Arabidopsis AUGMIN8 Contains Two Independent Microtubule Association Domains

Plant cells create a plasma membrane-associated network of microtubules that are nucleated by γ-tubulin ring complexes primarily through microtubule-dependent microtubule nucleation (MDMN). This dynamic array organizes into specific patterns in response to developmental and environmental cues to influence primary cell wall construction. The molecular mechanisms directing the creation of cortical microtubule array patterns are largely unknown. The hetero-octameric AUGMIN complex facilitates mitotic spindle formation by associating γ-tubulin ring complexes with existing spindle microtubules and creating parallel branched microtubules through MDMN. AUGMIN8, the key linker protein connecting the AUGMIN complex to the parent microtubule, is encoded by a paralogous family of QWRF genes in flowering plants. Members of the QWRF family are distinguished by an unstructured N-terminal half encoded in a single 5' exon. We hypothesize that the QWRF paralogs form interchangeable AUGMIN microtubule binding subunits that confer specific roles to the AUGMIN complex in mitotic and non-mitotic microtubule arrays. We identify four QWRF family members expressed in Arabidopsis hypocotyl cells and investigate the sites of QWRF interaction with cortical microtubules using transient transformation of fluorescently tagged constructs in the heterologous Nicotiana benthamiana system. We show that full-length QWRF8 and QWRF4 associate with non-mitotic, cortical microtubules as distributed puncta where QWRF8 shows evidence for two independent sites of microtubule association. Sequence comparisons and in vivo assay with homologous fragments from QWRF1, 2, 4, and 5 define a shared N-terminal conserved microtubule association domain. We additionally identify protein regions leading to the formation of microtubule-associated "QWRF bodies" potentially linked to discontinuous localization on microtubules. We identify the "QWRF" protein motif as a conserved domain associating the AUGMIN8 paralogs with AUGMIN6, part of the larger AUGMIN complex.