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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. 

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.