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An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria.

Rompolas P, Patel-King RS, King SM - Mol. Biol. Cell (2010)

Bottom Line: The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination.In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia.We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305, USA.

ABSTRACT
Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.

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Cilia are required for gliding locomotion in planarians. (a) Cilia from Smed-ift88(RNAi) and control-treated animals were observed using DIC optics (top panels). In addition, planaria were stained with anti-α-tubulin antibody (clone B-5-1-2) and visualized using confocal microscopy (side view, middle panel; top view, bottom panel). Analysis showed a substantial decrease in the number of cilia in the Smed-ift88(RNAi) flatworms compared with control. The dotted lines in the middle panels mark the apical surface of the ventral epithelium. The numerous bright puncta visible on the ventral surface of Smed-ift88(RNAi) animals derive from nonspecific binding of the secondary antibody to mucus associated with the gland cells. (b) Initial frame (t = 0; top panel) and overlays of sequential frames (t = 60 s; bottom panel) from 60-s video segments, illustrating the distance traveled by individual flatworms in Smed-ift88(RNAi) and control-treated groups. (c) Quantification of gliding velocity in RNAi- and control-treated animals. Smed-ift88(RNAi) animals moved at ∼0.46 mm/s (n = 23, sem = 0.013) compared with ∼1.47 mm/s (n = 27, sem = 0.02) for the control group. (d) Sequential video frames (1.5 s apart) illustrating the differences in the general mode of movement between Smed-ift88(RNAi) and control-treated animals. Smed-ift88(RNAi) flatworms display no gliding locomotion and travel using peristaltic muscular movements. Arrowheads show peristaltic muscular waves as they propagate along the body (also see Movies S3 and S4).
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Figure 5: Cilia are required for gliding locomotion in planarians. (a) Cilia from Smed-ift88(RNAi) and control-treated animals were observed using DIC optics (top panels). In addition, planaria were stained with anti-α-tubulin antibody (clone B-5-1-2) and visualized using confocal microscopy (side view, middle panel; top view, bottom panel). Analysis showed a substantial decrease in the number of cilia in the Smed-ift88(RNAi) flatworms compared with control. The dotted lines in the middle panels mark the apical surface of the ventral epithelium. The numerous bright puncta visible on the ventral surface of Smed-ift88(RNAi) animals derive from nonspecific binding of the secondary antibody to mucus associated with the gland cells. (b) Initial frame (t = 0; top panel) and overlays of sequential frames (t = 60 s; bottom panel) from 60-s video segments, illustrating the distance traveled by individual flatworms in Smed-ift88(RNAi) and control-treated groups. (c) Quantification of gliding velocity in RNAi- and control-treated animals. Smed-ift88(RNAi) animals moved at ∼0.46 mm/s (n = 23, sem = 0.013) compared with ∼1.47 mm/s (n = 27, sem = 0.02) for the control group. (d) Sequential video frames (1.5 s apart) illustrating the differences in the general mode of movement between Smed-ift88(RNAi) and control-treated animals. Smed-ift88(RNAi) flatworms display no gliding locomotion and travel using peristaltic muscular movements. Arrowheads show peristaltic muscular waves as they propagate along the body (also see Movies S3 and S4).

Mentions: Comparative analysis of the S. mediterranea genome revealed a great number of highly conserved genes with known ciliary function. To study the role of individual proteins in ciliary motility, we used RNAi to inhibit the expression of genes that are known to be essential for cilia formation. Using the S. mediterranea Genome Database (SmedGD; Robb et al., 2008), we identified a conserved gene that encodes the IFT protein IFT88 (Figure 4a). IFT88 is a component of IFT complex B, and mutations that affect the normal levels of this protein result in short or diminished cilia (Marszalek et al., 1999; Pazour et al., 2000). A sequence of 527 base pairs from the coding region of Smed-ift88 was inserted into plasmid L4440 (Timmons et al., 2001) to produce the vector for RNAi. Groups of planarians were fed with bacteria expressing Smed-ift88(RNAi) or an empty vector, twice a week over a period of 2 wk; at which time a significant drop in mRNA was achieved, as evidenced by semi-quantitative RT-PCR and verified by Northern blot analysis (Figure 4, b and c). Both DIC microscopy and confocal imaging of animals prepared for tubulin immunofluorescence revealed that the ventral surface of Smed-ift88(RNAi) animals was almost completely devoid of cilia, whereas cilia on flatworms that were fed with the empty vector remained essentially unchanged (Figure 5a).


An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria.

Rompolas P, Patel-King RS, King SM - Mol. Biol. Cell (2010)

Cilia are required for gliding locomotion in planarians. (a) Cilia from Smed-ift88(RNAi) and control-treated animals were observed using DIC optics (top panels). In addition, planaria were stained with anti-α-tubulin antibody (clone B-5-1-2) and visualized using confocal microscopy (side view, middle panel; top view, bottom panel). Analysis showed a substantial decrease in the number of cilia in the Smed-ift88(RNAi) flatworms compared with control. The dotted lines in the middle panels mark the apical surface of the ventral epithelium. The numerous bright puncta visible on the ventral surface of Smed-ift88(RNAi) animals derive from nonspecific binding of the secondary antibody to mucus associated with the gland cells. (b) Initial frame (t = 0; top panel) and overlays of sequential frames (t = 60 s; bottom panel) from 60-s video segments, illustrating the distance traveled by individual flatworms in Smed-ift88(RNAi) and control-treated groups. (c) Quantification of gliding velocity in RNAi- and control-treated animals. Smed-ift88(RNAi) animals moved at ∼0.46 mm/s (n = 23, sem = 0.013) compared with ∼1.47 mm/s (n = 27, sem = 0.02) for the control group. (d) Sequential video frames (1.5 s apart) illustrating the differences in the general mode of movement between Smed-ift88(RNAi) and control-treated animals. Smed-ift88(RNAi) flatworms display no gliding locomotion and travel using peristaltic muscular movements. Arrowheads show peristaltic muscular waves as they propagate along the body (also see Movies S3 and S4).
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Figure 5: Cilia are required for gliding locomotion in planarians. (a) Cilia from Smed-ift88(RNAi) and control-treated animals were observed using DIC optics (top panels). In addition, planaria were stained with anti-α-tubulin antibody (clone B-5-1-2) and visualized using confocal microscopy (side view, middle panel; top view, bottom panel). Analysis showed a substantial decrease in the number of cilia in the Smed-ift88(RNAi) flatworms compared with control. The dotted lines in the middle panels mark the apical surface of the ventral epithelium. The numerous bright puncta visible on the ventral surface of Smed-ift88(RNAi) animals derive from nonspecific binding of the secondary antibody to mucus associated with the gland cells. (b) Initial frame (t = 0; top panel) and overlays of sequential frames (t = 60 s; bottom panel) from 60-s video segments, illustrating the distance traveled by individual flatworms in Smed-ift88(RNAi) and control-treated groups. (c) Quantification of gliding velocity in RNAi- and control-treated animals. Smed-ift88(RNAi) animals moved at ∼0.46 mm/s (n = 23, sem = 0.013) compared with ∼1.47 mm/s (n = 27, sem = 0.02) for the control group. (d) Sequential video frames (1.5 s apart) illustrating the differences in the general mode of movement between Smed-ift88(RNAi) and control-treated animals. Smed-ift88(RNAi) flatworms display no gliding locomotion and travel using peristaltic muscular movements. Arrowheads show peristaltic muscular waves as they propagate along the body (also see Movies S3 and S4).
Mentions: Comparative analysis of the S. mediterranea genome revealed a great number of highly conserved genes with known ciliary function. To study the role of individual proteins in ciliary motility, we used RNAi to inhibit the expression of genes that are known to be essential for cilia formation. Using the S. mediterranea Genome Database (SmedGD; Robb et al., 2008), we identified a conserved gene that encodes the IFT protein IFT88 (Figure 4a). IFT88 is a component of IFT complex B, and mutations that affect the normal levels of this protein result in short or diminished cilia (Marszalek et al., 1999; Pazour et al., 2000). A sequence of 527 base pairs from the coding region of Smed-ift88 was inserted into plasmid L4440 (Timmons et al., 2001) to produce the vector for RNAi. Groups of planarians were fed with bacteria expressing Smed-ift88(RNAi) or an empty vector, twice a week over a period of 2 wk; at which time a significant drop in mRNA was achieved, as evidenced by semi-quantitative RT-PCR and verified by Northern blot analysis (Figure 4, b and c). Both DIC microscopy and confocal imaging of animals prepared for tubulin immunofluorescence revealed that the ventral surface of Smed-ift88(RNAi) animals was almost completely devoid of cilia, whereas cilia on flatworms that were fed with the empty vector remained essentially unchanged (Figure 5a).

Bottom Line: The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination.In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia.We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305, USA.

ABSTRACT
Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.

Show MeSH