Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

Abstract

bLike many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue.

MLT1 links cytoskeletal asymmetry to organelle placement in chlamydomonas

“…Rootlet microtubule polymerization and MLT1 association prior to acetylation is consistent with recent data suggestive that acetylation levels are a function of microtubule lifetime, and acetylation appears minutes to hours after microtubule polymerization [Szyk et al, 2014]. As significant accumulation of the photoreceptors and EYE2 at the nascent eyespot occurs within the same time frame as rootlet acetylation [Mittelmeier et al, 2013], prior association of MLT1 with the rootlet is consistent with the hypothesis that MLT1 promotes directed movement of the photoreceptors.…”

“…The eyespots in mlt1 cells typically occupy more anterior positions than that of the equatorial wild-type eyespot. In dividing mlt1 cells, ChR1 and ChR2 accumulate at the anterior end of the cell and localization to the primary eyespot is delayed, suggestive that the MLT1 protein promotes photoreceptor movement along the D4 rootlet [Mittelmeier et al, 2013]. Here we identify MLT1 as a large, lowcomplexity protein that is specifically associated with D4 microtubules from their minus ends near the basal bodies to the position of the eyespot.…”

“…Like flagellar microtubules, the rootlet microtubules are highly acetylated [LeDizet and Piperno, 1986]. The daughter basal body matured just prior to the most recent cell division, assembly of the associated D4 and D2 rootlets began during metaphase, and the rootlet microtubules were acetylated shortly thereafter [Gould, 1975;Gaffal, 1988;Mittelmeier et al, 2013;O'Toole and Dutcher, 2014]. The mother basal body and rootlets were assembled and acetylated during a previous division.…”

“…This precise placement of the eyespot at an asymmetric position relative to the flagella is crucial for phototaxis [Witman, 1993]. Eyespot proteins such as the channelrhodopsin photoreceptors, ChR1 and ChR2 [Schmidt et al, 2006;Berthold et al, 2008;Sineshchekov et al, 2009], first begin to accumulate at the distal (plus end) tips of the newly polymerized D4 microtubules during cell division, and ChR1 has been observed along the outer surface of the D4 rootlet in interphase cells [Mittelmeier et al, 2011[Mittelmeier et al, , 2013. These data prompted the hypothesis that D4 microtubules direct trafficking of the photoreceptors from the anterior end of the cell to the position of the eyespot, but the identity of putative trafficking components and the basis for their specificity for D4 microtubules remains unknown.…”

A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom

“…Parallel cortical arrays of microtubules in interphase give way to microtubules lining the cytokinetic cleavage site (Johnson and Porter, 1968;Piasecki et al, 2008;Mittelmeier et al, 2013). We employed antimicrotubule immunofluorescence to assess the status of microtubules in arrested mutants, using deconvolution microscopy ( Figure 6).…”