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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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p38 MAPK activation accelerates the liberation of ciliated plates in transforming S. mansoni larvae. Cultures of larvae were exposed to (A) SB 203580 or (B) anisomycin, during transformation in vitro and the proportion of fully-ciliated parasites determined by microscopic examination over 50 h, compared to CBSS controls. Values shown are means (± SEM, n = 90 for each time point and each treatment from three independent experiments). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
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Figure 5: p38 MAPK activation accelerates the liberation of ciliated plates in transforming S. mansoni larvae. Cultures of larvae were exposed to (A) SB 203580 or (B) anisomycin, during transformation in vitro and the proportion of fully-ciliated parasites determined by microscopic examination over 50 h, compared to CBSS controls. Values shown are means (± SEM, n = 90 for each time point and each treatment from three independent experiments). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Mentions: When miracidia penetrate a suitable intermediate snail host they shed their ciliated plates and develop into the next larval stage, the mother sporocyst. This transformation can also be performed in vitro [52] during which miracidia stop swimming before the plates are released. As p38 MAPK activation attenuates miracidia swim speed whereas inhibition accelerates it we explored the effects of anisomycin and SB 203580 on deciliation and thus swimming during miracidium-to-mother sporocyst transformation in vitro. DMSO did not affect the deciliation rate when compared to Chernin's balanced salt solution (CBSS) controls and none of the treatments affected the survival of the developing larvae (data not shown). Anisomycin accelerated the shedding of ciliary plates considerably (Figure 5B); after only 2 h transformation 52 % of parasites had stopped swimming having shed at least some cilia in contrast to only 18 % of parasites in the CBSS control group (P ≤ 0.001; Figure 5B). At this time point 20 % of anisomycin-treated larvae had shed all their ciliated plates compared to none in CBSS alone (data not shown). This effect of anisomycin persisted throughout larval transformation (Figure 5B). Although the effects of SB 203580 were somewhat less marked, at 21 h, 25 h and 29 h significantly more miracidia were observed swimming (with all cilia attached) than were present in the CBSS control group (Figure 5A). Thus, p38 MAPK activation appears to accelerate the initial rate of S. mansoni miracidium transformation by attenuating cilia-mediated swimming behaviour leading to early release of ciliated plates.


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)

p38 MAPK activation accelerates the liberation of ciliated plates in transforming S. mansoni larvae. Cultures of larvae were exposed to (A) SB 203580 or (B) anisomycin, during transformation in vitro and the proportion of fully-ciliated parasites determined by microscopic examination over 50 h, compared to CBSS controls. Values shown are means (± SEM, n = 90 for each time point and each treatment from three independent experiments). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040701&req=5

Figure 5: p38 MAPK activation accelerates the liberation of ciliated plates in transforming S. mansoni larvae. Cultures of larvae were exposed to (A) SB 203580 or (B) anisomycin, during transformation in vitro and the proportion of fully-ciliated parasites determined by microscopic examination over 50 h, compared to CBSS controls. Values shown are means (± SEM, n = 90 for each time point and each treatment from three independent experiments). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
Mentions: When miracidia penetrate a suitable intermediate snail host they shed their ciliated plates and develop into the next larval stage, the mother sporocyst. This transformation can also be performed in vitro [52] during which miracidia stop swimming before the plates are released. As p38 MAPK activation attenuates miracidia swim speed whereas inhibition accelerates it we explored the effects of anisomycin and SB 203580 on deciliation and thus swimming during miracidium-to-mother sporocyst transformation in vitro. DMSO did not affect the deciliation rate when compared to Chernin's balanced salt solution (CBSS) controls and none of the treatments affected the survival of the developing larvae (data not shown). Anisomycin accelerated the shedding of ciliary plates considerably (Figure 5B); after only 2 h transformation 52 % of parasites had stopped swimming having shed at least some cilia in contrast to only 18 % of parasites in the CBSS control group (P ≤ 0.001; Figure 5B). At this time point 20 % of anisomycin-treated larvae had shed all their ciliated plates compared to none in CBSS alone (data not shown). This effect of anisomycin persisted throughout larval transformation (Figure 5B). Although the effects of SB 203580 were somewhat less marked, at 21 h, 25 h and 29 h significantly more miracidia were observed swimming (with all cilia attached) than were present in the CBSS control group (Figure 5A). Thus, p38 MAPK activation appears to accelerate the initial rate of S. mansoni miracidium transformation by attenuating cilia-mediated swimming behaviour leading to early release of ciliated plates.

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