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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 Light, temperature, water, and nutrient availability influence how plants grow to maximize access to resources. Axial growth, the linear extension of tissues by coordinated axial cell expansion, plays a central role in these adaptive morphological responses. Using Arabidopsis (Arabidopsis thaliana) hypocotyl cells to explore axial growth control mechanisms, we investigated WAVE-DAMPENED2-LIKE4 (WDL4), an auxin-induced, microtubule-associated protein and member of the larger WDL gene family shown to modulate hypocotyl growth under changing environmental conditions. Loss-of-function wdl4 seedlings exhibited a hyper-elongation phenotype under light conditions, continuing to elongate when wild-type Col-0 hypocotyls arrested and reaching 150% to 200% of wild-type length before shoot emergence. wdl4 seedling hypocotyls showed dramatic hyper-elongation (500%) in response to temperature elevation, indicating an important role in morphological adaptation to environmental cues. WDL4 was associated with microtubules under both light and dark growth conditions, and no evidence was found for altered microtubule array patterning in loss-of-function wdl4 mutants under various conditions. Examination of hormone responses showed altered sensitivity to ethylene and evidence for changes in the spatial distribution of an auxin-dependent transcriptional reporter. Our data provide evidence that WDL4 regulates hypocotyl cell elongation without substantial changes to microtubule array patterning, suggesting an unconventional role in axial growth control. 

Abstract Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid‐shapedStreptococcus pneumoniae(Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examinedSpncells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D‐SIM (structured‐illumination microscopy). Labeling with fluorescent D‐amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin‐binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading‐edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA‐marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division. 

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. 

Ovoid-shaped bacteria, such asStreptococcus pneumoniae(pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body. 

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/β as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/β interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/β increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/β have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/β spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal–containing spindle assembly factors. 

Microtubules are essential components of eukaryotic cells. Myriad proteins associate with microtubules to facilitate the organization and operation of microtubule arrays. Various Microtubule Associated Proteins (MAPs) assist the assembly and function of mitotic spindles and interphase arrays. Nine MAP65 genes exist in the genome of the acentrosomal model plant, Arabidopsis thaliana, and the function of majority of these proteins is unclear. To address this knowledge gap, we demonstrate the localization of A. thaliana MAP65-6 and MAP65-7 fusion proteins expressed from native promoters in interphase cells of developing A. thaliana seedlings. Analyses of these fusion proteins co-expressed with alpha-tubulin 6 reporters indicate that MAP65-6 and MAP65-7 bind a subset of interphase microtubules. Co-expression of GFP:MAP65-6 with mCherry:MAP65-2 from native promoters in A. thaliana showed overlapping localization patterns on interphase microtubule bundles. Collectively, these data suggested that MAP65-2, -6, and -7 bind cortical microtubule bundles in plant interphase microtubule arrays.