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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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Caldesmon and Rsu-1 localisation in IACs. (a,b) U2OS cells were spread on FN for 2 h and visualised using antibodies against caldesmon (green) (a) and Rsu-1 (green) (b). IACs were visualised by immunofluorescence staining for vinculin (red) and the actin cytoskeleton was visualised by staining with fluorophore-conjugated phalloidin (blue). Graphs show fluorescence intensity values for each channel across line segments in corresponding zoomed areas above each graph. In addition, colocalisation with vinculin-positive areas was quantified for caldesmon (MOC27 = 0.51 ± 0.19) and Rsu-1 (MOC = 0.98 ± 0.03). Values are mean ± s.d. (n = 20 cells from one independent experiment; see Supplementary Table 15 for source data). Scale bars, 20 μm.
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Figure 5: Caldesmon and Rsu-1 localisation in IACs. (a,b) U2OS cells were spread on FN for 2 h and visualised using antibodies against caldesmon (green) (a) and Rsu-1 (green) (b). IACs were visualised by immunofluorescence staining for vinculin (red) and the actin cytoskeleton was visualised by staining with fluorophore-conjugated phalloidin (blue). Graphs show fluorescence intensity values for each channel across line segments in corresponding zoomed areas above each graph. In addition, colocalisation with vinculin-positive areas was quantified for caldesmon (MOC27 = 0.51 ± 0.19) and Rsu-1 (MOC = 0.98 ± 0.03). Values are mean ± s.d. (n = 20 cells from one independent experiment; see Supplementary Table 15 for source data). Scale bars, 20 μm.

Mentions: To verify that underappreciated proteins identified in the consensus adhesome localise to IACs, we visualised by immunofluorescence two consensus adhesome proteins that are not literature-curated adhesome4 members (Fig. 5). Caldesmon26 localised to actin within vinculin-positive areas (Fig. 5a; Mander’s overlap coefficient27 (MOC) = 0.51 ± 0.19, mean ± s.d.). Rsu-1, which has been reported to associate with IACs in other cell types28, co-localised with vinculin (Fig. 5b; MOC = 0.98 ± 0.03, mean ± s.d.). These data confirm IAC localisation of caldesmon and Rsu-1 and suggest that underappreciated consensus adhesome proteins may participate in regulating the integrin-actin connection.


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)

Caldesmon and Rsu-1 localisation in IACs. (a,b) U2OS cells were spread on FN for 2 h and visualised using antibodies against caldesmon (green) (a) and Rsu-1 (green) (b). IACs were visualised by immunofluorescence staining for vinculin (red) and the actin cytoskeleton was visualised by staining with fluorophore-conjugated phalloidin (blue). Graphs show fluorescence intensity values for each channel across line segments in corresponding zoomed areas above each graph. In addition, colocalisation with vinculin-positive areas was quantified for caldesmon (MOC27 = 0.51 ± 0.19) and Rsu-1 (MOC = 0.98 ± 0.03). Values are mean ± s.d. (n = 20 cells from one independent experiment; see Supplementary Table 15 for source data). Scale bars, 20 μm.
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Related In: Results  -  Collection

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Figure 5: Caldesmon and Rsu-1 localisation in IACs. (a,b) U2OS cells were spread on FN for 2 h and visualised using antibodies against caldesmon (green) (a) and Rsu-1 (green) (b). IACs were visualised by immunofluorescence staining for vinculin (red) and the actin cytoskeleton was visualised by staining with fluorophore-conjugated phalloidin (blue). Graphs show fluorescence intensity values for each channel across line segments in corresponding zoomed areas above each graph. In addition, colocalisation with vinculin-positive areas was quantified for caldesmon (MOC27 = 0.51 ± 0.19) and Rsu-1 (MOC = 0.98 ± 0.03). Values are mean ± s.d. (n = 20 cells from one independent experiment; see Supplementary Table 15 for source data). Scale bars, 20 μm.
Mentions: To verify that underappreciated proteins identified in the consensus adhesome localise to IACs, we visualised by immunofluorescence two consensus adhesome proteins that are not literature-curated adhesome4 members (Fig. 5). Caldesmon26 localised to actin within vinculin-positive areas (Fig. 5a; Mander’s overlap coefficient27 (MOC) = 0.51 ± 0.19, mean ± s.d.). Rsu-1, which has been reported to associate with IACs in other cell types28, co-localised with vinculin (Fig. 5b; MOC = 0.98 ± 0.03, mean ± s.d.). These data confirm IAC localisation of caldesmon and Rsu-1 and suggest that underappreciated consensus adhesome proteins may participate in regulating the integrin-actin connection.

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