The daughter four‐membered microtubule rootlet determines anterior–posterior positioning of the eyespot in Chlamydomonas reinhardtii

Abstract

The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely-defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths. The length of the acetylated daughter four-membered microtubule rootlet correlates with the position of the eyespot, which appears in a posterior position in the majority of cells. The correlation of rootlet length with eyespot positioning was also observed in the cmu1 mutant, which has longer acetylated microtubules, and the mlt1 mutant, in which the rootlet microtubules are shorter. Observation of eyespot positioning after depolymerization of rootlet microtubules indicated that eyespot position is fixed early in eyespot development and becomes independent of the rootlet. Our data demonstrate that the length of the daughter four-membered rootlet is the major determinant of eyespot positioning on the anterior-posterior axis and are suggestive that the gene product of the PEY1 locus is a novel regulator of acetylated microtubule length.

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

“…8D). Eventually, ChR1 moves to the primary eyespot, which in interphase mlt1 cells is at a more anterior position than the wildtype eyespot (36,70). These data suggest that the MLT1 protein promotes the D4-directed movement of the photoreceptors away from the anterior end of the cell.…”

“…Previously, we hypothesized that MLT1 primarily affects microtubule rootlet asymmetry, which results in a shorter D4 rootlet and more anterior eyespots in mlt1 cells (36). The hypothesis that D4 rootlet length determines the position of the eyespot along the anterior-posterior axis would also explain the more posterior eyespots in pey1 and cmu1 mutant cells, which have correspondingly long D4 rootlets (70,71). If, however, the primary effect of the MLT1 protein is on photoreceptor localization, then the shorter D4 rootlet in mlt1 cells may be the result, rather than the cause, of the more anterior position of the primary eyespot.…”

New insights into eyespot placement and assembly in Chlamydomonas

“…26 Conversely, longer microtubule rootlets in the cmu1 (cytoplasmic microtubules unorganized) mutant and the recently-identified pey1 (posterior eyespot) mutant correlated with posterior placement of the eyespot. 27 This cytoskeleton-mediated positioning process, however, may be more complex than it initially appears. Preliminary observations of dividing cells indicate that acetylation of the D4 rootlets extends to the extreme posterior of the cells before receding, while the nascent eyespots remain in an approximately equatorial position along the rootlet.…”

“…The mlt1 mutant exhibits enhanced sensitivity to the microtubule-destabilizing drug colchicine, lending support to the hypothesis that MLT1 affects the stability of the microtubule cytoskeleton. 27 Association of MLT1, a low-complexity protein with no identifiable domains, with either the mother or daughter basal body (or associated structures) may promote association of photoreceptor with the daughter rootlet and/or block localization to the mother hemisphere (Fig. 1A), although other rootlet-associated factors or modifications likely play roles in establishing the specificity of the D4 rootlet.…”

Statistical method for comparing the level of intracellular organization between cells

“…In Chlamydomonas cells, a pair of centrioles is located at one pole of the cell and these centrioles organize a set of four microtubule rootlets that run from the anterior pole of the cell around the cell cortex. These rootlets are known to be important for localization of cellular structures, such as the eyespot (24). Using a combination of genetic screening and image analysis, we have previously identified mutants in which centrioles lose their consistent polarized location in Chlamydomonas (8,25).…”