Figure 5.
Figure 5. The Rootletin conserved domain is essential for Root function and rootlet assembly, but not for localization to basal bodies. (A) Schematic view showing RootDEL, which has a deletion of the N-terminal 333 amino acids, including the entire conserved Root domain. (B) elav-GAL4 driving RFP-RootDEL expression does not rescue Root66 locomotor defects. Flies with RFP-RootDEL expression driven by Insc-GAL4 performed better than the Root66, but the level of rescue is low compared with that of GFP-Root (full length). RootDEL does not introduce dominant effects, as flies with RFP-RootDEL expression in the Root66/TM6B background do not show locomotor defects. (C) RFP-RootDEL expression driven by Insc- or elav-GAL4 failed to rescue the larval touch insensitivity phenotype in Root66. RFP-RootDEL expression in Root66/TM6B background does not cause defects, indicating no dominant effects are associated with RootDEL. (D) RFP-RootDEL localizes to basal bodies but does not assemble normal rootlets. GFP-Root is included as a positive control. When expressed in control Root66/TM6B Ch neurons, RFP-RootDEL localizes to rootlets and does not affect their assembly (solid arrows in top panels), similar to GFP-Root (solid arrows in lower panels). For unknown reason, in Root66/TM6B Es neurons, RootDEL localizes to rootlets shorter than those labeled by GFP-Root (open arrows with asterisks), see also E. While GFP-Root supports assembly of short rootlets in Root66 Ch neurons (solid arrowheads) and normal ones in Es neurons (open arrows), RFP-RootDEL localizes to basal bodies but does not form proper rootlets in both Ch and Es neurons (solid and empty arrowheads). Note that the signal of RFP-RootDEL may not be a proper rootlet but rather a focus of RFP-RootDEL accumulated at the basal body. The black and white panels are live images of the signal for GFP-Root or RFP-RootDEL taken through the pupal cuticle. Bars, 10 µm. (E) Quantification of rootlet lengths in control and Root66 neurons expressing GFP-Root or RFP-RootDEL. Rootlets are measured from at least four antennae for each genotype. Rightmost panels in D show images of the antenna squash. For all graphs, numbers of males/larvae/rootlets assayed are indicated in/near the bars, and error bars represent SEM. ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. See also Fig. S2.

The Rootletin conserved domain is essential for Root function and rootlet assembly, but not for localization to basal bodies. (A) Schematic view showing RootDEL, which has a deletion of the N-terminal 333 amino acids, including the entire conserved Root domain. (B) elav-GAL4 driving RFP-RootDEL expression does not rescue Root66 locomotor defects. Flies with RFP-RootDEL expression driven by Insc-GAL4 performed better than the Root66, but the level of rescue is low compared with that of GFP-Root (full length). RootDEL does not introduce dominant effects, as flies with RFP-RootDEL expression in the Root66/TM6B background do not show locomotor defects. (C) RFP-RootDEL expression driven by Insc- or elav-GAL4 failed to rescue the larval touch insensitivity phenotype in Root66. RFP-RootDEL expression in Root66/TM6B background does not cause defects, indicating no dominant effects are associated with RootDEL. (D) RFP-RootDEL localizes to basal bodies but does not assemble normal rootlets. GFP-Root is included as a positive control. When expressed in control Root66/TM6B Ch neurons, RFP-RootDEL localizes to rootlets and does not affect their assembly (solid arrows in top panels), similar to GFP-Root (solid arrows in lower panels). For unknown reason, in Root66/TM6B Es neurons, RootDEL localizes to rootlets shorter than those labeled by GFP-Root (open arrows with asterisks), see also E. While GFP-Root supports assembly of short rootlets in Root66 Ch neurons (solid arrowheads) and normal ones in Es neurons (open arrows), RFP-RootDEL localizes to basal bodies but does not form proper rootlets in both Ch and Es neurons (solid and empty arrowheads). Note that the signal of RFP-RootDEL may not be a proper rootlet but rather a focus of RFP-RootDEL accumulated at the basal body. The black and white panels are live images of the signal for GFP-Root or RFP-RootDEL taken through the pupal cuticle. Bars, 10 µm. (E) Quantification of rootlet lengths in control and Root66 neurons expressing GFP-Root or RFP-RootDEL. Rootlets are measured from at least four antennae for each genotype. Rightmost panels in D show images of the antenna squash. For all graphs, numbers of males/larvae/rootlets assayed are indicated in/near the bars, and error bars represent SEM. ns, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. See also Fig. S2.

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