GCP-WD mediates γ-TuRC recruitment and the geometry of microtubule nucleation in interphase arrays of Arabidopsis.

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis, but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1, associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

Arabidopsis TANGLED identifies the division plane throughout mitosis and cytokinesis.

BACKGROUND: In premitotic plant cells, the future division plane is predicted by a cortical ring of microtubules and F-actin called the preprophase band (PPB). The PPB persists throughout prophase, but is disassembled upon nuclear-envelope breakdown as the mitotic spindle forms. Following nuclear division, a cytokinetic phragmoplast forms between the daughter nuclei and expands laterally to attach the new cell wall at the former PPB site. A variety of observations suggest that expanding phragmoplasts are actively guided to the former PPB site, but little is known about how plant cells "remember" this site after PPB disassembly. RESULTS: In premitotic plant cells, Arabidopsis TANGLED fused to YFP (AtTAN::YFP) colocalizes at the future division plane with PPBs. Strikingly, cortical AtTAN::YFP rings persist after PPB disassembly, marking the division plane throughout mitosis and cytokinesis. The AtTAN::YFP ring is relatively broad during preprophase/prophase and mitosis; narrows to become a sharper, more punctate ring during cytokinesis; and then rapidly disassembles upon completion of cytokinesis. The initial recruitment of AtTAN::YFP to the division plane requires microtubules and the kinesins POK1 and POK2, but subsequent maintenance of AtTAN::YFP rings appears to be microtubule independent. Consistent with the localization data, analysis of Arabidopsis tan mutants shows that AtTAN plays a role in guidance of expanding phragmoplasts to the former PPB site. CONCLUSIONS: AtTAN is implicated as a component of a cortical guidance cue that remains behind when the PPB is disassembled and directs the expanding phragmoplast to the former PPB site during cytokinesis.