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The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons.

Styczynska-Soczka K, Jarman AP - Cilia (2015)

Bottom Line: Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function.No evidence was found for a defect in cell division.Although our evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD UK.

ABSTRACT

Background: In vertebrates, rootletin is the major structural component of the ciliary rootlet and is also part of the tether linking the centrioles of the centrosome. Various functions have been ascribed to the rootlet, including maintenance of ciliary integrity through anchoring and facilitation of transport to the cilium or at the base of the cilium. In Drosophila, Rootletin function has not been explored.

Results: In the Drosophila embryo, Rootletin is expressed exclusively in cell lineages of type I sensory neurons, the only somatic cells bearing a cilium. Expression is strongest in mechanosensory chordotonal neurons. Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function. However, the sensory cilium appears largely normal in structure and in localisation of proteins suggesting no strong defect in ciliogenesis. No evidence was found for a defect in cell division.

Conclusions: The role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although our evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself.

No MeSH data available.


Rootletin knock-down severely disrupts Ch neuron function. a Larval locomotion assay showing that Rootletin knock-down larvae move less due to uncoordination. Larvae were tested at two temperatures as the Gal4 driving the RNAi construct is more active at higher temperatures (RNAi predicted to be more complete). Larvae from an atonal mutant stock were tested for comparison; such larvae lack Ch neurons. b Adult climbing assay show a significant disruption of climbing behaviour in adult Rootletin knock-down flies. Results plotted are averages of three trials of 15 flies. c Larval hearing assay. Larvae are tested in groups of five and the retraction score refers to the average number of larvae that contract. n = 40, error bars represent standard deviation. d Grooming assay for behaviour in response to sensory bristle deflection. Proportion represents an average response proportion from six replicas each testing five flies. Error bars represent standard error. e Adult climbing assay performed ageing flies. Results are plotted as averages from four replicas of 15 flies; the same flies were used in each time point, error bars represent standard error, two-way Anova p > 0.003, f Plot of ratio of wild-type/RNAi knockdown for data shown in (e). The rate of performance decrease over time is greater for Rootletin knock-down flies
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Fig5: Rootletin knock-down severely disrupts Ch neuron function. a Larval locomotion assay showing that Rootletin knock-down larvae move less due to uncoordination. Larvae were tested at two temperatures as the Gal4 driving the RNAi construct is more active at higher temperatures (RNAi predicted to be more complete). Larvae from an atonal mutant stock were tested for comparison; such larvae lack Ch neurons. b Adult climbing assay show a significant disruption of climbing behaviour in adult Rootletin knock-down flies. Results plotted are averages of three trials of 15 flies. c Larval hearing assay. Larvae are tested in groups of five and the retraction score refers to the average number of larvae that contract. n = 40, error bars represent standard deviation. d Grooming assay for behaviour in response to sensory bristle deflection. Proportion represents an average response proportion from six replicas each testing five flies. Error bars represent standard error. e Adult climbing assay performed ageing flies. Results are plotted as averages from four replicas of 15 flies; the same flies were used in each time point, error bars represent standard error, two-way Anova p > 0.003, f Plot of ratio of wild-type/RNAi knockdown for data shown in (e). The rate of performance decrease over time is greater for Rootletin knock-down flies

Mentions: To test whether the loss of Rootletin and the rootlet affects Ch sensory neuron function, we analysed behaviour of larvae and adults. Mutations that affect Ch neuron function cause impairments of larval and adult locomotory behaviours due to their proprioceptive function [25]. Compared with control larvae, knock-down larvae show reduced crawling path lengths (Fig. 5a). The reduction is at least as severe as that exhibited by atonal mutant larvae, which lack Ch neurons. Similarly, knock-down adult flies show impaired behaviour in a climbing assay (Fig. 5b). This phenotype was strengthened by greater Gal4 activity (higher incubation temperature) and by enhancement of RNAi with UAS-Dcr-2 and was observed for both Rootletin RNAi lines. Given that Rootletin expression is confined to sensory cells, the results are consistent with defective sensory neuron function.Fig. 5


The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons.

Styczynska-Soczka K, Jarman AP - Cilia (2015)

Rootletin knock-down severely disrupts Ch neuron function. a Larval locomotion assay showing that Rootletin knock-down larvae move less due to uncoordination. Larvae were tested at two temperatures as the Gal4 driving the RNAi construct is more active at higher temperatures (RNAi predicted to be more complete). Larvae from an atonal mutant stock were tested for comparison; such larvae lack Ch neurons. b Adult climbing assay show a significant disruption of climbing behaviour in adult Rootletin knock-down flies. Results plotted are averages of three trials of 15 flies. c Larval hearing assay. Larvae are tested in groups of five and the retraction score refers to the average number of larvae that contract. n = 40, error bars represent standard deviation. d Grooming assay for behaviour in response to sensory bristle deflection. Proportion represents an average response proportion from six replicas each testing five flies. Error bars represent standard error. e Adult climbing assay performed ageing flies. Results are plotted as averages from four replicas of 15 flies; the same flies were used in each time point, error bars represent standard error, two-way Anova p > 0.003, f Plot of ratio of wild-type/RNAi knockdown for data shown in (e). The rate of performance decrease over time is greater for Rootletin knock-down flies
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4489026&req=5

Fig5: Rootletin knock-down severely disrupts Ch neuron function. a Larval locomotion assay showing that Rootletin knock-down larvae move less due to uncoordination. Larvae were tested at two temperatures as the Gal4 driving the RNAi construct is more active at higher temperatures (RNAi predicted to be more complete). Larvae from an atonal mutant stock were tested for comparison; such larvae lack Ch neurons. b Adult climbing assay show a significant disruption of climbing behaviour in adult Rootletin knock-down flies. Results plotted are averages of three trials of 15 flies. c Larval hearing assay. Larvae are tested in groups of five and the retraction score refers to the average number of larvae that contract. n = 40, error bars represent standard deviation. d Grooming assay for behaviour in response to sensory bristle deflection. Proportion represents an average response proportion from six replicas each testing five flies. Error bars represent standard error. e Adult climbing assay performed ageing flies. Results are plotted as averages from four replicas of 15 flies; the same flies were used in each time point, error bars represent standard error, two-way Anova p > 0.003, f Plot of ratio of wild-type/RNAi knockdown for data shown in (e). The rate of performance decrease over time is greater for Rootletin knock-down flies
Mentions: To test whether the loss of Rootletin and the rootlet affects Ch sensory neuron function, we analysed behaviour of larvae and adults. Mutations that affect Ch neuron function cause impairments of larval and adult locomotory behaviours due to their proprioceptive function [25]. Compared with control larvae, knock-down larvae show reduced crawling path lengths (Fig. 5a). The reduction is at least as severe as that exhibited by atonal mutant larvae, which lack Ch neurons. Similarly, knock-down adult flies show impaired behaviour in a climbing assay (Fig. 5b). This phenotype was strengthened by greater Gal4 activity (higher incubation temperature) and by enhancement of RNAi with UAS-Dcr-2 and was observed for both Rootletin RNAi lines. Given that Rootletin expression is confined to sensory cells, the results are consistent with defective sensory neuron function.Fig. 5

Bottom Line: Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function.No evidence was found for a defect in cell division.Although our evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD UK.

ABSTRACT

Background: In vertebrates, rootletin is the major structural component of the ciliary rootlet and is also part of the tether linking the centrioles of the centrosome. Various functions have been ascribed to the rootlet, including maintenance of ciliary integrity through anchoring and facilitation of transport to the cilium or at the base of the cilium. In Drosophila, Rootletin function has not been explored.

Results: In the Drosophila embryo, Rootletin is expressed exclusively in cell lineages of type I sensory neurons, the only somatic cells bearing a cilium. Expression is strongest in mechanosensory chordotonal neurons. Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function. However, the sensory cilium appears largely normal in structure and in localisation of proteins suggesting no strong defect in ciliogenesis. No evidence was found for a defect in cell division.

Conclusions: The role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although our evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself.

No MeSH data available.