Limits...
Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium.

Wilson CW, Chen MH, Chuang PT - PLoS ONE (2009)

Bottom Line: Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation.Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium.We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

Show MeSH
Hh pathway agonists and antagonists stimulate Smo translocation to the primary cilium.(A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2664476&req=5

pone-0005182-g001: Hh pathway agonists and antagonists stimulate Smo translocation to the primary cilium.(A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.

Mentions: We generated antibodies against the C-terminal domain of mouse Smo [17] to examine the ciliary localization of endogenous Smo in response to known Hh pathway agonists and antagonists. When exposed to conditioned media (CM) collected from cells expressing the N-terminal signaling fragment of Sonic hedgehog (ShhN), wild-type mouse embryonic fibroblasts (MEFs) accumulated Smo in primary cilia, and nearly 100% of cilia were positive for Smo (Smo+) after 6 hours of treatment (Fig. 1A, 1B). In agreement with previously published results [7], a reduced number of cilia were Smo+ after brief (1 hour) treatment with cyclopamine, a teratogen derived from the Veratrum genus of plants and a well-defined Smo antagonist known to bind to the heptahelical bundle of Smo (Fig. 1A, 1B) [18]. Surprisingly, prolonged treatment of MEFs with cyclopamine resulted in a significant number of Smo+ cilia, with roughly 70% displaying strong Smo signal along the entire length of the cilium after 24 hours of exposure to cyclopamine (Fig. 1A, 1B). We speculate that the increased time of cyclopamine treatment required to generate a high number of Smo+ cilia underlies the difference between our observation and prior reports. This finding also provided a unique opportunity to examine the relationship between ciliary localization of Smo and Hh pathway activation.


Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium.

Wilson CW, Chen MH, Chuang PT - PLoS ONE (2009)

Hh pathway agonists and antagonists stimulate Smo translocation to the primary cilium.(A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664476&req=5

pone-0005182-g001: Hh pathway agonists and antagonists stimulate Smo translocation to the primary cilium.(A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.
Mentions: We generated antibodies against the C-terminal domain of mouse Smo [17] to examine the ciliary localization of endogenous Smo in response to known Hh pathway agonists and antagonists. When exposed to conditioned media (CM) collected from cells expressing the N-terminal signaling fragment of Sonic hedgehog (ShhN), wild-type mouse embryonic fibroblasts (MEFs) accumulated Smo in primary cilia, and nearly 100% of cilia were positive for Smo (Smo+) after 6 hours of treatment (Fig. 1A, 1B). In agreement with previously published results [7], a reduced number of cilia were Smo+ after brief (1 hour) treatment with cyclopamine, a teratogen derived from the Veratrum genus of plants and a well-defined Smo antagonist known to bind to the heptahelical bundle of Smo (Fig. 1A, 1B) [18]. Surprisingly, prolonged treatment of MEFs with cyclopamine resulted in a significant number of Smo+ cilia, with roughly 70% displaying strong Smo signal along the entire length of the cilium after 24 hours of exposure to cyclopamine (Fig. 1A, 1B). We speculate that the increased time of cyclopamine treatment required to generate a high number of Smo+ cilia underlies the difference between our observation and prior reports. This finding also provided a unique opportunity to examine the relationship between ciliary localization of Smo and Hh pathway activation.

Bottom Line: Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation.Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium.We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

Show MeSH