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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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Temporal profiling of the consensus adhesome during IAC assembly. IACs were isolated from K562 cells in biological duplicate after 3, 9 and 32 min incubation with FN-coated beads and analysed by MS (data are from 2 independent experiments; see Supplementary Table 11). Throughout IAC assembly, 39 of the 60 consensus adhesome proteins were identified and were analysed by unsupervised hierarchical clustering, revealing distinct temporal profiles of protein recruitment to IACs. Six clusters, labelled A1–6, were chosen on the basis of a Pearson correlation threshold greater than 0.9 and are indicated by blue and green bars. Clusters are shown alongside corresponding profile plots, with the mean temporal profile for each cluster indicated by a red line. Quantitative heat map displays mean spectral counts as a proportion of the maximum spectral count for each given protein. Proteins are labelled with gene names for clarity. Proteins also identified during IAC disassembly (Fig. 7, Supplementary Table 12) are indicated by an asterisk. Literature-curated adhesome4 proteins and their isoforms are in bold. Proteins able to bind actin or integrin are indicated by black bars.
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Figure 6: Temporal profiling of the consensus adhesome during IAC assembly. IACs were isolated from K562 cells in biological duplicate after 3, 9 and 32 min incubation with FN-coated beads and analysed by MS (data are from 2 independent experiments; see Supplementary Table 11). Throughout IAC assembly, 39 of the 60 consensus adhesome proteins were identified and were analysed by unsupervised hierarchical clustering, revealing distinct temporal profiles of protein recruitment to IACs. Six clusters, labelled A1–6, were chosen on the basis of a Pearson correlation threshold greater than 0.9 and are indicated by blue and green bars. Clusters are shown alongside corresponding profile plots, with the mean temporal profile for each cluster indicated by a red line. Quantitative heat map displays mean spectral counts as a proportion of the maximum spectral count for each given protein. Proteins are labelled with gene names for clarity. Proteins also identified during IAC disassembly (Fig. 7, Supplementary Table 12) are indicated by an asterisk. Literature-curated adhesome4 proteins and their isoforms are in bold. Proteins able to bind actin or integrin are indicated by black bars.

Mentions: To examine the core adhesion machinery, hierarchical clustering revealed that different consensus adhesome components display distinct dynamics (Figs. 6, 7). β1, α5 and αV integrins reached maximum abundance by 30 minutes in this system. Integrins were relatively stable throughout IAC disassembly, and this was also the case for other cell-surface molecules (e.g. annexin A1, transglutaminase-2 and the CD98 heavy chain (SLC3A2)). Most consensus components, although distributed in different clusters (Fig. 6), were detected in high abundance late in IAC assembly here, indicating distinct dynamics of protein recruitment. Integrin-binding proteins decreased during IAC disassembly but with different kinetics (clusters D1, D4; Fig. 7). Most of the adaptors in the consensus adhesome were almost completely absent from IACs after 15 minutes (cluster D1, Fig. 7), while 13 of the 17 actin-binding proteins, five of which were integrin-binding, decreased in abundance less rapidly (cluster D4, Fig. 7). These data suggest that adaptor proteins located between actin and integrins are lost earlier and at a faster rate than actin-binding proteins and that the integrin-actin linkage is disrupted late 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)

Temporal profiling of the consensus adhesome during IAC assembly. IACs were isolated from K562 cells in biological duplicate after 3, 9 and 32 min incubation with FN-coated beads and analysed by MS (data are from 2 independent experiments; see Supplementary Table 11). Throughout IAC assembly, 39 of the 60 consensus adhesome proteins were identified and were analysed by unsupervised hierarchical clustering, revealing distinct temporal profiles of protein recruitment to IACs. Six clusters, labelled A1–6, were chosen on the basis of a Pearson correlation threshold greater than 0.9 and are indicated by blue and green bars. Clusters are shown alongside corresponding profile plots, with the mean temporal profile for each cluster indicated by a red line. Quantitative heat map displays mean spectral counts as a proportion of the maximum spectral count for each given protein. Proteins are labelled with gene names for clarity. Proteins also identified during IAC disassembly (Fig. 7, Supplementary Table 12) are indicated by an asterisk. Literature-curated adhesome4 proteins and their isoforms are in bold. Proteins able to bind actin or integrin are indicated by black bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4663675&req=5

Figure 6: Temporal profiling of the consensus adhesome during IAC assembly. IACs were isolated from K562 cells in biological duplicate after 3, 9 and 32 min incubation with FN-coated beads and analysed by MS (data are from 2 independent experiments; see Supplementary Table 11). Throughout IAC assembly, 39 of the 60 consensus adhesome proteins were identified and were analysed by unsupervised hierarchical clustering, revealing distinct temporal profiles of protein recruitment to IACs. Six clusters, labelled A1–6, were chosen on the basis of a Pearson correlation threshold greater than 0.9 and are indicated by blue and green bars. Clusters are shown alongside corresponding profile plots, with the mean temporal profile for each cluster indicated by a red line. Quantitative heat map displays mean spectral counts as a proportion of the maximum spectral count for each given protein. Proteins are labelled with gene names for clarity. Proteins also identified during IAC disassembly (Fig. 7, Supplementary Table 12) are indicated by an asterisk. Literature-curated adhesome4 proteins and their isoforms are in bold. Proteins able to bind actin or integrin are indicated by black bars.
Mentions: To examine the core adhesion machinery, hierarchical clustering revealed that different consensus adhesome components display distinct dynamics (Figs. 6, 7). β1, α5 and αV integrins reached maximum abundance by 30 minutes in this system. Integrins were relatively stable throughout IAC disassembly, and this was also the case for other cell-surface molecules (e.g. annexin A1, transglutaminase-2 and the CD98 heavy chain (SLC3A2)). Most consensus components, although distributed in different clusters (Fig. 6), were detected in high abundance late in IAC assembly here, indicating distinct dynamics of protein recruitment. Integrin-binding proteins decreased during IAC disassembly but with different kinetics (clusters D1, D4; Fig. 7). Most of the adaptors in the consensus adhesome were almost completely absent from IACs after 15 minutes (cluster D1, Fig. 7), while 13 of the 17 actin-binding proteins, five of which were integrin-binding, decreased in abundance less rapidly (cluster D4, Fig. 7). These data suggest that adaptor proteins located between actin and integrins are lost earlier and at a faster rate than actin-binding proteins and that the integrin-actin linkage is disrupted late 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