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Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta.

Krylova O, Messenger MJ, Salinas PC - J. Cell Biol. (2000)

Bottom Line: The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta.These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics.This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

View Article: PubMed Central - PubMed

Affiliation: The Randall Institute, King's College London, London, United Kingdom.

ABSTRACT
Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta. These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

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DVL-1 prevents the loss of stable microtubules in nocodazole treated COS-7 cells. COS-7 expressing DVL-HA (A–C and G–I) or GFP (D–F) were examined for the level of MTs (B, E, and H). COS-7 cells expressing DVL-HA (A) have a normal level of acetylated MTs (B and C). COS-7 cells expressing GFP (D–F) or DVL-HA (G–I) were treated with nocodazole to depolymerize MTs. GFP-expressing cells have no acetylated MTs, similar to neighboring untransfected cells (D–F). Expression of DVL-HA prevents MT depolymerization by nocodazole (G–I). Arrows denote MTs resistant to nocodazole in the cell expressing DVL-HA. *Neighboring untransfected cell, which has lost stable MTs. (Inset) Small vesicle-like structures are present on some MTs. Bar, 50 μM.
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Figure 5: DVL-1 prevents the loss of stable microtubules in nocodazole treated COS-7 cells. COS-7 expressing DVL-HA (A–C and G–I) or GFP (D–F) were examined for the level of MTs (B, E, and H). COS-7 cells expressing DVL-HA (A) have a normal level of acetylated MTs (B and C). COS-7 cells expressing GFP (D–F) or DVL-HA (G–I) were treated with nocodazole to depolymerize MTs. GFP-expressing cells have no acetylated MTs, similar to neighboring untransfected cells (D–F). Expression of DVL-HA prevents MT depolymerization by nocodazole (G–I). Arrows denote MTs resistant to nocodazole in the cell expressing DVL-HA. *Neighboring untransfected cell, which has lost stable MTs. (Inset) Small vesicle-like structures are present on some MTs. Bar, 50 μM.

Mentions: The interaction of endogenous DVL-1 with neuronal MTs suggests its possible role in controlling MT organization or, alternatively, that MTs are just used to transport DVL-1 along the axon. We obtained evidence in favor of the former possibility when we examined the effect of expression of Dvl-HA on MTs in COS cells. Consistent with previous studies, DVL-HA forms vesicle-like structures in transfected cells (Fig. 5A and Fig. G; Fagotto et al. 1999; Smalley et al. 1999). Cells expressing DVL-1 have similar levels of acetylated MTs than neighboring nonexpressing cells (Fig. 5, A–C). Similar results were obtained with DVL-1 without the epitope tag HA (data not shown). We then examined the effect of DVL-1 on MTs in cells that have been treated with 10 μM nocodazole, an MT depolymerizing drug (Hoebeke et al. 1976; Lee 1990). Expression of GFP (control) does not prevent nocodazole-induced MT depolymerization as GFP-expressing cells, like untransfected cells, have no stable MTs (Fig. 5, D–F). However, cells expressing DVL-HA have a significant number of stable MTs after nocodazole treatment (Fig. 5, G–I). We often observed that DVL-1 induces the formation of processes (Fig. 5, G–I). Interestingly, most of the DVL-HA that colocalizes with MTs is in small vesicle-like structures (Fig. 5 I, insert). The stabilizing effect of DVL-1 was quantified by counting the number of transfected cells displaying stable MTs after nocodazole treatment. While expression of GFP does not stabilize microtubules, DVL-HA driven by the cytomegalovirus promoter stabilizes MTs in 74.7% of transfected cells after nocodazole treatment (Fig. 6 C). These results show that DVL-1 can protect MTs from nocodazole and can therefore act as an MT stabilizer.


Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta.

Krylova O, Messenger MJ, Salinas PC - J. Cell Biol. (2000)

DVL-1 prevents the loss of stable microtubules in nocodazole treated COS-7 cells. COS-7 expressing DVL-HA (A–C and G–I) or GFP (D–F) were examined for the level of MTs (B, E, and H). COS-7 cells expressing DVL-HA (A) have a normal level of acetylated MTs (B and C). COS-7 cells expressing GFP (D–F) or DVL-HA (G–I) were treated with nocodazole to depolymerize MTs. GFP-expressing cells have no acetylated MTs, similar to neighboring untransfected cells (D–F). Expression of DVL-HA prevents MT depolymerization by nocodazole (G–I). Arrows denote MTs resistant to nocodazole in the cell expressing DVL-HA. *Neighboring untransfected cell, which has lost stable MTs. (Inset) Small vesicle-like structures are present on some MTs. Bar, 50 μM.
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Related In: Results  -  Collection

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Figure 5: DVL-1 prevents the loss of stable microtubules in nocodazole treated COS-7 cells. COS-7 expressing DVL-HA (A–C and G–I) or GFP (D–F) were examined for the level of MTs (B, E, and H). COS-7 cells expressing DVL-HA (A) have a normal level of acetylated MTs (B and C). COS-7 cells expressing GFP (D–F) or DVL-HA (G–I) were treated with nocodazole to depolymerize MTs. GFP-expressing cells have no acetylated MTs, similar to neighboring untransfected cells (D–F). Expression of DVL-HA prevents MT depolymerization by nocodazole (G–I). Arrows denote MTs resistant to nocodazole in the cell expressing DVL-HA. *Neighboring untransfected cell, which has lost stable MTs. (Inset) Small vesicle-like structures are present on some MTs. Bar, 50 μM.
Mentions: The interaction of endogenous DVL-1 with neuronal MTs suggests its possible role in controlling MT organization or, alternatively, that MTs are just used to transport DVL-1 along the axon. We obtained evidence in favor of the former possibility when we examined the effect of expression of Dvl-HA on MTs in COS cells. Consistent with previous studies, DVL-HA forms vesicle-like structures in transfected cells (Fig. 5A and Fig. G; Fagotto et al. 1999; Smalley et al. 1999). Cells expressing DVL-1 have similar levels of acetylated MTs than neighboring nonexpressing cells (Fig. 5, A–C). Similar results were obtained with DVL-1 without the epitope tag HA (data not shown). We then examined the effect of DVL-1 on MTs in cells that have been treated with 10 μM nocodazole, an MT depolymerizing drug (Hoebeke et al. 1976; Lee 1990). Expression of GFP (control) does not prevent nocodazole-induced MT depolymerization as GFP-expressing cells, like untransfected cells, have no stable MTs (Fig. 5, D–F). However, cells expressing DVL-HA have a significant number of stable MTs after nocodazole treatment (Fig. 5, G–I). We often observed that DVL-1 induces the formation of processes (Fig. 5, G–I). Interestingly, most of the DVL-HA that colocalizes with MTs is in small vesicle-like structures (Fig. 5 I, insert). The stabilizing effect of DVL-1 was quantified by counting the number of transfected cells displaying stable MTs after nocodazole treatment. While expression of GFP does not stabilize microtubules, DVL-HA driven by the cytomegalovirus promoter stabilizes MTs in 74.7% of transfected cells after nocodazole treatment (Fig. 6 C). These results show that DVL-1 can protect MTs from nocodazole and can therefore act as an MT stabilizer.

Bottom Line: The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta.These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics.This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

View Article: PubMed Central - PubMed

Affiliation: The Randall Institute, King's College London, London, United Kingdom.

ABSTRACT
Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3beta (GSK-3beta) and blocked by expression of GSK-3beta. These findings suggest that DVL-1, through GSK-3beta, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.

Show MeSH
Related in: MedlinePlus