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Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

Bloch W, Fan Y, Han J, Xue S, Schöneberg T, Ji G, Lu ZJ, Walther M, Fässler R, Hescheler J, Addicks K, Fleischmann BK - J. Cell Biol. (2001)

Bottom Line: Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes.A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling.We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy I, University of Cologne, Germany.

ABSTRACT
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

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Gαi, talin, and vinculin colocalize in wt cells. (a and b) Deconvolution microscopy of ES cell–derived cells exhibited no overlap of Gαi and β1 integrins (a), but colocalization of Gαi and talin (b). (c) Higher magnification showing colocalization of Gαi and talin. (d and e) Immunogold staining for Gαi and vinculin in murine embryonic cardiomyocytes (d) and in adult mouse heart (e). Vinculin and Gαi were labeled with 5- and 15-nm gold particles, respectively. The distance between pairs of gold particles was 40–150 nm. Bars: (a and b) 6 μm; (c) 2.5 μm; (d and e) 90 nm.
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fig6: Gαi, talin, and vinculin colocalize in wt cells. (a and b) Deconvolution microscopy of ES cell–derived cells exhibited no overlap of Gαi and β1 integrins (a), but colocalization of Gαi and talin (b). (c) Higher magnification showing colocalization of Gαi and talin. (d and e) Immunogold staining for Gαi and vinculin in murine embryonic cardiomyocytes (d) and in adult mouse heart (e). Vinculin and Gαi were labeled with 5- and 15-nm gold particles, respectively. The distance between pairs of gold particles was 40–150 nm. Bars: (a and b) 6 μm; (c) 2.5 μm; (d and e) 90 nm.

Mentions: A similar network-like distribution of Gαi has been reported for β1 integrin focal adhesion–associated molecules such as talin and vinculin (Kostin et al., 1998; Imanaka-Yoshida et al., 1999). We have identified a unique architecture for Gαi in the β1 integrin−/− cells and found that talin and vinculin exhibit a similar diffuse distribution as Gαi (data not shown). We subsequently analyzed the interaction between β1 integrins, talin, and vinculin with Gαi using deconvolution and electron microscopy. These approaches failed to demonstrate a close colocalization of β1 integrins and Gαi (Fig. 6 a). Deconvolution microscopy did reveal, however, spatial colocalization between Gαi and both vinculin and talin (Fig. 6, b and c) in wt cells. Electron microscopy of double immunogold–labeled cells further revealed a relatively close colocalization between Gαi and vinculin (40–150 nm) in embryonic (Fig. 6 d) and adult mouse/rat cardiomyocytes (Fig. 6 e), suggesting that direct protein–protein interaction is highly unlikely.


Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

Bloch W, Fan Y, Han J, Xue S, Schöneberg T, Ji G, Lu ZJ, Walther M, Fässler R, Hescheler J, Addicks K, Fleischmann BK - J. Cell Biol. (2001)

Gαi, talin, and vinculin colocalize in wt cells. (a and b) Deconvolution microscopy of ES cell–derived cells exhibited no overlap of Gαi and β1 integrins (a), but colocalization of Gαi and talin (b). (c) Higher magnification showing colocalization of Gαi and talin. (d and e) Immunogold staining for Gαi and vinculin in murine embryonic cardiomyocytes (d) and in adult mouse heart (e). Vinculin and Gαi were labeled with 5- and 15-nm gold particles, respectively. The distance between pairs of gold particles was 40–150 nm. Bars: (a and b) 6 μm; (c) 2.5 μm; (d and e) 90 nm.
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Related In: Results  -  Collection

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fig6: Gαi, talin, and vinculin colocalize in wt cells. (a and b) Deconvolution microscopy of ES cell–derived cells exhibited no overlap of Gαi and β1 integrins (a), but colocalization of Gαi and talin (b). (c) Higher magnification showing colocalization of Gαi and talin. (d and e) Immunogold staining for Gαi and vinculin in murine embryonic cardiomyocytes (d) and in adult mouse heart (e). Vinculin and Gαi were labeled with 5- and 15-nm gold particles, respectively. The distance between pairs of gold particles was 40–150 nm. Bars: (a and b) 6 μm; (c) 2.5 μm; (d and e) 90 nm.
Mentions: A similar network-like distribution of Gαi has been reported for β1 integrin focal adhesion–associated molecules such as talin and vinculin (Kostin et al., 1998; Imanaka-Yoshida et al., 1999). We have identified a unique architecture for Gαi in the β1 integrin−/− cells and found that talin and vinculin exhibit a similar diffuse distribution as Gαi (data not shown). We subsequently analyzed the interaction between β1 integrins, talin, and vinculin with Gαi using deconvolution and electron microscopy. These approaches failed to demonstrate a close colocalization of β1 integrins and Gαi (Fig. 6 a). Deconvolution microscopy did reveal, however, spatial colocalization between Gαi and both vinculin and talin (Fig. 6, b and c) in wt cells. Electron microscopy of double immunogold–labeled cells further revealed a relatively close colocalization between Gαi and vinculin (40–150 nm) in embryonic (Fig. 6 d) and adult mouse/rat cardiomyocytes (Fig. 6 e), suggesting that direct protein–protein interaction is highly unlikely.

Bottom Line: Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes.A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling.We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy I, University of Cologne, Germany.

ABSTRACT
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Show MeSH
Related in: MedlinePlus