Kim HS et al. (2018), Fibronectin type III and intracellular domains ...

XB-ART-54419

Mol Biol Cell 2018 Mar 01;295:523-531. doi: 10.1091/mbc.E17-07-0446.

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Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling.

Kim HS , McKnite A , Xie Y , Christian JL .

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Toll-like receptor 4 interactor with leucine-rich repeats (Tril) functions as a coreceptor for Toll-like receptors (Tlrs) to mediate innate immune responses in adults. In embryos, Tril signals to promote degradation of the Bmp inhibitor, Smad7, to allow for blood formation. It is not known whether this function requires, or is independent of, Tlrs. In the current studies, we performed a structure-function analysis, which indicated that the fibronectin type III (FN) domain and the intracellular domain of Tril are required to trigger Smad7 degradation in Xenopus embryos. Furthermore, we found evidence suggesting that a Tril deletion mutant lacking the FN domain (Tril∆FN) can dominantly inhibit signaling by endogenous Tril when overexpressed. This finding raises the possibility that the FN domain functions to bind endogenous Tril ligands. We also show that Tril cycles between the cell surface and endosomes and that the Tril extracellular domain, as well as cadherin based cell-cell adhesion, are required for cell surface retention, while the intracellular domain is required for internalization in Xenopus ectodermal explants. Using a CHO cell aggregation assay, we show that, unlike other transmembrane proteins that contain leucine-rich repeats, Tril is not sufficient to mediate homophilic adhesion.

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Species referenced: Xenopus laevis
Genes referenced: fn1 hba3 myc smad1 smad7 tlr3 tlr4 tril
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	FIGURE 1: Structure–function analysis of Tril. (A) Embryos were injected with RNA (100 pg) encoding Smad7myc together with control or Tril MOs (35 ng). Immunoblots of lysates from stage 11 embryos (10 per group) were probed with anti-Myc antibodies and then reprobed for β-actin. Relative level of Smad7myc, normalized to actin and reported relative to that in control embryos is indicated below each lane. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for Myc (all images taken under identical conditions). (B, E, I, J) RNA (100 pg) encoding Smad7Myc was injected into two-cell embryos alone or together with RNA encoding wild-type or deletion mutant forms of Tril. Immunoblots of lysates from stage 11 embryos (15 per group) were probed with anti-Myc antibodies and then reprobed for β-actin. Representative blots are shown. The relative level of Smad7myc, normalized to actin and reported relative to that in embryos injected with Smad7myc alone is indicated below each lane and quantitated and graphed below each blot (mean ± SD, n is the number of replicates as indicated on each column). In E,
	FIGURE 2: Overexpression of Tril or Tril∆FN inhibits blood formation. (A, B) RNA encoding mRFP or wild-type or deletion mutant forms of Tril was injected into both ventral cells of four-cell embryos, and expression of hba3.L was analyzed by WMISH at stage 34 in three experiments. hba3.L staining in the posterior VBI (pVBI) was scored as absent or very weak (+/–), weak (++), or strong (+++), as illustrated. aVBI, anterior VBI. n represents the number of embryos analyzed in each group, pooled from three independent biological replicates. (C) RNA encoding mRFP or wild-type or deletion mutant forms of Tril was injected into both ventral cells of four-cell embryos and expression of hba3.L was analyzed in 15 pooled embryos from each group by qPCR at stage 34. Mean ± SD are shown. n is the number of biological replicates, indicated on each column. ***p < 0.01 by two-tailed t test.
	FIGURE 3: The extracellular domain of Tril is required to retain it at the plasma membrane while the intracellular domain is required for internalization. (A–D‴) RNAs encoding wild-type or deletion mutant forms of Tri-HA (illustrated to the left of each panel) and membrane-localized RFP (memRFP) were injected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and RFP. (A–D″) Projections viewed from the xy-axis. (A‴–D‴) Projections viewed from the z-axis. Results were replicated in three independent experiments. (E) CHO cells transfected with GFP (negative control), cadherin6-GFP (Cad6-GFP) (positive control), or Tril-GFP were tested for adhesion. Only cells expressing cadherin6 formed aggregates. The aggregation index was calculated by dividing the total GFP fluorescence in cell aggregates by the total GFP fluorescence in the well. Mean ± SD are shown, n = 3, ***p < 0.01 by two-tailed t test.
	FIGURE 4: Tril is retrieved from the cell surface into endosomes in HeLa cells. (A) HeLa cells were transiently transfected with DNA encoding human Tril-GFP. The plasma membrane was stained with CellMask 24 h later, and GFP was imaged in unfixed cells. (B) HeLa cells were transfected with DNA encoding human Tril-Flag. Cells were fixed 24 h later and double-label immunostained with antibodies specific for Flag and Rab. All results were replicated in three experiments. (C–F) Hela cells were transiently transfected with DNA encoding HA- and Flag-epitope tagged Xenopus Tril (illustrated above panels). Cells were either permeabilized and double-label immunostained with antibodies specific for both epitopes (C–C″) or were incubated at 4°C with anti-HA antibodies for 1 h and then either permeabilized immediately (D–D″) or incubated at 4°C (E–E″) or at 37°C (F–F″) for 1 h before permeabilization and addition of antibodies specific for the Flag tag. HA and Flag epitopes were visualized with species specific fluorescent secondary antibodies. The white arrowheads in D–D″ denote Tril present at the cell surface while the white arrows in F–F″ indicate Tril present in intracellular compartments. Results were replicated in three independent experiments.
	FIGURE 5: Cadherin-mediated cell–cell adhesion is required for cell surface retention of Tril in Xenopus explants. (A–D‴) RNA encoding Tril-HA (100 pg) was injected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and Fibronectin (A–A‴) or E-cadherin (B–B‴). (C–C‴) RNA encoding Tri-HA (100 pg) and N-cad∆Emyc was coinjected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and myc. Results were replicated in three independent experiments. A–C″″ show projections viewed from the xy-axis and A‴–D‴ viewed from the z-axis.
	FIGURE 1:. Structure–function analysis of Tril. (A) Embryos were injected with RNA (100 pg) encoding Smad7myc together with control or Tril MOs (35 ng). Immunoblots of lysates from stage 11 embryos (10 per group) were probed with anti-Myc antibodies and then reprobed for β-actin. Relative level of Smad7myc, normalized to actin and reported relative to that in control embryos is indicated below each lane. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for Myc (all images taken under identical conditions). (B, E, I, J) RNA (100 pg) encoding Smad7Myc was injected into two-cell embryos alone or together with RNA encoding wild-type or deletion mutant forms of Tril. Immunoblots of lysates from stage 11 embryos (15 per group) were probed with anti-Myc antibodies and then reprobed for β-actin. Representative blots are shown. The relative level of Smad7myc, normalized to actin and reported relative to that in embryos injected with Smad7myc alone is indicated below each lane and quantitated and graphed below each blot (mean ± SD, n is the number of replicates as indicated on each column). In E, all lanes are from the same immunoblot, aligned following removal of an intervening lane (following the third lane, marked by a black bar). (C, D, F, G) smad7myc RNA was injected alone or together with increasing doses of tril (C, D) or 500 pg of tril∆FN RNA (F, G) into one cell of two-cell embryos. Ectoderm was explanted from 7–15 embryos in each group at stage 11 and immunostained for Myc. Representative immunostaining is shown in C and F (all images taken under identical conditions), and results are quantified in D and G. Results are pooled from three independent experiments. The total number of explants analyzed is indicated on each column. (H) Embryos were injected with RNA (100 pg) encoding Smad7myc together with control or Tril MOs (35 ng) and tril or tril∆fn RNA (100 pg) as indicated at the top of each lane. Immunoblots of lysates from stage 11 embryos (10 per group) were probed with anti-Myc antibodies and then reprobed for β-actin. Relative level of Smad7myc, normalized to actin and reported relative to that in control embryos is indicated below each lane. Results were replicated in three independent experiments.
	FIGURE 2:. Overexpression of Tril or Tril∆FN inhibits blood formation. (A, B) RNA encoding mRFP or wild-type or deletion mutant forms of Tril was injected into both ventral cells of four-cell embryos, and expression of hba3.L was analyzed by WMISH at stage 34 in three experiments. hba3.L staining in the posterior VBI (pVBI) was scored as absent or very weak (+/–), weak (++), or strong (+++), as illustrated. aVBI, anterior VBI. n represents the number of embryos analyzed in each group, pooled from three independent biological replicates. (C) RNA encoding mRFP or wild-type or deletion mutant forms of Tril was injected into both ventral cells of four-cell embryos and expression of hba3.L was analyzed in 15 pooled embryos from each group by qPCR at stage 34. Mean ± SD are shown. n is the number of biological replicates, indicated on each column. ***p < 0.01 by two-tailed t test.
	FIGURE 3:. The extracellular domain of Tril is required to retain it at the plasma membrane while the intracellular domain is required for internalization. (A–D‴) RNAs encoding wild-type or deletion mutant forms of Tri-HA (illustrated to the left of each panel) and membrane-localized RFP (memRFP) were injected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and RFP. (A–D″) Projections viewed from the xy-axis. (A‴–D‴) Projections viewed from the z-axis. Results were replicated in three independent experiments. (E) CHO cells transfected with GFP (negative control), cadherin6-GFP (Cad6-GFP) (positive control), or Tril-GFP were tested for adhesion. Only cells expressing cadherin6 formed aggregates. The aggregation index was calculated by dividing the total GFP fluorescence in cell aggregates by the total GFP fluorescence in the well. Mean ± SD are shown, n = 3, ***p < 0.01 by two-tailed t test.
	FIGURE 4:. Tril is retrieved from the cell surface into endosomes in HeLa cells. (A) HeLa cells were transiently transfected with DNA encoding human Tril-GFP. The plasma membrane was stained with CellMask 24 h later, and GFP was imaged in unfixed cells. (B) HeLa cells were transfected with DNA encoding human Tril-Flag. Cells were fixed 24 h later and double-label immunostained with antibodies specific for Flag and Rab. All results were replicated in three experiments. (C–F) Hela cells were transiently transfected with DNA encoding HA- and Flag-epitope tagged Xenopus Tril (illustrated above panels). Cells were either permeabilized and double-label immunostained with antibodies specific for both epitopes (C–C″) or were incubated at 4°C with anti-HA antibodies for 1 h and then either permeabilized immediately (D–D″) or incubated at 4°C (E–E″) or at 37°C (F–F″) for 1 h before permeabilization and addition of antibodies specific for the Flag tag. HA and Flag epitopes were visualized with species specific fluorescent secondary antibodies. The white arrowheads in D–D″ denote Tril present at the cell surface while the white arrows in F–F″ indicate Tril present in intracellular compartments. Results were replicated in three independent experiments.
	FIGURE 5:. Cadherin-mediated cell–cell adhesion is required for cell surface retention of Tril in Xenopus explants. (A–D‴) RNA encoding Tril-HA (100 pg) was injected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and Fibronectin (A–A‴) or E-cadherin (B–B‴). (C–C‴) RNA encoding Tri-HA (100 pg) and N-cad∆Emyc was coinjected into a single animal pole blastomere of four-cell embryos. Ectoderm was explanted from 5–10 embryos in each group at stage 11 and immunostained for HA and myc. Results were replicated in three independent experiments. A–C″″ show projections viewed from the xy-axis and A‴–D‴ viewed from the z-axis.

References [+] :

Brubaker, Innate immune pattern recognition: a cell biological perspective. 2015, Pubmed