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Bacterial peptidoglycan (PG) synthesis requires strict spatial and temporal organization to reproduce specific cell shapes. In the ovoid-shaped, pathogenic bacterium Streptococcus pneumoniae (Spn), septal and peripheral (sidewall-like) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Using fluorescent D-amino acid (FDAA) probes, we show 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. Short pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Control experiments in wild-type and mutant strains support the interpretation of nodal spacing of TP activity, and statistical analysis confirmed that the number of nodes correlates with different ring diameters. This nodal pattern of FDAA labeling is conserved in other ovoid-shaped species. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal a highly ordered PG synthesis apparatus in ovococcal bacteria at different stages of division.SIGNIFICANCEThe spatial organization of PBPs and their TP activity at division septa is not well understood. In some bacteria, TP activity and PBP localization seem to be nodal (also called punctate), whereas in other bacteria, discrete foci of PBP activity are infrequently or not observed. Here we report two basic properties of the organization of PBPs and TP activity in the ovoid-shaped bacterium Spn. First, there is distinct spatial separation of the sPG machine, including FtsZ, from the pPG synthesis machine at the midcell of dividing Spn cells. Second, in predivisional cells, PBPs and TP activity are organized heterogeneously into regularly spaced nodes, whose number and dynamic distribution are likely driven by the PG synthesis of PBP:SEDS complexes.

Auxin plays a central role in controlling plant cell growth and morphogenesis. Application of auxin to light-grown seedlings elicits both axial growth and transverse patterning of the cortical microtubule cytoskeleton in hypocotyl cells. Microtubules respond to exogenous auxin within 5 min, although repatterning of the array does not initiate until 30 min after application and is complete by 2 h. To examine the requirements for auxin-induced microtubule array patterning, we used an Arabidopsis (Arabidopsis thaliana) double auxin f-box (afb) receptor mutant, afb4-8 afb5-5, that responds to conventional auxin (indole-3-acetic acid) but has a strongly diminished response to the auxin analog, picloram. We show that 5 µm picloram induces immediate changes to microtubule density and later transverse microtubule patterning in wild-type plants, but does not cause microtubule array reorganization in the afb4-8 afb5-5 mutant. Additionally, a dominant mutant (axr2-1) for the auxin coreceptor AUXIN RESPONSIVE2 (AXR2) was strongly suppressed for auxin-induced microtubule array reorganization, providing additional evidence that auxin functions through a transcriptional pathway for transverse patterning. We observed that brassinosteroid application mimicked the auxin response, showing both early and late microtubule array effects, and induced transverse patterning in the axr2-1 mutant. Application of auxin to the brassinosteroid synthesis mutant, diminuto1, induced transverse array patterning but did not produce significant axial growth. Thus, exogenous auxin induces transverse microtubule patterning through the TRANSPORT INHIBITOR 1/AUXIN F-BOX (TIR1/AFB) transcriptional pathway and can act independently of brassinosteroids.

The growth properties of individual cells within a tissue determine plant morphology, and the organization of the cytoskeleton, particularly the microtubule arrays, determines cellular growth properties. We investigated the mechanisms governing the formation of transverse microtubule array patterns in axially growing Arabidopsis (Arabidopsis thaliana) epidermal hypocotyl cells. Using quantitative imaging approaches, we mapped the transition of the cortical microtubule arrays into a transverse coaligned pattern after induction with auxin and gibberellic acid. Hormone induction led to an early loss of microtubule plus end density and a rotation toward oblique patterns. Beginning 30 min after induction, transverse microtubules appeared at the cell’s midzone concurrently with the loss of longitudinal polymers, eventually progressing apically and basally to remodel the array pattern. Based on the timing and known hormone-signaling pathways, we tested the hypothesis that the later events require de novo gene expression and, thus, constitute a level of genetic control over transverse patterning. We found that the presence of the translation inhibitor cycloheximide (CHX) resulted in a selective and reversible loss of transverse patterns that were replaced with radial-like pinwheel arrays exhibiting a split bipolar architecture centered at the cell’s midzone. Experiments using hormone induction and CHX revealed that pinwheel arrays occur when transverse microtubules increase at the midzone but longitudinal microtubules in the split bipolar architecture are not suppressed. We propose that a key regulatory mechanism for creating the transverse microtubule coalignment in axially growing hypocotyls involves the expression of a CHX-sensitive factor that acts to suppress the nucleation of the longitudinally oriented polymers.

Cortical microtubules play a critical role in plant morphogenesis by creating array patterns that template the deposition of cellulose microfibrils.

Acentrosomal plant microtubule arrays form patterns at the cell cortex that influence cellular morphogenesis by templating the deposition of cell wall materials, but the molecular basis by which the microtubules form the cortical array patterns remains largely unknown. Loss of the Arabidopsis (Arabidopsis thaliana) microtubule-associated protein, CYTOPLASMIC LINKER ASSOCIATED PROTEIN (AtCLASP), results in cellular growth anisotropy defects in hypocotyl cells. We examined the microtubule array patterning in atclasp-1 null mutants and discovered a significant defect in the timing of transitions between array patterns but no substantive defect in the array patterns per se. Detailed analysis and computational modeling of the microtubule dynamics in two atclasp-1 fluorescent tubulin marker lines revealed marker-dependent effects on depolymerization and catastrophe frequency predicted to alter the steady-state microtubule population. Quantitative in vivo analysis of the underlying microtubule array architecture showed that AtCLASP is required to maintain the number of growing microtubule plus ends during transitions between array patterns. We propose that AtCLASP plays a critical role in cellular morphogenesis through actions on new microtubules that facilitate array transitions.

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn). Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase binding partner relative to FtsZ treadmilling in Spn cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in Spn.SignificanceThis study answers two long-standing questions about FtsZ dynamics and its relationship to septal PG synthesis in Spn for the first time. In previous models, FtsZ concertedly moves from midcell septa to MapZ rings that have reached the equators of daughter cells. Instead, the results presented here show that FtsZ, FtsA, and EzrA filaments/bundles move continuously out from early septa as part of MapZ rings. In addition, this study establishes that the movement of bPBP2x:FtsW complexes in septal PG synthesis depends on and likely mirrors new PG synthesis and is not correlated with the treadmilling of FtsZ filaments/bundles. These findings are consistent with a mechanism where septal FtsZ rings organize directional movement of bPBP2x:FtsW complexes dependent on PG substrate availability.

The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the mid-cell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centres of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centres of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodelling.

Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the resilient peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low-GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin-binding proteins (PBPs) between septal and side-wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent-vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2D and 3D-SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3D-SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.

Advances in live-cell imaging technology have provided an unprecedented look at the dynamic behaviors of the plant microtubule cytoskeleton. Recent studies revisit the classic question of how plants create cell shape through the patterned construction of the cell wall. Visualization of the cellulose synthase complex traveling in the plasma membrane has brought a watershed of new information about cellulose deposition. Observation of the cellulose synthase complex tracking precisely over the underlying cortical microtubules has provided clear evidence that the microtubule array pattern serves as a spatial template for cellulose microfibril extrusion. Understanding how the microtubules are organized into specific array patterns remains a challenge, though new ideas are arising from genetic and cell biological studies. Long-term time-lapse observations of the microtubule arrays in light-grown hypocotyl cells have revealed a striking process of microtubule patterning possibly linked to the creation of polylamellate cell walls.

Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. Here, we characterize early calcium responses of wild-type and mutant Medicago truncatula plants to nodulation factors produced by the bacterial symbiont Sinorhizobium meliloti using a dual-dye ratiometric imaging technique. When presented with 1 nm Nod factor, root hair cells exhibited only the previously described calcium spiking response initiating 10 min after application. Nod factor (10 nm) elicited an immediate increase in calcium levels that was temporally earlier and spatially distinct from calcium spikes occurring later in the same cell. Nod factor analogs that were structurally related, applied at 10 nm, failed to initiate this calcium flux response. Cells induced to spike with low Nod factor concentrations show a calcium flux response when Nod factor is raised from 1 to 10 nm. Plant mutants previously shown to be deficient for the calcium spiking response (dmi1 and dmi2) exhibited an immediate, truncated calcium flux with 10 nm Nod factor, demonstrating a competence to respond to Nod factor but an impaired ability to generate a full biphasic response. These results demonstrate that the legume root hair cell exhibits two independent calcium responses to Nod factor triggered at different agonist concentrations and suggests an early branch point in the Nod factor signal transduction pathway.

Live-cell studies have brought fresh insight into the organizational activities of the plant cortical array. Plant interphase arrays organize in the absence of a discrete microtubule organizing center, having plus and minus ends distributed throughout the cell cortex. Microtubule nucleation occurs at the cell cortex, frequently followed by minus-end detachment from origin sites. Microtubules associate tightly with the cell cortex, resisting lateral and axial translocation. Slow, intermitant loss of dimers from minus ends, coupled with growth-biased dynamic instability at the plus ends, results in the migration of cortically attached microtubules across the cell via polymer treadmilling. Microtubule-microtubule interactions, a direct consequence of treadmilling, result in polymer reorientation and creation of polymer bundles. The combined properties of microtubule dynamics and interactions among polymers constitute a system with predicted properties of self-organization.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

We have used a fluorescently-labeled dihydropyridine (FL-DHP) to vitally stain living Fucus zygotes during the establishment of cell polarity. Localization of FL-DHP is primarily at the plasma membrane and FL-DHP binding is competitively blocked by an unlabeled dihydropyridine. Distribution of FL-DHP is initially symmetrical before fixation of the polar axis, but becomes asymmetrical in response to a unilateral light gradient. The distribution of FL-DHP receptors can be relocalized when the direction of the photopolarizing stimulus is changed. Treatment of cells with cytochalasin B prior to axis fixation reversibly prevents localization of FL-DHP receptors. Observation of FL-DHP labeling by time-lapse fluorescence microscopy indicates that the existing receptors are redistributed during polar axis formation. The asymmetric distribution of FL-DHP receptors coincides temporally and spatially with increased local intracellular calcium ion concentrations, as measured by calcium green dextran. Based on the site, timing, photo-reversibility, and actin dependence of the asymmetric localization of FL-DHP receptors, we conclude that FL-DHP is a vital probe for the later stage of polar axis formation in Fucus zygotes. Furthermore, we propose that FL-DHP receptors correspond to ion channels that are transported to the future site of polar growth to create the changes in local calcium concentration required for polarity establishment.