Search for: All records

ABSTRACT 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 inArabidopsishypocotyl cells and investigate the sites of QWRF interaction with cortical microtubules using transient transformation of fluorescently tagged constructs in the heterologousNicotiana benthamianasystem. 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. 

ABSTRACT Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation inBacillus subtilisusing a fluorescent fusion to the C-ring protein FliM. We find thatB. subtilisbasal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site.Bacillus subtilisassembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show thatB. subtilisbasal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella ofB. subtilisis different from the polar patterning of other bacteria, and we infer that theB. subtilisrod probes the PG for holes that can accommodate the machine. 

Cortical microtubules influence plant cell shape by guiding cellulose deposition. Epidermal hypocotyl cells in Arabidopsis thaliana create distinct cortical microtubule array patterns to enable axial cell growth. How these array patterns are created and maintained during cell wall formation is a critical and unsolved problem in cell biology. Previous work showed that arrays aligned longitudinally with the cell's growth axis have a “split bipolar” organization, with microtubules treadmilling toward the apical or basal ends of the cell from a region of antiparallel overlap at the cell's midzone. The underlying order or architecture of these coaligned arrays prompted us to ask whether microtubules oriented transversely to the cell's axis are organized to a similar degree. Creating new fluorescently tagged End-Binding Protein 1b (EB1b) probes to circumvent gain-of-function effects observed for GFP-EB1b, we found that transverse arrays form persistent, nearly unipolar domains of microtubules treadmilling around the short axis of the cell, independent of the EB1b probe used. Our findings reveal an organizational strategy for transverse arrays distinct from that of longitudinal arrays, with implications for the mechanisms of array pattern creation and maintenance.