|Phototaxis requires the two anterior flagella and the single, asymmetrically localized eyespot. Two anterior basal bodies/centrosomes (mother and daughter) nucleate assembly of the flagella and four acetylated microtubule bundles (two from the mother basal body, and two from the daughter), termed "rootlets." The eyespot, which comprises carotenoid pigment granule layers in the chloroplast underlying rhodopsin-family photoreceptors in the plasma membrane, is associated specifically with the daughter four-membered microtubule rootlet, prompting the hypothesis that localization of the eyespot is determined by cytoskeletal structures. This hypothesis predicts that in cells with mutations that disrupt rootlet organization, the eyespot will be associated with aberrant rootlets as opposed to maintaining a specific location relative to other cellular structures such as the chloroplast. bld2 (epsilon-tubulin), vfl2 (centrin), asq2 (tubulin co-chaperone Tbccd1) and mlt1 (multiple eyespot phenotype) mutants have cytoskeletal defects that affect the basal bodies and/or microtubule rootlets. By light microscopy, between 14% (bld2) and 65% (mlt1) of mutant cells contain two eyespots, suggestive that at least one was "misplaced." To determine whether the eyespots remain associated with rootlets, we used indirect immunofluorescence to label the ChR1 photoreceptor and acetylated microtubules. In the majority of vfl2, asq2 and mlt1 cells with two ChR1 patches, both were associated with acetylated microtubules, consistent with the hypothesis that acetylated microtubules direct ChR1 localization. A functional relationship between assembly of the cytoskeleton and of the eyespot is also supported by the phenotype of mlt1uni1 double mutant cells, which are bald (as opposed to uniflagellate) and contain only a single eyespot, as determined by both light and immunofluorescence microscopy. Collectively, our results suggest that longitudinal photo
receptor localization is dictated by the position of the daughter four-membered microtubule rootlet in wild-type cells. Supported by the NSF.