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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 and inhibition affects S. mansoni miracidia swim velocity. Freshly-hatched swimming miracidia were collected and incubated with (A) SB 203580 (1 μM) or (B) anisomycin (20 μM) in spring water, or spring water alone, and the effects on miracidia swim behaviour over 60 min captured by digital video microscopy. Calculation of swim velocities (mm/s) for 30 miracidia for each treatment and each time point was achieved using ImageJ and mean swim speeds (± SEM) were calculated. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
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Figure 4: p38 MAPK activation and inhibition affects S. mansoni miracidia swim velocity. Freshly-hatched swimming miracidia were collected and incubated with (A) SB 203580 (1 μM) or (B) anisomycin (20 μM) in spring water, or spring water alone, and the effects on miracidia swim behaviour over 60 min captured by digital video microscopy. Calculation of swim velocities (mm/s) for 30 miracidia for each treatment and each time point was achieved using ImageJ and mean swim speeds (± SEM) were calculated. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Mentions: Because p38 MAPK was active in stationary miracidia within eggs but inactive in swimming miracidia, and because p38 MAPK localized to cilia, we next explored whether pharmacological activation or inhibition of p38 MAPK phenotypically affected ciliary function by performing swim velocity analyses. Miracidia were exposed to anisomycin or SB 203580 and their swim behaviour recorded by digital video microscopy (see additional file 1: Supplementary movie file). Determination of swim velocities revealed that freshly-hatched miracidia swam at speeds between 2.17 and 2.38 mm/s over 60 min (Figure 4A and B); DMSO treatment did not affect swim velocities over this duration (data not shown). However, anisomycin (20 μM) significantly attenuated miracidia swim velocity over time when compared to untreated (spring water) controls (P ≤ 0.001) (Figure 4B); after 5 min and 30 min mean swim velocity was reduced by 25% to 1.70 mm/s (P ≤ 0.01), and by 80% to 0.43 mm/s (P ≤ 0.001), respectively. After 60 min anisomycin treatment swimming essentially ceased. On the other hand, SB 203580 (1 μM) significantly accelerated miracidia swim velocity (P ≤ 0.001), particularly after 30 and 45 min treatment when it increased ~15 % (P ≤ 0.001). Importantly, following 60 min anisomycin treatment, when spring water (to dilute the anisomycin concentration 10-fold) or SB 203580 (1 μM) were added, the miracidia started swimming displaying swim velocities of between 0.4 and 0.6 mm/s after 20 min (data not shown; see additional file 1: Supplementary movie file). That the effects of anisomycin could be reversed demonstrates that cessation of swimming was not due to larval death. Thus, the contrasting effects of SB 203580 and anisomycin on swim velocity and the finding that p38 MAPK localizes to the cilia are consistent with p38 MAPK playing a role in ciliary beat and thus miracidia swimming. Anisomycin is thought to indirectly activate p38 MAPK via cdc42/rac, MAPKKKs, MAPKK3 (MKK3) and MKK6 which ultimately phosphorylate p38 MAPK [46]. Importantly, orthologues of these pathway components have been identified in S. mansoni [33] suggesting that the mechanisms by which anisomycin exerts its effect on p38 MAPK are conserved between schistosomes and higher eukaryotes. P38 MAPK phosphatases have also been identified in S. mansoni [33], allowing rapid dephosphorylation of p38 MAPK when required. Although aspects of sperm flagella make them distinct from other motile cilia it is interesting that p38 MAPK inhibition by SB 203580 was recently shown to stimulate forward and hyperactivated motility of human sperm [45].


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 and inhibition affects S. mansoni miracidia swim velocity. Freshly-hatched swimming miracidia were collected and incubated with (A) SB 203580 (1 μM) or (B) anisomycin (20 μM) in spring water, or spring water alone, and the effects on miracidia swim behaviour over 60 min captured by digital video microscopy. Calculation of swim velocities (mm/s) for 30 miracidia for each treatment and each time point was achieved using ImageJ and mean swim speeds (± SEM) were calculated. *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 4: p38 MAPK activation and inhibition affects S. mansoni miracidia swim velocity. Freshly-hatched swimming miracidia were collected and incubated with (A) SB 203580 (1 μM) or (B) anisomycin (20 μM) in spring water, or spring water alone, and the effects on miracidia swim behaviour over 60 min captured by digital video microscopy. Calculation of swim velocities (mm/s) for 30 miracidia for each treatment and each time point was achieved using ImageJ and mean swim speeds (± SEM) were calculated. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
Mentions: Because p38 MAPK was active in stationary miracidia within eggs but inactive in swimming miracidia, and because p38 MAPK localized to cilia, we next explored whether pharmacological activation or inhibition of p38 MAPK phenotypically affected ciliary function by performing swim velocity analyses. Miracidia were exposed to anisomycin or SB 203580 and their swim behaviour recorded by digital video microscopy (see additional file 1: Supplementary movie file). Determination of swim velocities revealed that freshly-hatched miracidia swam at speeds between 2.17 and 2.38 mm/s over 60 min (Figure 4A and B); DMSO treatment did not affect swim velocities over this duration (data not shown). However, anisomycin (20 μM) significantly attenuated miracidia swim velocity over time when compared to untreated (spring water) controls (P ≤ 0.001) (Figure 4B); after 5 min and 30 min mean swim velocity was reduced by 25% to 1.70 mm/s (P ≤ 0.01), and by 80% to 0.43 mm/s (P ≤ 0.001), respectively. After 60 min anisomycin treatment swimming essentially ceased. On the other hand, SB 203580 (1 μM) significantly accelerated miracidia swim velocity (P ≤ 0.001), particularly after 30 and 45 min treatment when it increased ~15 % (P ≤ 0.001). Importantly, following 60 min anisomycin treatment, when spring water (to dilute the anisomycin concentration 10-fold) or SB 203580 (1 μM) were added, the miracidia started swimming displaying swim velocities of between 0.4 and 0.6 mm/s after 20 min (data not shown; see additional file 1: Supplementary movie file). That the effects of anisomycin could be reversed demonstrates that cessation of swimming was not due to larval death. Thus, the contrasting effects of SB 203580 and anisomycin on swim velocity and the finding that p38 MAPK localizes to the cilia are consistent with p38 MAPK playing a role in ciliary beat and thus miracidia swimming. Anisomycin is thought to indirectly activate p38 MAPK via cdc42/rac, MAPKKKs, MAPKK3 (MKK3) and MKK6 which ultimately phosphorylate p38 MAPK [46]. Importantly, orthologues of these pathway components have been identified in S. mansoni [33] suggesting that the mechanisms by which anisomycin exerts its effect on p38 MAPK are conserved between schistosomes and higher eukaryotes. P38 MAPK phosphatases have also been identified in S. mansoni [33], allowing rapid dephosphorylation of p38 MAPK when required. Although aspects of sperm flagella make them distinct from other motile cilia it is interesting that p38 MAPK inhibition by SB 203580 was recently shown to stimulate forward and hyperactivated motility of human sperm [45].

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