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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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Sea urchin aPKC localizes to a disk situated between the basal body and the elongating axoneme of motile cilia. (A–E, G–I) Confocal sections of cells on the surface of swimming blastulas stained for microtubules (red) and aPKC (green). (A, B, D, E) Surface views; (C, G–I) sagittal views. The arrowhead and arrow in C show the absence of aPKC in the basal body and in the elongating axoneme, respectively. (D′, E) Higher-magnification views of aPKC ring-shaped labeling at the bases of two different cilia. (F) Relative intensity of the aPKC signal observed in (E) using the surface plot quantitation function of the ImageJ program. (J) Relative quantitation of intensity of the fluorescence signals (y-axis) over distance (x-axis) obtained in the green (top) and red (bottom) channels along the line drawn in I using the plot profile function of the ImageJ image processing program. All bars, 2 μm.
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Figure 5: Sea urchin aPKC localizes to a disk situated between the basal body and the elongating axoneme of motile cilia. (A–E, G–I) Confocal sections of cells on the surface of swimming blastulas stained for microtubules (red) and aPKC (green). (A, B, D, E) Surface views; (C, G–I) sagittal views. The arrowhead and arrow in C show the absence of aPKC in the basal body and in the elongating axoneme, respectively. (D′, E) Higher-magnification views of aPKC ring-shaped labeling at the bases of two different cilia. (F) Relative intensity of the aPKC signal observed in (E) using the surface plot quantitation function of the ImageJ program. (J) Relative quantitation of intensity of the fluorescence signals (y-axis) over distance (x-axis) obtained in the green (top) and red (bottom) channels along the line drawn in I using the plot profile function of the ImageJ image processing program. All bars, 2 μm.

Mentions: The localization of aPKC was examined in later stages of embryogenesis. Sea urchin embryos start swimming right after hatching due to the metachronic beating of hundreds of cilia, which lie all around the blastula (Figure 1H) and can be observed under dark-field or phase-contrast microscopy (Figure 4A). A handful of cilia located at the animal pole appear twice as long (∼40 μm) as beating cilia but are mostly immobile and often rolled up all around each other (Figure 4B). This “apical tuft” acts as a rudder to allow the linear swimming of the embryo. Labeling with antitubulin antibody shows that the cilia are preserved after methanol fixation in blastula-, gastrula-, and pluteus-stage embryos (Figure 4, D, G, and J). The immunofluorescence signal obtained for aPKC labeling was essentially the same in blastula and gastrula: the aPKC antibody stains the apical and lateral surface of all blastomeres (see Figure 5, H and I, for lateral view). The apical membrane of the internal archenteron is also labeled in gastrula-stage embryos (Figure 4C), closely correlating with a localized burst in zygotic expression (Supplemental Figure S2, F–H). Of interest, aPKC signal also concentrates at the animal pole, at the basis of the apical tuft (Figure 4, H and I, arrow in C). This strong enrichment of aPKC protein is likely due to accumulation of preexisting maternal protein because no zygotic expression of aPKC mRNA was observed in this region in P. lividus (Supplemental Figure S2) or in H. pulcherrimus (Shiomi and Yamaguchi, 2008). In late pluteus larva, aPKC protein is also detected at the basis of very long cilia nearby the mouth orifice, as well as in the ciliary band (Figure 4K, arrow), and in the stomach endoderm (Figure 4L, arrow).


Atypical protein kinase C controls sea urchin ciliogenesis.

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

Sea urchin aPKC localizes to a disk situated between the basal body and the elongating axoneme of motile cilia. (A–E, G–I) Confocal sections of cells on the surface of swimming blastulas stained for microtubules (red) and aPKC (green). (A, B, D, E) Surface views; (C, G–I) sagittal views. The arrowhead and arrow in C show the absence of aPKC in the basal body and in the elongating axoneme, respectively. (D′, E) Higher-magnification views of aPKC ring-shaped labeling at the bases of two different cilia. (F) Relative intensity of the aPKC signal observed in (E) using the surface plot quantitation function of the ImageJ program. (J) Relative quantitation of intensity of the fluorescence signals (y-axis) over distance (x-axis) obtained in the green (top) and red (bottom) channels along the line drawn in I using the plot profile function of the ImageJ image processing program. All bars, 2 μm.
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Related In: Results  -  Collection

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Figure 5: Sea urchin aPKC localizes to a disk situated between the basal body and the elongating axoneme of motile cilia. (A–E, G–I) Confocal sections of cells on the surface of swimming blastulas stained for microtubules (red) and aPKC (green). (A, B, D, E) Surface views; (C, G–I) sagittal views. The arrowhead and arrow in C show the absence of aPKC in the basal body and in the elongating axoneme, respectively. (D′, E) Higher-magnification views of aPKC ring-shaped labeling at the bases of two different cilia. (F) Relative intensity of the aPKC signal observed in (E) using the surface plot quantitation function of the ImageJ program. (J) Relative quantitation of intensity of the fluorescence signals (y-axis) over distance (x-axis) obtained in the green (top) and red (bottom) channels along the line drawn in I using the plot profile function of the ImageJ image processing program. All bars, 2 μm.
Mentions: The localization of aPKC was examined in later stages of embryogenesis. Sea urchin embryos start swimming right after hatching due to the metachronic beating of hundreds of cilia, which lie all around the blastula (Figure 1H) and can be observed under dark-field or phase-contrast microscopy (Figure 4A). A handful of cilia located at the animal pole appear twice as long (∼40 μm) as beating cilia but are mostly immobile and often rolled up all around each other (Figure 4B). This “apical tuft” acts as a rudder to allow the linear swimming of the embryo. Labeling with antitubulin antibody shows that the cilia are preserved after methanol fixation in blastula-, gastrula-, and pluteus-stage embryos (Figure 4, D, G, and J). The immunofluorescence signal obtained for aPKC labeling was essentially the same in blastula and gastrula: the aPKC antibody stains the apical and lateral surface of all blastomeres (see Figure 5, H and I, for lateral view). The apical membrane of the internal archenteron is also labeled in gastrula-stage embryos (Figure 4C), closely correlating with a localized burst in zygotic expression (Supplemental Figure S2, F–H). Of interest, aPKC signal also concentrates at the animal pole, at the basis of the apical tuft (Figure 4, H and I, arrow in C). This strong enrichment of aPKC protein is likely due to accumulation of preexisting maternal protein because no zygotic expression of aPKC mRNA was observed in this region in P. lividus (Supplemental Figure S2) or in H. pulcherrimus (Shiomi and Yamaguchi, 2008). In late pluteus larva, aPKC protein is also detected at the basis of very long cilia nearby the mouth orifice, as well as in the ciliary band (Figure 4K, arrow), and in the stomach endoderm (Figure 4L, 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