Application of Force to a Syndecan-4 Containing Complex With Thy-1-αVβ3 Integrin Accelerates Neurite Retraction - PubMed (original) (raw)
Application of Force to a Syndecan-4 Containing Complex With Thy-1-αVβ3 Integrin Accelerates Neurite Retraction
Francesca Burgos-Bravo et al. Front Mol Biosci. 2020.
Abstract
Inflammation contributes to the genesis and progression of chronic diseases, such as cancer and neurodegeneration. Upregulation of integrins in astrocytes during inflammation induces neurite retraction by binding to the neuronal protein Thy-1, also known as CD90. Additionally, Thy-1 alters astrocyte contractility and movement by binding to the mechano-sensors αVβ3 integrin and Syndecan-4. However, the contribution of Syndecan-4 to neurite shortening following Thy-1-αVβ3 integrin interaction remains unknown. To further characterize the contribution of Syndecan-4 in Thy-1-dependent neurite outgrowth inhibition and neurite retraction, cell-based assays under pro-inflammatory conditions were performed. In addition, using Optical Tweezers, we studied single-molecule binding properties between these proteins, and their mechanical responses. Syndecan-4 increased the lifetime of Thy-1-αVβ3 integrin binding by interacting directly with Thy-1 and forming a ternary complex (Thy-1-αVβ3 integrin + Syndecan-4). Under _in vitro_-generated pro-inflammatory conditions, Syndecan-4 accelerated the effect of integrin-engaged Thy-1 by forming this ternary complex, leading to faster neurite retraction and the inhibition of neurite outgrowth. Thus, Syndecan-4 controls neurite cytoskeleton contractility by modulating αVβ3 integrin mechano-receptor function. These results suggest that mechano-transduction, cell-matrix and cell-cell interactions are likely critical events in inflammation-related disease development.
Keywords: cell adhesion molecules; cell–cell adhesion; inflammation; mechano-sensor; mechano-transduction; single-molecule analysis; trimolecular adhesion complex.
Copyright © 2020 Burgos-Bravo, Martínez-Meza, Quest, Wilson and Leyton.
Figures
FIGURE 1
Characterization at the single-molecule level reveals that Syndecan-4 increases the lifetime of the Thy-1–αVβ3 integrin binding by forming a ternary complex. (A) Scheme of the assay using miniTweezers. Two different sizes of protein G-coated beads were used; the smaller bead (Bead 1; 2.1 μm) contained purified Thy-1-Fc and was attached to a micropipette by suction; the larger bead (Bead 2; 3.1 μm) containing the αVβ3-Fc, Syndecan-4-Fc or both molecules, was trapped by a laser beam and held in the focus of the microscope (Figure adapted from Burgos-Bravo et al., 2018.
https://doi.org/10.1091/mbc.E17-03-0133
). (B) Adhesion frequency of Syndecan-4 (Synd-4-Fc) with TRAIL-R2-Fc (control protein), wild-type Thy-1-Fc, Thy-1(RLE)-Fc mutated in the integrin binding-site, and Thy-1-(AEAAA)-Fc mutated in the heparin binding domain, was assessed using force-ramp assays at a loading rate of 10 pN/s. The total number of binding events in at least 50 approaching-retraction cycles per 4–5 pairs of beads were measured. Non-specific interactions were evaluated using Thy-1-Fc- and Hepes buffer-treated beads (Buffer). Data are expressed as the mean ± SEM (*p < 0.05; n.s. non-significant, assessed by Mann–Whitney’s test). (C) Representative force-time trace obtained by force-constant assay at 30 pN between Thy-1-Fc and Syndecan-4-Fc. The force is ramped to and sustained at a constant force until the interaction is disrupted. (D) Lifetime of bi-molecular Thy-1-Fc interactions with Syndecan-4-Fc (Thy-1-Fc/Syndecan-4-Fc) or αVβ3-Fc integrin (Thy-1-Fc/αVβ3-Fc) as a function of the constant force. (E) Lifetime of tri-molecular Thy-1-Fc interactions with αVβ3-Fc integrin and Syndecan-4-Fc (Thy-1-Fc/αVβ3-Fc + Syndecan-4-Fc) plotted versus constant force. As a control for the tri-molecular interactions, lifetime data were evaluated for Thy-1-Fc binding with TRAIL-R2-Fc in the presence of Syndecan-4-Fc (Thy-1-Fc/Syndecan-4-Fc + TRAIL-R2-Fc) or αVβ3-Fc integrin (Thy-1-Fc/αVβ3-Fc + TRAIL-R2-Fc). Lifetime data plotted against constant forces were fitted to the Bell model (see Materials and Methods) to calculate the unbinding parameters at zero force for each interaction (F), including lifetime (τ0), off-rate constants (koff0; inversely related to the lifetime), and the distance to the transition state (Δx‡). Lifetime data in (D,E) are expressed as the mean ± SEM from at least 60 binding events obtained using 3 pairs of different beads. Force-ramp and constant-force results were analyzed by a Matlab program.
FIGURE 2
Syndecan-4 accelerates neurite retraction promoted by the αVβ3 integrin. (A) Thy-1–αVβ3 integrin binding induces cell signaling events resulting in the retraction of neuronal processes. On the other hand, Thy-1–αVβ3 integrin + Syndecan-4 association promotes astrocyte migration. Here, the effect of Syndecan-4 on αVβ3 integrin-induced neurite retraction was tested. (B) Serum-free medium containing Syndecan-4-Fc fusion protein was incubated (+) or not (–) with an excess of protein-A-sepharose beads and then centrifuged to obtain a precipitated Syndecan-4-Fc-protein-A-sepharose complex and Syndecan-4-depleted supernatant, respectively. All these samples were treated (+) or not (–) with Heparitinase III, separated by SDS-PAGE and analyzed by immunoblotting with anti-Syndecan-4 antibodies. (C) A microplate coated with human bFGF (1 μg/ml) or BSA (1 μg/ml) was incubated with serum-free medium containing Syndecan-4-Fc or TRAIL-R2-Fc (control Fc-protein), followed by incubation with anti-Fc-HRP conjugated antibody. Specific binding was measured in a colorimetric method with TMB substrate solution (Absorbance at 450 nm). (D) Quantification of the neurite length of differentiated CAD cells (1 × 105 cells/cm2) over a 24-well plate after 5, 10, 20, and 40 min of incubation with control medium (without fusion proteins), Syndecan-4-Fc or αVβ3-Fc in serum-free medium, αVβ3-Fc/Protein-A, or αVβ3-Fc/Syndecan-4-Fc (ratio 1:1). (E) Quantification of the neurite length of differentiated CAD cells over a 24-well plate (1 × 105 cells/cm2) after 40 min incubation with serum-free medium containing αVβ3-Fc (1:10 of the total volume, 100 μl) in the absence or presence of different volumes of serum-free medium containing Syndecan-4-Fc. In (D,E) the neurites of at least 100 cells were measured per condition by using NeuronJ plug-in for ImageJ. In all graphs, data are expressed as the mean ± SEM (n = 3; *p < 0.05; n.s. non-significant, assessed by Mann–Whitney’s test). In (D) *p < 0.05 compared to the control situation at the respective times analyzed.
FIGURE 3
The inhibitory effect of astrocytes on neurite extension requires Syndecan-4. Cell tracker green-labeled CAD cells (5 × 104 cells/cm2) were seeded onto a plate or co-cultured on top of a fixed-monolayer of DITNC1 astrocytes. Neurite extension was then induced by serum deprivation for 24 h (1 × 105 cells/cm2). To evaluate the participation of heparan sulfate chains in the inhibition of neurite outgrowth, DITNC1 cells were pre-treated with Heparitinase III (Hase III; 0.5 mU; 3 h at 37°C) or pre-incubated with Heparin (Hep; 400 μg/ml; 30 min). To block αVβ3-integrin, astrocytes were incubated with anti-β3 integrin antibodies (anti-β3; 5 μg/ml; 1 h; 37°C). (A) Representative microphotographs of different conditions. Quantification of neurite length (μm) after (B) Hase III treatment or (C) Hep incubation. (D) siRNA silencing of Syndecan-4 protein in whole cell lysates. DITNC1 cells that were either non-transfected (NT), transfected with siRNA control (siCTRL) or with siRNA targeting Syndecan-4 (siSDC4) were evaluated by immunoblotting. Actin was used as a loading control. The band intensities were quantified by ImageJ software and normalized to actin. (E) Quantification of neurite length (μm) after Syndecan-4 silencing. For each quantification (B,C,E), neurites of at least 100 cells per condition were evaluated by using NeuronJ plug-in for ImageJ. Arrowheads in (A) indicate neurites growing over the DITNC1 astrocytes. In all graphs data are expressed as mean ± SEM (n = 3; *p < 0.05; **p < 0.01; n.s. non-significant, assessed by Mann–Whitney’s test).
FIGURE 4
Primary astrocytes under pro-inflammatory conditions inhibit neurite outgrowth in an αVβ3 integrin- and Syndecan-4-dependent manner. (A,C) Protein levels of β3 integrin and Syndecan-4 in primary astrocytes treated or not with TNF were evaluated by immunoblotting. Actin was used as a loading control. The band intensities were quantified by ImageJ software and normalized to actin. (B,C) CAD cells (5 × 104 cells/cm2) were seeded on top of a monolayer of primary astrocytes pre-treated or not with TNF (10 ng/ml; 48 h) and neurite extension was induced by serum deprivation. Primary astrocytes were (B) pre-treated with Heparitinase (Hase III; 0.5 mU) for 3 h at 37°C or (D) transfected with siRNA against Syndecan-4 (siSDC4) to evaluate heparan sulfate chains and Syndecan-4 participation in the inhibition of neurite outgrowth, respectively. To block αVβ3 integrin, astrocytes were also incubated with anti-β3 integrin antibodies (5 μg/ml; 1 h; 37°C). (B,D) Quantification of neurite length (μm) in co-culture assays. Neurites of at least 100 CAD cells per condition were evaluated by using NeuronJ plug-in for ImageJ. Data are expressed as mean ± SEM (n = 3; *p < 0.05, assessed by Mann–Whitney’s test).
FIGURE 5
Model showing Syndecan-4 as a key player in neuron-astrocyte interactions under inflammation accelerating axonal retraction. The illustrated behavior is particularly relevant under inflammatory conditions where the expression of both Syndecan-4 and αVβ3 integrin increase on astrocytes and exacerbate the detrimental effects of Thy-1–αVβ3 integrin association, on neurons.
References
- Avalos A. M., Valdivia A. D., Muñoz N., Herrera-Molina R., Tapia J. C., Lavandero S., et al. (2009). Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with αvβ3 integrin that activates PKCα and RhoA. J. Cell Sci. 122 3462–3471. 10.1242/jcs.034827 - DOI - PMC - PubMed
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