Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells

Abstract

Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labelled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blasticidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1 μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.

Keywords: Actin; Cross-linked actin networks; Glaucoma; Latrunculin; Phagocytosis; Stiffness; Trabecular meshwork.

Figure 1.. Spontaneous CLAN formation in GTM3-LifeAct-GFP cells.

A) Live imaging of the GTM3-LifeAct-GFP cells. Arrows: CLANs. B) The cells were fixed with paraformaldehyde and stained with Phalloidin-488 (Green, for F-actin) and DAPI (Blue). C) Morphology of CLANs in fixed primary human trabecular meshwork (pHTM) cells. Arrows: CLANs. White triangles: definition of CLANs. Scale bars: 20μm.

Figure 2.. CLANs increased GTM3-LifeAct-GFP cell stiffness and lowered cell viscosity.

CLAN+/− GTM3-LifeAct-GFP cells were identified using epifluorescent microscopy coupled to the AFM and characterized biomechanically. (A) Elastic modulus (stiffness) and (B) the factor of viscosity (ratio of viscous energy to sum of viscous and elastic energy) were determined. CLAN+ GTM3-LifeAct-GFP cells showed significantly higher elastic modulus and lower factor of viscosity compared to CLAN− GTM3-LifeAct-GFP cells or naïve GTM3 cells. CLAN− GTM3-LifeAct-GFP cells and naïve GTM3 cells were similar (P>0.05). N=3 independent experiments. In each experiment, 5 CLAN+, 7 CLAN−, and 7 naïve cells were studied. The different color represents each replicate. One-way ANOVA and Tukey posthoc tests were used. **: p<0.01, ****: p<0.0001.

Figure 3.. DEX or TGFβ2 treatment increased pHTM cell stiffness via CLAN formation.

(A) Untreated pHTM cells and those treated with 100 nM DEX or 5 ng/ml TGFβ2 were labelled with SiR-actin and the presence or absence of CLANs were visualized in live cells. White arrows: CLAN− cells. Yellow arrows: CLANs. Inserts (lower right corner): detailed views of CLANs. (B) Presence of CLANs above the nuclear region were confirmed by imaging in peak force tapping mode with AFM. (C) Elastic modulus of CLAN +/− cells were determined from three independent donor strains (N=3; biological repeats/experiments). In each experiment, 5 CLAN+, 7 CLAN−, and 7 naïve cells were used. Data are represented as a cello plot. The different color represents each replicate. One-Way ANOVA and Tukey’s multiple comparison were used. *: p<0.05.

Figure 4.. CLANs in GTM3-LifeAct-GFP cells were resistant to latrunculin B.

GTM3-LifeAct-GFP cells with or without CLANs were treated with 1μM latrunculin B and live imaged. (A) Before treatment. (B) 1 min after treatment. (C) 90 min after treatment but right before latrunculin B washout. (D) 20 hr from the adding of latrunculin B/18.5 hr after latrunculin B washout. Green: LifeAct-GFP, which binds to F-actin. Arrows: the CLAN+ cell. Triangles: stress fibers (Due to focal plane differences, images were focused on CLANs, not stress fibers). Multiple cells were studied in at least 3 different experiments, and a representative image series is shown. Please see supplemental videos 1 and 2 for more information. Scale bar: 20μm.

Figure 5.. CLANs decreased GTM3-LifeAct-GFP cell phagocytosis.

GTM3-LifeAct-GFP cells (Green) were incubated with pHrodo Red bioparticles (red dots). Arrow: a CLAN+ cell; triangles: CLAN− cells. Multiple cells were studied in at least 3 different experiments , and a representative image is shown. Please see supplemental Figure 1 for more information. Scale bar: 20μm.

Figure 6.. CLANs decreased pHTM cell phagocytosis.

Primary HTM cells were treated with 5ng/ml TGFβ2 for 14 days to induce CLAN formation. The cells were treated with pHrodo bioparticles (red; B and F). After incubation with bioparticles overnight, the cells were fixed and stained with DAPI (blue; A and E) and phalloidin (green; C and G). (D) and (H): color merged images of (A-C) and (E-G), respectively. (I): enlarged view of circled region in (H). A representative image is shown. Please see supplemental Figure 2 for more information. Scale bar: 20μm.

Figure 7.. TGFβ2 or DEX did not induce CLAN formation in GTM3-LifeAct-GFP cells.

GTM3-LifeAct-GFP cells were seeded onto 96-well glass bottom plates, treated with or without 5ng/ml TGFβ2, 0.1%EtOH, or 100nM DEX for 7 days. CLAN+ cells and nuclei (stained with DAPI or Hoechst) were counted and the percentage of CLAN+ cells over nuclear numbers were compared using Student’s t-test. N=24. NS: not significant. **: p<0.01.