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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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Muscarinic modulation of basal ICa is absent in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Time course of peak ICa in a representative wt EDS ES cell–derived cardiomyocyte. Prominent inhibition of basal ICa was observed after application of carbachol (1 μM). The CCh effect could be reversed by washout. Each data point in the time course was evoked by a 20-ms depolarization from a holding potential of −50 mV to a test potential of 0 mV. The upper trace indicates the holding current. (b) A representative β1 integrin−/− EDS cardiomyocyte demonstrates the absence of the CCh-induced inhibition of basal ICa (50 ms depolarization from −50 mV to 0 mV). The time of current recordings (1 and 2, inset) is indicated. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced inhibition of basal ICa. (d) ICa density in wt cells with CCh response. (e) ICa density in β1 integrin−/− cells in absence and presence of CCh. (f) Traces (left) and I/V relationship (right) of ICa recorded in an ES cell–derived EDS β1 integrin−/− cardiomyocyte (1, control; 2, in presence of CCh). ICa was evoked by 50-ms depolarizations from −40 mV to +40 mV in 10-mV increments (holding potential, −50 mV). *Indicates statistical significance (paired t test, p-value < 0.05).
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fig1: Muscarinic modulation of basal ICa is absent in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Time course of peak ICa in a representative wt EDS ES cell–derived cardiomyocyte. Prominent inhibition of basal ICa was observed after application of carbachol (1 μM). The CCh effect could be reversed by washout. Each data point in the time course was evoked by a 20-ms depolarization from a holding potential of −50 mV to a test potential of 0 mV. The upper trace indicates the holding current. (b) A representative β1 integrin−/− EDS cardiomyocyte demonstrates the absence of the CCh-induced inhibition of basal ICa (50 ms depolarization from −50 mV to 0 mV). The time of current recordings (1 and 2, inset) is indicated. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced inhibition of basal ICa. (d) ICa density in wt cells with CCh response. (e) ICa density in β1 integrin−/− cells in absence and presence of CCh. (f) Traces (left) and I/V relationship (right) of ICa recorded in an ES cell–derived EDS β1 integrin−/− cardiomyocyte (1, control; 2, in presence of CCh). ICa was evoked by 50-ms depolarizations from −40 mV to +40 mV in 10-mV increments (holding potential, −50 mV). *Indicates statistical significance (paired t test, p-value < 0.05).

Mentions: As previously reported (Ji et al., 1999), prominent inhibition (44.7 ± 8%; Fig. 1 d) of basal ICa density was observed upon application of the muscarinic agonist carbachol (CCh) (1 μM) (Fig. 1 a) in a large percentage of early developmental stage (EDS) cardiomyocytes (69%, n = 13; Fig. 1 c). Similarly, in the β1 integrin+/− ES cell–derived EDS cardiomyocytes, basal ICa density was depressed in half of the cells tested by 44 ± 8% (n = 10). In contrast, β1 integrin−/− cells did not exhibit CCh-mediated inhibition of basal ICa (n = 37) (Fig. 1, b, c, and e). To rule out that a shift in the voltage dependence of ICa underlies the CCh effect after the step to 0 mV, the current–voltage (I/V) relationship was defined in the presence and absence of CCh (n = 5). As depicted in Fig. 1 f, no CCh-mediated depression of ICa was observed at the chosen potentials.


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)

Muscarinic modulation of basal ICa is absent in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Time course of peak ICa in a representative wt EDS ES cell–derived cardiomyocyte. Prominent inhibition of basal ICa was observed after application of carbachol (1 μM). The CCh effect could be reversed by washout. Each data point in the time course was evoked by a 20-ms depolarization from a holding potential of −50 mV to a test potential of 0 mV. The upper trace indicates the holding current. (b) A representative β1 integrin−/− EDS cardiomyocyte demonstrates the absence of the CCh-induced inhibition of basal ICa (50 ms depolarization from −50 mV to 0 mV). The time of current recordings (1 and 2, inset) is indicated. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced inhibition of basal ICa. (d) ICa density in wt cells with CCh response. (e) ICa density in β1 integrin−/− cells in absence and presence of CCh. (f) Traces (left) and I/V relationship (right) of ICa recorded in an ES cell–derived EDS β1 integrin−/− cardiomyocyte (1, control; 2, in presence of CCh). ICa was evoked by 50-ms depolarizations from −40 mV to +40 mV in 10-mV increments (holding potential, −50 mV). *Indicates statistical significance (paired t test, p-value < 0.05).
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Related In: Results  -  Collection

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fig1: Muscarinic modulation of basal ICa is absent in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Time course of peak ICa in a representative wt EDS ES cell–derived cardiomyocyte. Prominent inhibition of basal ICa was observed after application of carbachol (1 μM). The CCh effect could be reversed by washout. Each data point in the time course was evoked by a 20-ms depolarization from a holding potential of −50 mV to a test potential of 0 mV. The upper trace indicates the holding current. (b) A representative β1 integrin−/− EDS cardiomyocyte demonstrates the absence of the CCh-induced inhibition of basal ICa (50 ms depolarization from −50 mV to 0 mV). The time of current recordings (1 and 2, inset) is indicated. (c) Percentage of wt and β1 integrin−/− cardiomyocytes displaying CCh-induced inhibition of basal ICa. (d) ICa density in wt cells with CCh response. (e) ICa density in β1 integrin−/− cells in absence and presence of CCh. (f) Traces (left) and I/V relationship (right) of ICa recorded in an ES cell–derived EDS β1 integrin−/− cardiomyocyte (1, control; 2, in presence of CCh). ICa was evoked by 50-ms depolarizations from −40 mV to +40 mV in 10-mV increments (holding potential, −50 mV). *Indicates statistical significance (paired t test, p-value < 0.05).
Mentions: As previously reported (Ji et al., 1999), prominent inhibition (44.7 ± 8%; Fig. 1 d) of basal ICa density was observed upon application of the muscarinic agonist carbachol (CCh) (1 μM) (Fig. 1 a) in a large percentage of early developmental stage (EDS) cardiomyocytes (69%, n = 13; Fig. 1 c). Similarly, in the β1 integrin+/− ES cell–derived EDS cardiomyocytes, basal ICa density was depressed in half of the cells tested by 44 ± 8% (n = 10). In contrast, β1 integrin−/− cells did not exhibit CCh-mediated inhibition of basal ICa (n = 37) (Fig. 1, b, c, and e). To rule out that a shift in the voltage dependence of ICa underlies the CCh effect after the step to 0 mV, the current–voltage (I/V) relationship was defined in the presence and absence of CCh (n = 5). As depicted in Fig. 1 f, no CCh-mediated depression of ICa was observed at the chosen potentials.

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