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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 ICa is restored in β1 integrin rescue ES cell–derived cardiomyocytes. Other signaling cascades are intact in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Basal ICa in a representative β1 integrin rescue EDS cardiomyocyte was inhibited by CCh. The CCh effect could be reversed by wash out. (b) ISO-prestimulated ICa in a representative β1 integrin rescue LDS cardiomyocyte was depressed by CCh. The CCh effect was reversed by ISO washout. (c) Percentage of β1 integrin rescue EDS and LDS cardiomyocytes responding to application of CCh or ISO and CCh. (d) Statistical analysis of ICa density in β1 integrin rescue EDS and LDS cardiomyocytes with CCh or ISO and CCh responses. (e) The NOS inhibitor L-NMMA (0.2 mM) augmented basal ICa in β1 integrin−/− EDS cardiomyocytes. This effect could be partially reversed by agonist wash out. (f) Atrial natriuretic peptide (ANP; 0.1 μM) induced a prominent depression of ICa in β1 integrin−/− LDS cells. *Indicates statistically significant difference (paired t test, p-value < 0.05).
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fig3: Muscarinic modulation of ICa is restored in β1 integrin rescue ES cell–derived cardiomyocytes. Other signaling cascades are intact in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Basal ICa in a representative β1 integrin rescue EDS cardiomyocyte was inhibited by CCh. The CCh effect could be reversed by wash out. (b) ISO-prestimulated ICa in a representative β1 integrin rescue LDS cardiomyocyte was depressed by CCh. The CCh effect was reversed by ISO washout. (c) Percentage of β1 integrin rescue EDS and LDS cardiomyocytes responding to application of CCh or ISO and CCh. (d) Statistical analysis of ICa density in β1 integrin rescue EDS and LDS cardiomyocytes with CCh or ISO and CCh responses. (e) The NOS inhibitor L-NMMA (0.2 mM) augmented basal ICa in β1 integrin−/− EDS cardiomyocytes. This effect could be partially reversed by agonist wash out. (f) Atrial natriuretic peptide (ANP; 0.1 μM) induced a prominent depression of ICa in β1 integrin−/− LDS cells. *Indicates statistically significant difference (paired t test, p-value < 0.05).

Mentions: To confirm that the muscarinic signaling defect was evoked specifically by the absence of the β1 integrin gene, we next investigated the hormonal modulation of ICa in β1 integrin rescue ES cell–derived cardiomyocytes. As depicted in Fig. 3 a, muscarinic receptor activation inhibited basal ICa by 32.3 ± 6% (Fig. 3 d) in almost half (42%, n = 19; Fig. 3 d) of the EDS cells. In 36% (n = 33; Fig. 3 c) of the LDS β1 integrin rescue cardiomyocytes, CCh (1-10 μM) suppressed ISO prestimulated ICa by 21 ± 6% (Fig. 3, b and d). The reduction in muscarinic inhibition and altered morphology of β1 integrin reconstituted cells (see Fig. 5 i) suggests that the rescue was incomplete.


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 ICa is restored in β1 integrin rescue ES cell–derived cardiomyocytes. Other signaling cascades are intact in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Basal ICa in a representative β1 integrin rescue EDS cardiomyocyte was inhibited by CCh. The CCh effect could be reversed by wash out. (b) ISO-prestimulated ICa in a representative β1 integrin rescue LDS cardiomyocyte was depressed by CCh. The CCh effect was reversed by ISO washout. (c) Percentage of β1 integrin rescue EDS and LDS cardiomyocytes responding to application of CCh or ISO and CCh. (d) Statistical analysis of ICa density in β1 integrin rescue EDS and LDS cardiomyocytes with CCh or ISO and CCh responses. (e) The NOS inhibitor L-NMMA (0.2 mM) augmented basal ICa in β1 integrin−/− EDS cardiomyocytes. This effect could be partially reversed by agonist wash out. (f) Atrial natriuretic peptide (ANP; 0.1 μM) induced a prominent depression of ICa in β1 integrin−/− LDS cells. *Indicates statistically significant difference (paired t test, p-value < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Muscarinic modulation of ICa is restored in β1 integrin rescue ES cell–derived cardiomyocytes. Other signaling cascades are intact in β1 integrin−/− ES cell–derived cardiomyocytes. (a) Basal ICa in a representative β1 integrin rescue EDS cardiomyocyte was inhibited by CCh. The CCh effect could be reversed by wash out. (b) ISO-prestimulated ICa in a representative β1 integrin rescue LDS cardiomyocyte was depressed by CCh. The CCh effect was reversed by ISO washout. (c) Percentage of β1 integrin rescue EDS and LDS cardiomyocytes responding to application of CCh or ISO and CCh. (d) Statistical analysis of ICa density in β1 integrin rescue EDS and LDS cardiomyocytes with CCh or ISO and CCh responses. (e) The NOS inhibitor L-NMMA (0.2 mM) augmented basal ICa in β1 integrin−/− EDS cardiomyocytes. This effect could be partially reversed by agonist wash out. (f) Atrial natriuretic peptide (ANP; 0.1 μM) induced a prominent depression of ICa in β1 integrin−/− LDS cells. *Indicates statistically significant difference (paired t test, p-value < 0.05).
Mentions: To confirm that the muscarinic signaling defect was evoked specifically by the absence of the β1 integrin gene, we next investigated the hormonal modulation of ICa in β1 integrin rescue ES cell–derived cardiomyocytes. As depicted in Fig. 3 a, muscarinic receptor activation inhibited basal ICa by 32.3 ± 6% (Fig. 3 d) in almost half (42%, n = 19; Fig. 3 d) of the EDS cells. In 36% (n = 33; Fig. 3 c) of the LDS β1 integrin rescue cardiomyocytes, CCh (1-10 μM) suppressed ISO prestimulated ICa by 21 ± 6% (Fig. 3, b and d). The reduction in muscarinic inhibition and altered morphology of β1 integrin reconstituted cells (see Fig. 5 i) suggests that the rescue was incomplete.

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