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Atypical protein kinase C controls sea urchin ciliogenesis.

Prulière G, Cosson J, Chevalier S, Sardet C, Chenevert J - Mol. Biol. Cell (2011)

Bottom Line: We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages.A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming.Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos.

View Article: PubMed Central - PubMed

Affiliation: Observatoire Océanologique, Biologie du Développement, Université Pierre et Marie Curie and CNRS, Villefranche-sur-Mer, France. pruliere@obs-vlfr.fr

ABSTRACT
The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.

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Cilia length is proportional to level of aPKC inhibition. Confocal surface views of embryos fixed and labeled for tubulin. Embryos were deciliated by osmotic stress and then allowed to reciliate in the absence (A) or in the presence (B–D) of myristoylated aPKC pseudosubstrate inhibitor. Inhibitor final concentrations used are indicated.
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Figure 7: Cilia length is proportional to level of aPKC inhibition. Confocal surface views of embryos fixed and labeled for tubulin. Embryos were deciliated by osmotic stress and then allowed to reciliate in the absence (A) or in the presence (B–D) of myristoylated aPKC pseudosubstrate inhibitor. Inhibitor final concentrations used are indicated.

Mentions: To quantify the effect of aPKC inhibitor on cilia growth, a “deciliation–reciliation” assay was used (Auclair and Siegel, 1966). This method generates a homogeneous, cilia-less starting point from which cilia growth measurements can be compared. Twenty-four-hour–old swimming gastrulas were deciliated using a high osmotic stress in hypersaline (∼1.3 M NaCl) seawater for 5 min. This treatment causes the clear cut of the axoneme from the basal body without affecting cell contacts (Stephens, 1995). Embryos were allowed to regrow their cilia in MFSW supplemented with different concentrations of pseudosubstrate inhibitor and then observed for swimming behavior or fixed for immunofluorescence and cilia length measurements (Figure 7). Embryos treated with a low concentration of aPKC inhibitor (0.5 μM) were able to regrow cilia slightly shorter than those of controls (Figure 7B and Table 1) and were able to swim linearly because of the presence of an adequate apical tuft at the animal cap. Such was not the case at higher inhibitor concentrations: average ciliary length dropped rather rapidly, reaching 0 for 5.0 μM inhibitor (Figure 7D and Table 1). This effective pseudosubstrate inhibitory concentration is lower than that generally used to inhibit aPKC in mammalian cells (10–50 μM) (Sun et al., 2005) but similar to the 4 μM used to inhibit aPKC during S. purpuratus early development (Alford et al., 2009). The effect of aPKC inhibition on ciliogenesis is almost fully reversible, as 85% of embryos washed after treatment with 2 μM pseudosubstrate inhibitor and left to reciliate showed a ciliary pattern similar to that of control embryos (compare Figure 8, A–C and G–I) and aPKC localization was restored (Figure 8I); note the normal apical aPKC enrichment at the animal pole (Figure 8I, arrow).


Atypical protein kinase C controls sea urchin ciliogenesis.

Prulière G, Cosson J, Chevalier S, Sardet C, Chenevert J - Mol. Biol. Cell (2011)

Cilia length is proportional to level of aPKC inhibition. Confocal surface views of embryos fixed and labeled for tubulin. Embryos were deciliated by osmotic stress and then allowed to reciliate in the absence (A) or in the presence (B–D) of myristoylated aPKC pseudosubstrate inhibitor. Inhibitor final concentrations used are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Cilia length is proportional to level of aPKC inhibition. Confocal surface views of embryos fixed and labeled for tubulin. Embryos were deciliated by osmotic stress and then allowed to reciliate in the absence (A) or in the presence (B–D) of myristoylated aPKC pseudosubstrate inhibitor. Inhibitor final concentrations used are indicated.
Mentions: To quantify the effect of aPKC inhibitor on cilia growth, a “deciliation–reciliation” assay was used (Auclair and Siegel, 1966). This method generates a homogeneous, cilia-less starting point from which cilia growth measurements can be compared. Twenty-four-hour–old swimming gastrulas were deciliated using a high osmotic stress in hypersaline (∼1.3 M NaCl) seawater for 5 min. This treatment causes the clear cut of the axoneme from the basal body without affecting cell contacts (Stephens, 1995). Embryos were allowed to regrow their cilia in MFSW supplemented with different concentrations of pseudosubstrate inhibitor and then observed for swimming behavior or fixed for immunofluorescence and cilia length measurements (Figure 7). Embryos treated with a low concentration of aPKC inhibitor (0.5 μM) were able to regrow cilia slightly shorter than those of controls (Figure 7B and Table 1) and were able to swim linearly because of the presence of an adequate apical tuft at the animal cap. Such was not the case at higher inhibitor concentrations: average ciliary length dropped rather rapidly, reaching 0 for 5.0 μM inhibitor (Figure 7D and Table 1). This effective pseudosubstrate inhibitory concentration is lower than that generally used to inhibit aPKC in mammalian cells (10–50 μM) (Sun et al., 2005) but similar to the 4 μM used to inhibit aPKC during S. purpuratus early development (Alford et al., 2009). The effect of aPKC inhibition on ciliogenesis is almost fully reversible, as 85% of embryos washed after treatment with 2 μM pseudosubstrate inhibitor and left to reciliate showed a ciliary pattern similar to that of control embryos (compare Figure 8, A–C and G–I) and aPKC localization was restored (Figure 8I); note the normal apical aPKC enrichment at the animal pole (Figure 8I, arrow).

Bottom Line: We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages.A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming.Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos.

View Article: PubMed Central - PubMed

Affiliation: Observatoire Océanologique, Biologie du Développement, Université Pierre et Marie Curie and CNRS, Villefranche-sur-Mer, France. pruliere@obs-vlfr.fr

ABSTRACT
The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.

Show MeSH
Related in: MedlinePlus