Root is essential for rootlet formation and basal body cohesion. (A) Schematic view of the rootlet and its connection to the basal bodies in the JO. (B) Root66 JO neurons lack striated rootlets. Representative transmission EM longitudinal section images show that in control w1118, the rootlet is organized as a characteristic striated fiber (arrows), but in Root66/Df, this organization is lost; instead, some disconnected electron-dense clumps are observed at the rootlet region (arrowheads). (C) Quantification of defective rootlets in Root66 JO. Rootlet structures were observed in longitudinal and cross-sections. Numbers of rootlets analyzed are indicated; ****, P ≤ 0.0001. (D) The connecting fibers are normally found between the pBB and the dBB as thread-like electron-dense structures in the control (black arrows); they are lost in Root66 (black arrowhead). The striated rootlet is present in wild type (white arrow) but disrupted in the mutant (white arrowhead). Examined by serial sections, 60% of mutant JO neurons appear to lack a pBB. (E) Quantification of the edge-to-edge distance between the dBB and pBB in JO Ch neurons. Single data points, mean, and standard deviation are indicated. There is no significant difference between mean values of w1118 and Root66, using Mann-Whitney test. But F-distribution analysis indicated that distances in Root66 are significantly more variable than in w1118. (F) Immunostaining of olfactory neurons for basal bodies (Ana1-GFP), the transition zone (Cby-Tomato), and Root. Root localizes into the space between dBB and pBB. (G) In the control olfactory neurons (upper panels), basal bodies (Ana1-GFP) are in tandem pairs (arrows) with Cby located near one of them. In Root66 (lower panels), one of the basal bodies is frequently more distant, or “free” (arrowheads). (H) Quantification of loss of basal body cohesion in Root66. A basal body (centriole), marked with Ana1-GFP, is scored as “free” if it is not directly associated with the Tz (Cby-Tomato) and located more than 800 nm (center-to-center distance, which is about twice the diameter of an Ana1-GFP dot) from the nearest dBB that is associated with Cby. The frequency of “free centrioles” is defined by the ratio of the number of “free” Ana1 dots to the number of Cby dots. Numbers near/in the bars indicate numbers of Cby dots assayed from at least four antennae for each genotype. **, P ≤ 0.01. F and G show images of the antenna squash. Bars: (B and D) 500 nm; (F and G) 5 µm; (F and G, zoom) 1 µm.
