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A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting.

Byron A, Askari JA, Humphries JD, Jacquemet G, Koper EJ, Warwood S, Choi CK, Stroud MJ, Chen CS, Knight D, Humphries MJ - Nat Commun (2015)

Bottom Line: Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner.Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions.Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling.

No MeSH data available.


Related in: MedlinePlus

Microtubule (MT) morphology and dynamics are dictated by integrin activation state.(a) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see Supplementary Fig. 10 for original blots). (b) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. (n=9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). (c) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). (d) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. ***P<0.001, ****P<0.0001; one-way analysis of variance with Tukey’s post hoc correction in b, two-way analysis of variance with Tukey’s post hoc correction in c and d (see Supplementary Table 4 for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.
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f4: Microtubule (MT) morphology and dynamics are dictated by integrin activation state.(a) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see Supplementary Fig. 10 for original blots). (b) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. (n=9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). (c) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). (d) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. ***P<0.001, ****P<0.0001; one-way analysis of variance with Tukey’s post hoc correction in b, two-way analysis of variance with Tukey’s post hoc correction in c and d (see Supplementary Table 4 for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.

Mentions: To assess the recruitment of +TIPs to integrin complexes, affinity-isolated proteins were subjected to western blotting (WB; Fig. 4a). As expected, talin was strongly enriched in complexes associated with active β1 integrin, confirming the MS data. Moreover, the +TIPs EB1, ACF7 and CKAP5 were also enriched in active integrin complexes (Fig. 4a). To test the integrin activation state dependence of +TIP recruitment, HFFs were spread on integrin activation state-specific mAbs and stained for tubulin. HFFs spread on stimulatory mAb contained microtubules that extended to the cell periphery in a similar manner to those on FN (Fig. 4b). In contrast, cells spread on inhibitory mAb contained microtubules that did not reach the edge of the cell (Fig. 4b). The integrin activation state-dependent growth of microtubules to the cell periphery was also observed in other cell types and for the localization of the +TIP EB1 (Supplementary Fig. 7), and with a variety of stimulatory mAbs (Supplementary Fig. 8), substantiating the correlation between active integrin and microtubule targeting to the cell periphery. Furthermore, quantification of the expression of endogenous EB1 showed that integrin activation state did not alter the level of EB1 in areas away from the cell periphery, indicating that the observed effect was not due to differential expression of EB1 (Supplementary Fig. 7d).


A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting.

Byron A, Askari JA, Humphries JD, Jacquemet G, Koper EJ, Warwood S, Choi CK, Stroud MJ, Chen CS, Knight D, Humphries MJ - Nat Commun (2015)

Microtubule (MT) morphology and dynamics are dictated by integrin activation state.(a) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see Supplementary Fig. 10 for original blots). (b) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. (n=9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). (c) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). (d) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. ***P<0.001, ****P<0.0001; one-way analysis of variance with Tukey’s post hoc correction in b, two-way analysis of variance with Tukey’s post hoc correction in c and d (see Supplementary Table 4 for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.
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f4: Microtubule (MT) morphology and dynamics are dictated by integrin activation state.(a) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see Supplementary Fig. 10 for original blots). (b) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. (n=9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). (c) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). (d) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b. Results are mean±s.d. (n=5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. ***P<0.001, ****P<0.0001; one-way analysis of variance with Tukey’s post hoc correction in b, two-way analysis of variance with Tukey’s post hoc correction in c and d (see Supplementary Table 4 for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.
Mentions: To assess the recruitment of +TIPs to integrin complexes, affinity-isolated proteins were subjected to western blotting (WB; Fig. 4a). As expected, talin was strongly enriched in complexes associated with active β1 integrin, confirming the MS data. Moreover, the +TIPs EB1, ACF7 and CKAP5 were also enriched in active integrin complexes (Fig. 4a). To test the integrin activation state dependence of +TIP recruitment, HFFs were spread on integrin activation state-specific mAbs and stained for tubulin. HFFs spread on stimulatory mAb contained microtubules that extended to the cell periphery in a similar manner to those on FN (Fig. 4b). In contrast, cells spread on inhibitory mAb contained microtubules that did not reach the edge of the cell (Fig. 4b). The integrin activation state-dependent growth of microtubules to the cell periphery was also observed in other cell types and for the localization of the +TIP EB1 (Supplementary Fig. 7), and with a variety of stimulatory mAbs (Supplementary Fig. 8), substantiating the correlation between active integrin and microtubule targeting to the cell periphery. Furthermore, quantification of the expression of endogenous EB1 showed that integrin activation state did not alter the level of EB1 in areas away from the cell periphery, indicating that the observed effect was not due to differential expression of EB1 (Supplementary Fig. 7d).

Bottom Line: Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner.Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions.Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling.

No MeSH data available.


Related in: MedlinePlus