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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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I. Domain structure of sea urchin aPKCs. (A) The short isoform found so far in P. lividus and S. purpuratus encodes an aPKC that lacks the PB1 protein interaction domain. Kinase: serine threonine kinase domain; C1: cysteine-rich domain, which binds InsPtd(3,4,5)P3. The three urchin species examined so far (P. lividus, H. pulcherrimus, and S. purpuratus) also contain a long isoform that is similar to most other known aPKCs in its domain composition (see Supplemental Figure S1 for alignment). II. Two maternal aPKC proteins that localize in the cortex in early embryos of P. lividus. (A) Western blot with the SC216 anti-aPKC antibody on P. lividus extracts from (a) unfertilized eggs, (b) 2-cell stage, (c) 8-cell stage, (d) 16- to 32-cell stage, (e) swimming blastula, (f) early gastrula, (g) prism, (h) plutei. Equal amounts of protein were loaded in each lane. (i) Standard molecular weight markers from top to bottom: 200, 120, 100, 70, 50, 37, and 20 kDa. (B–F) Immunolocalization of aPKC in early sea urchin embryos observed by confocal microscopy. (B) Unfertilized egg, (C) 2-cell stage, (D) 8-cell stage, (E, F) two confocal sections of the same 16-cell embryo showing (E) the interior and (F) the surface. Scale bar, 20 μm.
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Figure 2: I. Domain structure of sea urchin aPKCs. (A) The short isoform found so far in P. lividus and S. purpuratus encodes an aPKC that lacks the PB1 protein interaction domain. Kinase: serine threonine kinase domain; C1: cysteine-rich domain, which binds InsPtd(3,4,5)P3. The three urchin species examined so far (P. lividus, H. pulcherrimus, and S. purpuratus) also contain a long isoform that is similar to most other known aPKCs in its domain composition (see Supplemental Figure S1 for alignment). II. Two maternal aPKC proteins that localize in the cortex in early embryos of P. lividus. (A) Western blot with the SC216 anti-aPKC antibody on P. lividus extracts from (a) unfertilized eggs, (b) 2-cell stage, (c) 8-cell stage, (d) 16- to 32-cell stage, (e) swimming blastula, (f) early gastrula, (g) prism, (h) plutei. Equal amounts of protein were loaded in each lane. (i) Standard molecular weight markers from top to bottom: 200, 120, 100, 70, 50, 37, and 20 kDa. (B–F) Immunolocalization of aPKC in early sea urchin embryos observed by confocal microscopy. (B) Unfertilized egg, (C) 2-cell stage, (D) 8-cell stage, (E, F) two confocal sections of the same 16-cell embryo showing (E) the interior and (F) the surface. Scale bar, 20 μm.

Mentions: To clone the aPKC homologue from P. lividus, a DNA fragment encoding part of the well-conserved kinase domain was amplified by RT-PCR using degenerate oligonucleotide primers. The screen of a P. lividus egg cDNA library followed by 5′-RACE yielded two classes of clones, which differed in their 5′ termini and encoded proteins of 523 and 598 amino acids (Figure 2-I). The deduced protein sequence of the shorter P. lividus aPKC isoform (Pl-aPKC-1) is 98% identical to the predicted open reading frame encoding S. purpuratus Sp-aPKCι (National Center for Biotechnology Information [NCBI] Reference Sequence XP_780275.1) (Supplemental Figure S1). Pl-aPKC-1 and Sp-aPKC contain the characteristic cysteine-rich C1 and kinase domains, but, of interest, lack the Phox and Bem (PB1) protein interaction domain, which allows aPKC to bind PAR6 to form heterodimers (Hirano et al., 2005; Sumimoto et al., 2007). All other known aPKC proteins contain this PB1 domain, including those from nematodes, insects, and chordates. On the other hand, the long P. lividus aPKC isoform obtained in our screen (Pl-aPKC-2) is unlike Sp-aPKC at the amino-terminal region but is 95% identical to the H. pulcherrimus aPKC and exhibits 70% identity and 81% homology with its human counterpart (Supplemental Figure S1).


Atypical protein kinase C controls sea urchin ciliogenesis.

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

I. Domain structure of sea urchin aPKCs. (A) The short isoform found so far in P. lividus and S. purpuratus encodes an aPKC that lacks the PB1 protein interaction domain. Kinase: serine threonine kinase domain; C1: cysteine-rich domain, which binds InsPtd(3,4,5)P3. The three urchin species examined so far (P. lividus, H. pulcherrimus, and S. purpuratus) also contain a long isoform that is similar to most other known aPKCs in its domain composition (see Supplemental Figure S1 for alignment). II. Two maternal aPKC proteins that localize in the cortex in early embryos of P. lividus. (A) Western blot with the SC216 anti-aPKC antibody on P. lividus extracts from (a) unfertilized eggs, (b) 2-cell stage, (c) 8-cell stage, (d) 16- to 32-cell stage, (e) swimming blastula, (f) early gastrula, (g) prism, (h) plutei. Equal amounts of protein were loaded in each lane. (i) Standard molecular weight markers from top to bottom: 200, 120, 100, 70, 50, 37, and 20 kDa. (B–F) Immunolocalization of aPKC in early sea urchin embryos observed by confocal microscopy. (B) Unfertilized egg, (C) 2-cell stage, (D) 8-cell stage, (E, F) two confocal sections of the same 16-cell embryo showing (E) the interior and (F) the surface. Scale bar, 20 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113769&req=5

Figure 2: I. Domain structure of sea urchin aPKCs. (A) The short isoform found so far in P. lividus and S. purpuratus encodes an aPKC that lacks the PB1 protein interaction domain. Kinase: serine threonine kinase domain; C1: cysteine-rich domain, which binds InsPtd(3,4,5)P3. The three urchin species examined so far (P. lividus, H. pulcherrimus, and S. purpuratus) also contain a long isoform that is similar to most other known aPKCs in its domain composition (see Supplemental Figure S1 for alignment). II. Two maternal aPKC proteins that localize in the cortex in early embryos of P. lividus. (A) Western blot with the SC216 anti-aPKC antibody on P. lividus extracts from (a) unfertilized eggs, (b) 2-cell stage, (c) 8-cell stage, (d) 16- to 32-cell stage, (e) swimming blastula, (f) early gastrula, (g) prism, (h) plutei. Equal amounts of protein were loaded in each lane. (i) Standard molecular weight markers from top to bottom: 200, 120, 100, 70, 50, 37, and 20 kDa. (B–F) Immunolocalization of aPKC in early sea urchin embryos observed by confocal microscopy. (B) Unfertilized egg, (C) 2-cell stage, (D) 8-cell stage, (E, F) two confocal sections of the same 16-cell embryo showing (E) the interior and (F) the surface. Scale bar, 20 μm.
Mentions: To clone the aPKC homologue from P. lividus, a DNA fragment encoding part of the well-conserved kinase domain was amplified by RT-PCR using degenerate oligonucleotide primers. The screen of a P. lividus egg cDNA library followed by 5′-RACE yielded two classes of clones, which differed in their 5′ termini and encoded proteins of 523 and 598 amino acids (Figure 2-I). The deduced protein sequence of the shorter P. lividus aPKC isoform (Pl-aPKC-1) is 98% identical to the predicted open reading frame encoding S. purpuratus Sp-aPKCι (National Center for Biotechnology Information [NCBI] Reference Sequence XP_780275.1) (Supplemental Figure S1). Pl-aPKC-1 and Sp-aPKC contain the characteristic cysteine-rich C1 and kinase domains, but, of interest, lack the Phox and Bem (PB1) protein interaction domain, which allows aPKC to bind PAR6 to form heterodimers (Hirano et al., 2005; Sumimoto et al., 2007). All other known aPKC proteins contain this PB1 domain, including those from nematodes, insects, and chordates. On the other hand, the long P. lividus aPKC isoform obtained in our screen (Pl-aPKC-2) is unlike Sp-aPKC at the amino-terminal region but is 95% identical to the H. pulcherrimus aPKC and exhibits 70% identity and 81% homology with its human counterpart (Supplemental Figure S1).

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