Limits...
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.

Show MeSH

Related in: MedlinePlus

Absence of muscarinic inhibition of ISO-prestimulated ICa in β1 integrin−/− ES cell–derived cardiomyocytes. (a) ICa in a representative wt LDS ES cell–derived cardiomyocyte demonstrated prominent stimulation by ISO (0.1 μM) and subsequent inhibition by CCh. The CCh effect was reversed by ISO washout. (b) ICa in a representative β1 integrin−/− ES cell–derived LDS cardiomyocyte displayed strong stimulation by ISO; however, no inhibition by subsequent application of CCh was observed. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced depression of ISO-prestimulated ICa. (d) ICa density in wt cells with ISO and CCh response. (e) ICa density in β1 integrin−/− cells exposed to ISO and CCh. (f–g) Immunostaining for Gαi expression in wt (f) and β1 integrin−/− ES cell–derived cardiomyocytes (g). *Indicates statistical significance (paired t test, p-value < 0.05). Bar, 25 μM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196465&req=5

fig2: Absence of muscarinic inhibition of ISO-prestimulated ICa in β1 integrin−/− ES cell–derived cardiomyocytes. (a) ICa in a representative wt LDS ES cell–derived cardiomyocyte demonstrated prominent stimulation by ISO (0.1 μM) and subsequent inhibition by CCh. The CCh effect was reversed by ISO washout. (b) ICa in a representative β1 integrin−/− ES cell–derived LDS cardiomyocyte displayed strong stimulation by ISO; however, no inhibition by subsequent application of CCh was observed. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced depression of ISO-prestimulated ICa. (d) ICa density in wt cells with ISO and CCh response. (e) ICa density in β1 integrin−/− cells exposed to ISO and CCh. (f–g) Immunostaining for Gαi expression in wt (f) and β1 integrin−/− ES cell–derived cardiomyocytes (g). *Indicates statistical significance (paired t test, p-value < 0.05). Bar, 25 μM.

Mentions: The absence of muscarinic signaling could be explained by perturbed intracellular signaling components including AC, cAMP, or protein kinase A. Therefore, we determined whether additional regulatory pathways, and in particular the β-adrenoceptor–mediated stimulation of ICa, were defective in β1 integrin−/− cardiomyocytes. All wild-type (wt) late developmental stage (LDS) cardiomyocytes exhibited an increased current density (41.4 ± 6.8%, n = 11; Fig. 2 , a and d) after prestimulation with the β-adrenoceptor agonist isoprenaline (ISO) (0.1 μM). Similarly, ISO increased the ICa density by 64.6 ± 12% (n = 20) in all β1 integrin−/− cardiomyocytes tested (Fig. 2, b and e). This ISO-induced upregulation of ICa was accompanied by a slight left shift in the I/V relationship (n = 5). However, superfusion with varying concentrations (1–10 μM) of CCh after β-adrenergic prestimulation did not suppress ICa in these cardiomyocytes (n = 20; Fig. 2, c and e). By contrast, CCh-inhibited ISO prestimulated ICa by 37.3 ± 7% (n = 11; Fig. 2 c) in 82% of wt LDS cells.


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)

Absence of muscarinic inhibition of ISO-prestimulated ICa in β1 integrin−/− ES cell–derived cardiomyocytes. (a) ICa in a representative wt LDS ES cell–derived cardiomyocyte demonstrated prominent stimulation by ISO (0.1 μM) and subsequent inhibition by CCh. The CCh effect was reversed by ISO washout. (b) ICa in a representative β1 integrin−/− ES cell–derived LDS cardiomyocyte displayed strong stimulation by ISO; however, no inhibition by subsequent application of CCh was observed. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced depression of ISO-prestimulated ICa. (d) ICa density in wt cells with ISO and CCh response. (e) ICa density in β1 integrin−/− cells exposed to ISO and CCh. (f–g) Immunostaining for Gαi expression in wt (f) and β1 integrin−/− ES cell–derived cardiomyocytes (g). *Indicates statistical significance (paired t test, p-value < 0.05). Bar, 25 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196465&req=5

fig2: Absence of muscarinic inhibition of ISO-prestimulated ICa in β1 integrin−/− ES cell–derived cardiomyocytes. (a) ICa in a representative wt LDS ES cell–derived cardiomyocyte demonstrated prominent stimulation by ISO (0.1 μM) and subsequent inhibition by CCh. The CCh effect was reversed by ISO washout. (b) ICa in a representative β1 integrin−/− ES cell–derived LDS cardiomyocyte displayed strong stimulation by ISO; however, no inhibition by subsequent application of CCh was observed. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced depression of ISO-prestimulated ICa. (d) ICa density in wt cells with ISO and CCh response. (e) ICa density in β1 integrin−/− cells exposed to ISO and CCh. (f–g) Immunostaining for Gαi expression in wt (f) and β1 integrin−/− ES cell–derived cardiomyocytes (g). *Indicates statistical significance (paired t test, p-value < 0.05). Bar, 25 μM.
Mentions: The absence of muscarinic signaling could be explained by perturbed intracellular signaling components including AC, cAMP, or protein kinase A. Therefore, we determined whether additional regulatory pathways, and in particular the β-adrenoceptor–mediated stimulation of ICa, were defective in β1 integrin−/− cardiomyocytes. All wild-type (wt) late developmental stage (LDS) cardiomyocytes exhibited an increased current density (41.4 ± 6.8%, n = 11; Fig. 2 , a and d) after prestimulation with the β-adrenoceptor agonist isoprenaline (ISO) (0.1 μM). Similarly, ISO increased the ICa density by 64.6 ± 12% (n = 20) in all β1 integrin−/− cardiomyocytes tested (Fig. 2, b and e). This ISO-induced upregulation of ICa was accompanied by a slight left shift in the I/V relationship (n = 5). However, superfusion with varying concentrations (1–10 μM) of CCh after β-adrenergic prestimulation did not suppress ICa in these cardiomyocytes (n = 20; Fig. 2, c and e). By contrast, CCh-inhibited ISO prestimulated ICa by 37.3 ± 7% (n = 11; Fig. 2 c) in 82% of wt LDS cells.

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