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Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

Horton ER, Byron A, Askari JA, Ng DH, Millon-Frémillon A, Robertson J, Koper EJ, Paul NR, Warwood S, Knight D, Humphries JD, Humphries MJ - Nat. Cell Biol. (2015)

Bottom Line: Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome.Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins.The definition of this consensus view of integrin adhesome components provides a resource for the research community.

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 receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

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Changes in consensus adhesome components during IAC disassembly. (a) HFF cells treated with DMSO, 10 μM nocodazole or after nocodazole removal at different times were stained for vinculin, zyxin and α5 integrin. Representative images are shown. Scale bars, 20 μm. (b–d) Quantification of images in a. Vinculin, zyxin and α5 integrin levels were quantified as a proportion of total cell area. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box) and 5th and 95th percentiles (whiskers) (n = 10 cells per condition from one independent experiment). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test with Dunn’s post hoc correction (comparisons with the nocodazole treatment group are shown; see Supplementary Table 15 for statistics source data).
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Figure 8: Changes in consensus adhesome components during IAC disassembly. (a) HFF cells treated with DMSO, 10 μM nocodazole or after nocodazole removal at different times were stained for vinculin, zyxin and α5 integrin. Representative images are shown. Scale bars, 20 μm. (b–d) Quantification of images in a. Vinculin, zyxin and α5 integrin levels were quantified as a proportion of total cell area. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box) and 5th and 95th percentiles (whiskers) (n = 10 cells per condition from one independent experiment). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test with Dunn’s post hoc correction (comparisons with the nocodazole treatment group are shown; see Supplementary Table 15 for statistics source data).

Mentions: To confirm the temporal differences in IAC components revealed by MS, IAC proteins were visualised during nocodazole washout32. Upon nocodazole washout, the area of the ventral cell surface covered by α5 or β1 integrin did not change (Fig. 8, Supplementary Fig. 7). In support of the different rates of loss of IAC components, the decrease in vinculin (30 min; Fig. 8) was delayed compared with the loss of zyxin (10 min; Fig. 8) and other adhesion molecules (phospho-FAKY397, 10 min; paxillin and phospho-paxillinY118, 15 min; Supplementary Fig. 7). These data validate the findings obtained using MS that indicate different adhesion molecules display distinct temporal profiles during IAC disassembly.


Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

Horton ER, Byron A, Askari JA, Ng DH, Millon-Frémillon A, Robertson J, Koper EJ, Paul NR, Warwood S, Knight D, Humphries JD, Humphries MJ - Nat. Cell Biol. (2015)

Changes in consensus adhesome components during IAC disassembly. (a) HFF cells treated with DMSO, 10 μM nocodazole or after nocodazole removal at different times were stained for vinculin, zyxin and α5 integrin. Representative images are shown. Scale bars, 20 μm. (b–d) Quantification of images in a. Vinculin, zyxin and α5 integrin levels were quantified as a proportion of total cell area. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box) and 5th and 95th percentiles (whiskers) (n = 10 cells per condition from one independent experiment). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test with Dunn’s post hoc correction (comparisons with the nocodazole treatment group are shown; see Supplementary Table 15 for statistics source data).
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Related In: Results  -  Collection

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Figure 8: Changes in consensus adhesome components during IAC disassembly. (a) HFF cells treated with DMSO, 10 μM nocodazole or after nocodazole removal at different times were stained for vinculin, zyxin and α5 integrin. Representative images are shown. Scale bars, 20 μm. (b–d) Quantification of images in a. Vinculin, zyxin and α5 integrin levels were quantified as a proportion of total cell area. Box-and-whisker plot shows the median (line), mean (plus sign), 25th and 75th percentiles (box) and 5th and 95th percentiles (whiskers) (n = 10 cells per condition from one independent experiment). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test with Dunn’s post hoc correction (comparisons with the nocodazole treatment group are shown; see Supplementary Table 15 for statistics source data).
Mentions: To confirm the temporal differences in IAC components revealed by MS, IAC proteins were visualised during nocodazole washout32. Upon nocodazole washout, the area of the ventral cell surface covered by α5 or β1 integrin did not change (Fig. 8, Supplementary Fig. 7). In support of the different rates of loss of IAC components, the decrease in vinculin (30 min; Fig. 8) was delayed compared with the loss of zyxin (10 min; Fig. 8) and other adhesion molecules (phospho-FAKY397, 10 min; paxillin and phospho-paxillinY118, 15 min; Supplementary Fig. 7). These data validate the findings obtained using MS that indicate different adhesion molecules display distinct temporal profiles during IAC disassembly.

Bottom Line: Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome.Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins.The definition of this consensus view of integrin adhesome components provides a resource for the research community.

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 receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

Show MeSH
Related in: MedlinePlus