A tension-induced mechanotransduction pathway promotes epithelial morphogenesis

Journal name:
Nature
Volume:
471,
Pages:
99–103
Date published:
DOI:
doi:10.1038/nature09765
Received
Accepted
Published online

Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins1. Mechanotransduction is crucial for the physiology of several organs and for cell migration2, 3. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome: p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.

At a glance

Figures

  1. Muscle tension promotes C. elegans hemidesmosome maturation.
    Figure 1: Muscle tension promotes C. elegans hemidesmosome maturation.

    a, Schemes showing a C. elegans embryo (top) and a cross-section of the embryo (at the level of red lines; bottom) and its hemidesmosomes (CeHDs), numbered 1–4. Three epidermal cell types are found around the circumference: dorsal and ventral (which uniquely express elt-3); and lateral. A, anterior; D, dorsal; P, posterior; V, ventral. b, Actin bundles (white) in WT embryo imaged by following an actin-binding domain labelled with GFP20. The dashed box shows the region selected for the kymograph in c. Scale bar, 10μm. c, Kymographs showing the distance change between actin bundles (white) in WT embryos, unc-112(RNAi) mutant embryos (which are muscle deficient) and vab-10A(RNAi) embryos (which are CeHD deficient) (see also Supplementary Movies 1–3). Red circles indicate actin-anchoring points displaced by muscle contractions. C, contracted distance (orange); R, relaxed distance (green). d, Quantification of tension changes in terms of distance (contracted divided by relaxed) and time span per contraction. Individual data points (n = 15) and mean±s.e.m. (black crosses) are shown. eh, Immunostaining of WT embryos at the 1.5–2-fold stage of development (early; e, f) or the 3–4-fold stage (late; g, h): muscles (red) and VAB-10A (green). Dashed boxes in e and g demarcate the regions shown in f and h, respectively.

  2. PAK-1 function is required for CeHD maturation.
    Figure 2: PAK-1 function is required for CeHD maturation.

    a, b, Conserved domains of VAB-10A and PAK-1 proteins. The missense mutation e698 maps to the region predicted to bind to intermediate filaments10. The deletions tm403 and ok448 remove the PAK-1 CRIB domain and kinase domain, respectively. ABD, actin-binding domain. ce, Co-localization of PAK-1 with IFA-2 and IFA-3 in a WT larva, as determined by immunofluorescence: PAK-1 (green) and IFA (red). Scale bar, 10µm. fn, Immunostaining for muscle (red) and VAB-10A (green) of vab-10A(e698) (f, i, l), pak-1(ok448) (g, j, m) and vab-10A(e698); pak-1(ok448) (h, k, n) mutant embryos at early or late stages of development. Dashed boxes indicate area shown in panel below. Dashed line in k shows where muscles should be. Arrow in n shows area with muscles still attached. Arrowheads in h and n show areas with muscles detached. Scale bar, 10µm.

  3. PAK-1-induced intermediate-filament phosphorylation depends on muscle tension.
    Figure 3: PAK-1-induced intermediate-filament phosphorylation depends on muscle tension.

    ac, Two-dimensional immunoblotting analysis showing spots that indicate IFA proteins and their phosphorylated forms. Arrows point to phosphorylated proteins that are present in WT embryos but not phosphatase-treated WT embryos, pak-1 mutants or egl-19 mutants. Arrowheads point to isoelectric species, which are always visible in this type of analysis. CIP, calf intestinal phosphatase; IEF, isoelectric focusing; MW, molecular weight. di, Immunostaining of WT and mutant embryos for IFA proteins (green) and VAB-10A (red). Arrows point to ectopic intermediate-filament bundles (g, i). Scale bar, 10µm. j, Pull-down assay for two independent samples showing levels of GTP-bound CED-10 in WT embryos and a muscle mutant (egl-19), both expressing GFP-tagged CED-10 under an epidermal promoter (epi::gfp::ced-10). k, CED-10–GTP level, as determined by pull-down experiment in j, was normalized to total CED-10 levels after densitometry analysis (n = 13; mean, black bar). **, P = 0.0006 (Mann–Whitney U test). l, Two-dimensional immunoblotting analysis showing phosphorylated IFA (arrows) restored in egl-19 mutants by CED-10(G12V) in a PAK-1-dependent manner. Arrowheads point to isoelectric species. m, Body length of unc-112(RNAi) L1 larvae expressing constitutively active CED-10, MLC-4 or both (n>26; y axis, arbitrary units). Data are presented as mean±s.e.m. **, P<3×10−8 (Student’s t-test).

  4. GIT-1 maintenance at CeHDs in a tension-dependent manner and PIX-1 promote PAK-1 activation.
    Figure 4: GIT-1 maintenance at CeHDs in a tension-dependent manner and PIX-1 promote PAK-1 activation.

    a, b, Localization of translational PIX-1–GFP (a) and GIT-1–GFP (b) in WT embryos. c, d, Immunostaining for IFA proteins (green) in vab-10A(e698); pix-1(gk416) and vab-10A(e698); git-1(tm1962) double mutants in early-stage embryos. Arrows point to ectopic intermediate-filament bundles. e, f, Immunostaining for VAB-10A (green) and muscle (red) in late-stage embryos of listed mutants, showing muscle detachment (arrows). g, Diagram showing the set-up of force stimulation. h, Quantification of CeHD-localized GIT-1–GFP level compared with time zero (n = 12). Data are presented as mean±s.e.m. **, P = 0.009 (Mann–Whitney U test). il, Representative images showing GIT-1–GFP localization (arrows) in unc-112(RNAi) embryos (denoted pat) with (k, l) or without (i, j) external force stimulation. af, il, Scale bars, 10µm.

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Author information

  1. These authors contributed equally to this work.

    • Frédéric Landmann &
    • Hala Zahreddine

Affiliations

  1. Development and Stem Cells Program, IGBMC, CNRS (UMR7104), INSERM (U964), Université de Strasbourg, 1 rue Laurent Fries, BP10142, 67400 Illkirch, France

    • Huimin Zhang,
    • Frédéric Landmann,
    • Hala Zahreddine,
    • David Rodriguez &
    • Michel Labouesse
  2. Imaging Centre, IGBMC, CNRS (UMR7104), INSERM (U964), Université de Strasbourg, 1 rue Laurent Fries, BP10142, 67400 Illkirch, France

    • Marc Koch
  3. Present address: MCBD Department, University of California, Santa Cruz, California 95064, USA .

    • Frédéric Landmann

Contributions

H. Zhang and M.L. designed the study, analysed the data and wrote the paper. H. Zhang conducted most of the experiments. F.L. and H. Zahreddine made some initial observations (tension-change modification of the epidermis, and PAK-1 distribution and mutant phenotype) that proved to be essential for designing the study. D.R. provided technical help. M.K. helped to design and analyse the pressing experiment.

Competing financial interests

The authors declare no competing financial interests.

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