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A role for p38 MAPK in the regulation of ciliary motion in a eukaryote.

Ressurreição M, Rollinson D, Emery AM, Walker AJ - BMC Cell Biol. (2011)

Bottom Line: Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument.Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min.Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK.

ABSTRACT

Background: Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.

Results: Fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

Conclusions: This study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes.

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Biochemical characterization of S. mansoni p38 MAPK. (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.
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Figure 2: Biochemical characterization of S. mansoni p38 MAPK. (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.

Mentions: Anti-phospho p38 MAPK monoclonal antibodies were used in an attempt to detect phosphorylated p38 MAPK in S. mansoni. These antibodies, that bind p38 MAPK only when dually phosphorylated on Thr/Tyr of the TGY motif, have been used to detect phosphorylated p38 MAPK in many multicellular eukaryotes including C. elegans [38] and D. melanogaster [39]. Because phosphorylation on these residues results in activation of the enzyme, immunoreactivity directly correlates with p38 MAPK activity. The p38 MAPK amino acid sequence surrounding the TGY motif to which these antibodies are raised is highly-conserved between both S. mansoni and S. japonicum, and between S. mansoni and C. elegans, D. melanogaster, D. rerio and human (Figure 1). Western blotting of adult worm homogenates revealed that anti-phospho p38 MAPK antibodies recognized a single protein band with apparent molecular weight of approximately 42 kDa, essentially co-migrating with phosphorylated p38 MAPK from human astrocytoma (U251 MG) cells (Figure 2A). In marked contrast to adult worms, freshly-hatched swimming miracidia did not possess detectable levels of phosphorylated p38 MAPK (Figure 2A). A second antibody (anti-p38 MAPK) that detects p38 MAPK in vertebrates irrespective of phosphorylation state did not react with the S. mansoni protein precluding its use in this study.


A role for p38 MAPK in the regulation of ciliary motion in a eukaryote.

Ressurreição M, Rollinson D, Emery AM, Walker AJ - BMC Cell Biol. (2011)

Biochemical characterization of S. mansoni p38 MAPK. (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Biochemical characterization of S. mansoni p38 MAPK. (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.
Mentions: Anti-phospho p38 MAPK monoclonal antibodies were used in an attempt to detect phosphorylated p38 MAPK in S. mansoni. These antibodies, that bind p38 MAPK only when dually phosphorylated on Thr/Tyr of the TGY motif, have been used to detect phosphorylated p38 MAPK in many multicellular eukaryotes including C. elegans [38] and D. melanogaster [39]. Because phosphorylation on these residues results in activation of the enzyme, immunoreactivity directly correlates with p38 MAPK activity. The p38 MAPK amino acid sequence surrounding the TGY motif to which these antibodies are raised is highly-conserved between both S. mansoni and S. japonicum, and between S. mansoni and C. elegans, D. melanogaster, D. rerio and human (Figure 1). Western blotting of adult worm homogenates revealed that anti-phospho p38 MAPK antibodies recognized a single protein band with apparent molecular weight of approximately 42 kDa, essentially co-migrating with phosphorylated p38 MAPK from human astrocytoma (U251 MG) cells (Figure 2A). In marked contrast to adult worms, freshly-hatched swimming miracidia did not possess detectable levels of phosphorylated p38 MAPK (Figure 2A). A second antibody (anti-p38 MAPK) that detects p38 MAPK in vertebrates irrespective of phosphorylation state did not react with the S. mansoni protein precluding its use in this study.

Bottom Line: Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument.Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min.Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK.

ABSTRACT

Background: Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.

Results: Fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

Conclusions: This study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes.

Show MeSH
Related in: MedlinePlus