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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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Related in: MedlinePlus

Activated p38 MAPK localizes to cilia in S. mansoni miracidia. (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.
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Figure 3: Activated p38 MAPK localizes to cilia in S. mansoni miracidia. (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.

Mentions: To determine the localization of activated p38 MAPK within S. mansoni miracidia, anti-phospho p38 MAPK antibodies and confocal laser scanning microscopy were used. Z-axis projections viewed in maximum pixel brightness mode revealed that freshly-hatched swimming miracidia possessed low levels of phosphorylated p38 MAPK, which is in agreement with western blotting analysis (Figures 2A and 2C). However where activation was evident, it appeared to be associated with the region occupied by the cilia (Figure 3C). Miracidia incubated with secondary antibodies alone possessed only background fluorescence (Figure 3B). When freshly-hatched swimming miracidia were incubated with 20 μM anisomycin for 30 min, a striking increase in p38 MAPK activation was observed (Figure 3G) whereas DMSO exposed (control) miracidia appeared similar to untreated miracidia (data not shown). Analysis of serial optical z-sections revealed activation largely at, or adjacent to, the ciliated surface of the parasite (e.g. Figure 3D). Fine z-sectioning and optical zooming revealed that activated p38 MAPK was associated with the shaft of the cilia (Figure 3E), further demonstrated through co-localization using anti-acetylated tubulin antibodies that permit visualization of these structures (Figure 3F). Immunoreactivity was present up to 6-7 μm from the tegument surface which correlates with the length (7-8 μm) of the cilia reported from transmission electron microscopy studies [10]. Staining for activated p38 MAPK was also observed in the tegument (Figures 3E); here the enzyme could either be associated with the base of the cilia, the microvilli (1 μm long [10]) that are known to exist among the cilia, the rootlet (2 μm long [10]) that is thought to provide support [44], or other structures. The semi-spherical terebratorium (anterior papilla) lacks cilia (Figure 3A) but possess filopodia-like sensory endings [10] thought to play a role in sensing the intermediate snail host. Although staining was observed at the terebratorium, it was noticeably less than that observed in the adjacent ciliated plates (Figure 3G).


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)

Activated p38 MAPK localizes to cilia in S. mansoni miracidia. (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Activated p38 MAPK localizes to cilia in S. mansoni miracidia. (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.
Mentions: To determine the localization of activated p38 MAPK within S. mansoni miracidia, anti-phospho p38 MAPK antibodies and confocal laser scanning microscopy were used. Z-axis projections viewed in maximum pixel brightness mode revealed that freshly-hatched swimming miracidia possessed low levels of phosphorylated p38 MAPK, which is in agreement with western blotting analysis (Figures 2A and 2C). However where activation was evident, it appeared to be associated with the region occupied by the cilia (Figure 3C). Miracidia incubated with secondary antibodies alone possessed only background fluorescence (Figure 3B). When freshly-hatched swimming miracidia were incubated with 20 μM anisomycin for 30 min, a striking increase in p38 MAPK activation was observed (Figure 3G) whereas DMSO exposed (control) miracidia appeared similar to untreated miracidia (data not shown). Analysis of serial optical z-sections revealed activation largely at, or adjacent to, the ciliated surface of the parasite (e.g. Figure 3D). Fine z-sectioning and optical zooming revealed that activated p38 MAPK was associated with the shaft of the cilia (Figure 3E), further demonstrated through co-localization using anti-acetylated tubulin antibodies that permit visualization of these structures (Figure 3F). Immunoreactivity was present up to 6-7 μm from the tegument surface which correlates with the length (7-8 μm) of the cilia reported from transmission electron microscopy studies [10]. Staining for activated p38 MAPK was also observed in the tegument (Figures 3E); here the enzyme could either be associated with the base of the cilia, the microvilli (1 μm long [10]) that are known to exist among the cilia, the rootlet (2 μm long [10]) that is thought to provide support [44], or other structures. The semi-spherical terebratorium (anterior papilla) lacks cilia (Figure 3A) but possess filopodia-like sensory endings [10] thought to play a role in sensing the intermediate snail host. Although staining was observed at the terebratorium, it was noticeably less than that observed in the adjacent ciliated plates (Figure 3G).

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