG staining in the outer area with TNFα. Cotreatment with CN03 + TNFα mitigated this GAG loss. Scale bar, 100 μm. (E) Quantification of GAG content (percentage of wet weight) measured by DMMB assay of explant digests, normalized to untreated control (means ± SD). Inflammatory treatment with TNFα reduced overall GAG content (*P = 0.04 versus untreated, n = 6 to 7). Scatter plot lines in (F) show means ± SD. (F) Heatmap (log2 fold change) of ECM genes and degradative/catabolic genes that were found to have significant change (Padj < 0.05) in TNFα versus untreated and CN03 + TNFα versus TNFα groups (color bar, log2 fold change).

  • Fig. 6 Hydraulic permeability during cell shrinking scales with pMLC levels and actomyosin contractility.

    (A) Trend for a positive correlation between pMLC intensity and cell circularity was observed. Across all treatment groups, we identified a strong inverse correlation between Lp during hyperosmotic loading and (B) pMLC intensity or (C) circularity. Linear regression R2 and P values are indicated in each subpanel. In each graph, data points represent the average value from each experimental group (error bars, SD).

Supplementary Materials

  • Supplementary Materials

    Actomyosin contractility confers mechanoprotection against TNFα-induced disruption of the intervertebral disc

    Paula A. Hernandez, Timothy D. Jacobsen, Nadeen O. Chahine

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