Mechanical force regulates integrin turnover in Drosophila in vivo

Journal name:
Nature Cell Biology
Volume:
14,
Pages:
935–943
Year published:
DOI:
doi:10.1038/ncb2555
Received
Accepted
Published online

Regulated assembly and disassembly, or turnover, of integrin-mediated cell–extracellular matrix (ECM) adhesions is essential for dynamic cell movements and long-term tissue maintenance. For example, in Drosophila, misregulation of integrin turnover disrupts muscle–tendon attachment at myotendinous junctions (MTJs). We demonstrate that mechanical force, which modulates integrin activity, also regulates integrin and intracellular adhesion complex (IAC) turnover in vivo. Using conditional mutants to alter the tensile force on MTJs, we found that the proportion of IAC components undergoing turnover inversely correlated with the force applied on MTJs. This effect was disrupted by point mutations in β-integrin that interfere with ECM-induced conformational changes and activation of β-integrin or integrin-mediated cytoplasmic signalling. These mutants also disrupted integrin dynamics at MTJs during larval development. Together, these data suggest that specific β-integrin-mediated signals regulate adhesion turnover in response to tension during tissue formation. We propose that integrin–ECM adhesive stability is continuously controlled by force in vivo through integrin-dependent auto-regulatory feedback mechanisms so that tissues can quickly adapt to and withstand mechanical stresses.

At a glance

Figures

  1. The effects of BrkdJ29 and parats2 temperature-sensitive mutations on muscle contraction.
    Figure 1: The effects of BrkdJ29 and parats2 temperature-sensitive mutations on muscle contraction.

    (ae) Fifteen-minute kymographs of larvae heat shocked (at 37 °C) demonstrate the effects of the temperature-sensitive BrkdJ29 and parats2 alleles on muscle contraction. Kymographs (bd) were generated from ROIs drawn across MTJs of larvae expressing ILK–GFP (as shown by the yellow line in a; the dotted line demarcates the outline of the anterior end of the larvae). Three representative images are shown per genotype. Changes in fluorescence intensity over time at the brightest portion of the MTJ (dotted line in d) were quantified and plotted in e (a.u., arbitrary units); the representative MTJ chosen for the trace of each genotype in e is indicated by an asterisk in bd. Intensity fluctuations reflect MTJ movements across the line drawn along the length of the MTJ and thereby represent muscle contraction. Compared with the WT at 37 °C (b, green in e), the BrkdJ29 allele (c, red in e) increased muscle contraction, and the parats2 allele (d, blue in e) inhibited muscle contraction. Scale bars, 200 μm (a) and 50 μm (bd). (fh) Examples of traces from force transduction experiments demonstrate differences in muscle contraction magnitude and frequency between WT (f), BrkdJ29 (g) and parats2 (h) larvae at 25 °C and 36 °C. (i,j) Average contraction magnitude (i) and frequency (j) were similar among all genotypes at 25 °C. At 36 °C, BrkdJ29 mutant muscle contractions were relatively greater in magnitude but similar in frequency to the WT, whereas parats2 muscles exhibited a strongly reduced contraction magnitude and frequency; n = number of larvae assayed (indicated above error bar). (k) Contraction rate of single muscles (n = number of muscles assayed) was quantified from 10-min time-lapse movies of larvae at 25 °C and 37 °C (Supplementary Movies S2–S7); at 37 °C, muscles contracted at a higher rate in BrkdJ29 larvae when compared with WT, whereas parats2 muscles contracted very little. (l) The average displacement of muscle ends at MTJs during individual muscle contractions was quantified in WT and BrkdJ29 larvae at 25 °C and 37 °C. There was no statistically significant difference between any of the genotypes or temperatures; n = number of muscle contractions assayed. (Results in il represent mean values±s.e.m; ***P<0.001; NS, not significant.)

  2. Force regulates integrin adhesion complex dynamics at Drosophila MTJs.
    Figure 2: Force regulates integrin adhesion complex dynamics at Drosophila MTJs.

    (ah) FRAP assays on intact, live first-instar (ad) and third-instar (eh) larvae reveal the turnover dynamics of fluorescently tagged integrin (a,b,e,f) and IAC components tensin (c,g) and ILK (d,h). (aa′′) In control first-instar larvae expressing β-integrin–YFP, FRAP curves recorded at 25 °C (grey curves in ah) and following a 1.5 h heat shock at 37 °C (black curves in ah) demonstrate that the integrin mobile fraction increases slightly (a′), and that the rate constants of β-integrin–YFP endocytosis from the membrane (kendo)and return to the membrane (kexo) both increase following heat shock (a′′). (bd) To increase the tensile force on integrin-mediated adhesions at MTJs, the temperature-sensitive allele BrkdJ29 was used to induce muscle hypercontraction in first-instar larvae. After heat shock at 37  °C, the mobile fractions of β-integrin–YFP (b′), tensin–GFP (c′) and ILK–GFP (d′) declined significantly when compared with control animals kept at 25 °C. (b′′) As for the control (see a′′), kexo increased on heat shock; however, unlike the control, kendo did not increase with the induction of muscle hypercontraction, suggesting that when the tensile force is increased, mechanisms act to prevent increases in kendo. (ee′′) In control third-instar larvae expressing β-integrin–YFP, FRAP curves recorded at 25 °C and following a 1.5 h heat shock at 37 °C reveal only a small change in mobile fraction (e′) and unchanged rate kinetics (e′′) of integrin–YFP following heat shock (e′′). (fh) To decrease the tensile force on integrin-mediated adhesions at MTJs, the temperature-sensitive allele parats2 was used to reduce muscle contractility in third-instar larvae. After heat shock at 37 °C, the mobile fraction of β-integrin–YFP (f′) exhibited a very small decline; however, both kendo and kexo increased (f′′). The mobile fractions of tensin–GFP (g′) and ILK–GFP (h′) showed statistically significant increases following heat shock at 37  °C when compared with control animals kept at 25 °C. *P<0.05; results in ah represent mean values±s.e.m.; results in a′–h′, a′′–b′′, and e′′–f′′ represent mean values±95% bootstrap confidence intervals (see Methods and Supplementary Fig. S4); NS, not significant.

  3. Mutant analyses reveal regulatory sites in integrin that regulate turnover in response to elevated tensile force.
    Figure 3: Mutant analyses reveal regulatory sites in integrin that regulate turnover in response to elevated tensile force.

    The temperature-sensitive BrkdJ29 allele was used to increase the tensile force on integrin adhesion sites at MTJs. FRAP assays on first-instar larval MTJs were conducted to determine the fluorescence recovery (ae), final mobile fractions (a′–e′) and rate constants kendo and kexo (a′′–e′′) of the WT β-integrin–YFP (a) and fluorescently tagged β-integrin point mutants (be) at 25 °C (grey) and 37 °C (black). Following heat shock to induce hypercontraction, the mobile fractions of the WT β-integrin–YFP (a′), the Y Y >FF point mutant (c′) or the N840>A mutant (e′) were significantly lower, whereas the mobile fractions of the S196>F and N828>A mutant integrins (b′,d′) did not change significantly. Similarly, no changes were observed in kendo and kexo for the S196>F nor N828>A mutants (b′′,d′′); thus, these mutants seemed unresponsive overall to elevated tension, whereas N840>A (e′′) exhibited abnormal recovery kinetics when compared with WT. In contrast, as for the WT β-integrin (a′′), the Y Y >FF mutant (c′′) exhibited an increase in kexo but no change in kendo. *P<0.05; results in ae represent mean values±s.e.m.; results in a′–e′ and a′′–e′′ represent mean values±95% bootstrap confidence intervals (see Methods); NS, not significant.

  4. Mutant analyses reveal regulatory sites in integrin that regulate turnover in response to reduced tensile force.
    Figure 4: Mutant analyses reveal regulatory sites in integrin that regulate turnover in response to reduced tensile force.

    The temperature-sensitive parats2 allele was used to reduce the tensile force on integrin adhesion sites at MTJs. FRAP assays on third-instar larval MTJs were conducted to determine the fluorescence recovery (ae), final mobile fractions (a′–e′) and rate constants kendo and kexo (a′′–e′′) of WT β-integrin–YFP (a) and fluorescently tagged β-integrin point mutants (be) at 25 °C (grey) and 37 °C (black). The mobile fraction of the WT β-integrin–YFP (a′) decreases slightly following heat shock to reduce contractility; in contrast, the mobile fractions of the mutant integrins (b′–d′) were significantly increased. These mutants also exhibited abnormal recovery kinetics (b′′–e′′) when compared with the WT (a′′), in which both kexo and kendo increased. *P<0.05; results in ae represent mean values±s.e.m.; results in a′–e′ and a′′–e′′ represent mean values±95% bootstrap confidence intervals (see Methods); NS, not significant.

  5. Outside-in integrin activation and signalling regulate integrin turnover dynamics at MTJs throughout development.
    Figure 5: Outside-in integrin activation and signalling regulate integrin turnover dynamics at MTJs throughout development.

    (ae) The dynamics of β-integrin–YFP mutants at larval MTJs were determined through four successive stages of development: embryonic stage 17 (e17), and larval instars 1, 2 and 3 (L1, L2 and L3). (a) The WT β-integrin–YFP turnover declined sharply through each successive stage of development. (be) In contrast, the integrin mutants S196>F (b), YY>FF (c), N828>A (d) and N840>A (e) underwent abnormal turnover dynamics, in that they failed to exhibit the characteristic decline in mobile fraction over the course of development. All experiments in ae were performed with one copy of the indicated integrin transgene and a heterozygous null mutant allele of the β-integrin gene (mysXG43; see Methods). Fluorescence recovery curves (f,g) and mobile fractions (f′,g′) for integrin–YFP at the first (L1, red) and third larval instar (L3, black). In the presence of the WT talin gene dosage (f), integrin turnover dynamics decrease between the first (L1, red) and third (L3, black) larval instars as indicated by the fluorescence recovery curves (f) and the final mobile fractions (f′). On the introduction of one copy of the talin null mutant allele (g, rhea79a, a stage-dependent drop in the integrin–YFP mobile fraction (g′) is still observed, although it is smaller than what is observed in a WT talin background. *P<0.05, results represent mean values±s.e.m. (h) Model for integrin mechanosensing at MTJs.

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Author information

Affiliations

  1. Department of Cellular and Physiological Sciences, University of British Columbia, Life Science Institute, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada

    • Mary Pines,
    • Stephanie J. Ellis,
    • Alexander Morin,
    • Stefan Czerniecki,
    • Lin Yuan,
    • Markus Klose &
    • Guy Tanentzapf
  2. Department of Mathematics and Institute of Applied Mathematics, 1984 Mathematics Road, University of British Columbia, Vancouver, British Columbia V6T 1Z2, Canada

    • Raibatak Das &
    • Daniel Coombs
  3. Department of Molecular and Cell Biology, 142 Life Sciences Addition, University of California, Berkeley, California, Berkeley 94720-3200, USA

    • Lin Yuan

Contributions

G.T. conceived, designed and supervised the project and participated in data analysis. M.P. carried out most of the genetics crosses, FRAP experiments and data analysis. S.J.E. performed genetic crosses, FRAP experiments and analysed data. L.Y. optimized the conditions and protocols for using the Brkd and para mutants. M.K. performed the force transducer experiments. A.M. and S.C. performed FRAP experiments and analysed data. A.M. carried out the stainings on larval flat preparations. M.P., S.J.E., A.M. and S.C. recorded movies for analysis. R.D and D.C. performed the mathematical modeling and data analysis. G.T., M.P., R.D. and S.J.E. wrote and revised the manuscript.

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