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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.

The critical regions of interaction between the Kif18B tail and its binding partners, importin α/β and EB1, are defined, and it is shown how these interactions control Kif18B function in cells and activity in vitro. A new mechanism is uncovered by which the spatial activity of Kif18B is controlled through the RanGTP pathway.

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.

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 bindingin 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 provides a spatial control mechanism to modulate MT binding of NLS-containing spindle assembly factors.

Flowering plants evolved away from creating centrosomes or conventional microtubule organizing centers. Therein, plants have posed a long-standing challenge to many of the conventional ideas for mitotic spindle construction and the process of chromosome segregation. The Arabidopsis seedling has emerged as a leading model for plant cell biological studies of the cytoskeleton and vesicle trafficking. Here we describe methods for creating a reusable chamber for mitotic studies in both seedling root and shoot cells with instruction for best practices with conventional microscopic techniques.

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.

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.

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

Microtubules at the plant cell cortex influence cell shape by patterning the deposition of cell wall materials. The elongated cells of the hypocotyl create a variety of microtubule array patterns with differing degrees of polymer coalignment and orientation to the cell's growth axis. To gain insight into the mechanisms driving array organization, we investigated the underlying microtubule array architecture in light-grown epidermal cells with explicit reference to array pattern. We discovered that all nontransverse patterns share a common underlying array architecture, having a core unimodal peak of coaligned microtubules in a split bipolarized arrangement. The growing microtubule plus ends extend toward the cell's apex and base with a region of antiparallel microtubule overlap at the cell's midzone. This core coalignment continuously shifts between ±30° from the cell's longitudinal growth axis, forming a continuum of longitudinal and oblique arrays. Transverse arrays exhibit the same unimodal core coalignment but form local domains of microtubules polymerizing in the same direction rather than a split bipolarized architecture. Quantitative imaging experiments and analysis of katanin mutants showed that the longitudinal arrays are created from microtubules originating on the outer periclinal cell face, pointing to a cell-directed, rather than self-organizing, mechanism for specifying the major array pattern classes in the hypocotyl cell.

Construction of the cell plate during plant cell division requires the precise insertion of materials around the circumferentially growing phragmoplast. New work shows that two kinesin-4 motor proteins act to shorten the domain of overlapping microtubules at the phragmoplast perimeter, limiting the site of material deposition.

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 advent of super-resolution techniques in biological microscopy has opened new frontiers for exploring the molecular distribution of proteins and small molecules in cells. Improvements in optical design and innovations in the approaches for the collection of fluorescence emission have produced substantial gains in signal from chemical labels and fluorescent proteins. Structuring the illumination to elicit fluorescence from specific or even random patterns allows the extraction of higher order spatial frequencies from specimens labeled with conventional probes. Application of this approach to plant systems for super-resolution imaging has been relatively slow owing in large part to aberrations incurred when imaging through the plant cell wall. In this brief review, we address the use of two prominent methods for generating super-resolution images in living plant specimens and discuss future directions for gaining better access to these techniques.

Parenting is a challenging activity and many parents report high stress and feelings of incompetence. Both of these factors (a) stress and (b) feelings of incompetence are associated with a variety of negative parenting outcomes. This study evaluated the effectiveness of a community-based, solution-focused, 2-session parent consultation intervention on parent perceptions of stress and competence. A pre-post quasi-experimental design was employed. Forty-five consecutive parents who sought consultation services were administered three preintervention questionnaires. Results included positive outcomes across all three outcome measures as well as high ratings on a satisfaction questionnaire. Although significant reductions in parenting stress and increased parenting self-efficacy were obtained, the study design and small and homogeneous sample limit generalization of these findings. Nevertheless, this study highlights the possibility that a straightforward, positive, brief, and community-based intervention may have the potential to decrease parental stress and increase parenting sense of competence.

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.

The therapeutic alliance is foundational to counseling practice and has amassed strong empirical support as being essential for successful counseling. Counselors generally rely on their own perspective when assessing the quality of the alliance, though the client's perspective has been found to be a better predictor of outcome. Formal methods for eliciting client feedback about the alliance and counseling outcomes have been strongly supported in the literature, yet such limitations as time constraints hinder counselor efforts to gather formal client feedback. Two ultra-brief measures of alliance and outcome, the Session Rating Scale and the Outcome Rating Scale, are feasible methods for counselors to secure client feedback. This article reviews the two measures and makes a case for using empirical means to understand adult clients' views of the therapeutic alliance.

Summary Background Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. Results Here, we develop the first Forster resonance energy transfer (FRET)-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging, we find that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. Conclusions Unlike motile kinesins, which are open when doing work, the high-affinity binding state for microtubule-depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule-depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of kinesin-13s can be rapidly modulated to control cellular microtubule dynamics.

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.

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly ( 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid-like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.

Graphical abstractDisplay Omitted Highlights? Monte Carlo simulations of microtubule dynamics with templated nucleation. ? Nucleation and polymer extinction form a second, interdependent steady state process in the array. ? Survey of microtubule dynamics parameter shifts shows sensitivity to catastrophe frequency. ? Starting subunit concentration and cell border show significant effects on array morphology. Microtubule polymers typically function through their collective organization into a patterned array. The formation of the pattern, whether it is a relatively simple astral array or a highly complex mitotic spindle, relies on controlled microtubule nucleation and the basal dynamics parameters governing polymer growth and shortening. We have investigated the interaction between the microtubule nucleation and dynamics parameters, using macroscopic Monte Carlo simulations, to determine how these parameters contribute to the underlying microtubule array morphology (i.e. polymer density and length distribution). In addition to the well-characterized steady state achieved between free tubulin subunits and microtubule polymer, we propose that microtubule nucleation and extinction constitute a second, interdependent steady state process. Our simulation studies show that the magnitude of both nucleation and extinction additively impacts the final steady state free subunit concentration. We systematically varied individual microtubule dynamics parameters to survey the effects on array morphology and find specific sensitivity to perturbations of catastrophe frequency. Altering the cellular context for the microtubule array, we find that nucleation template number plays a defining role in shaping the microtubule length distribution and polymer density.

The Arabidopsis thaliana MAP65-1 and MAP65-2 genes are members of the larger eukaryotic MAP65/ASE1/PRC gene family of microtubule-associated proteins. We created fluorescent protein fusions driven by native promoters that colocalized MAP65-1 and MAP65-2 to a subset of interphase microtubule bundles in all epidermal hypocotyl cells. MAP65-1 and MAP65-2 labeling was highly dynamic within microtubule bundles, showing episodes of linear extension and retraction coincident with microtubule growth and shortening. Dynamic colocalization of MAP65-1/2 with polymerizing microtubules provides in vivo evidence that plant cortical microtubules bundle through a microtubule-microtubule templating mechanism. Analysis of etiolated hypocotyl length in map65-1 and map65-2 mutants revealed a critical role for MAP65-2 in modulating axial cell growth. Double map65-1 map65-2 mutants showed significant growth retardation with no obvious cell swelling, twisting, or morphological defects. Surprisingly, interphase microtubules formed coaligned arrays transverse to the plant growth axis in dark-grown and GA4-treated light-grown map65-1 map65-2 mutant plants. We conclude that MAP65-1 and MAP65-2 play a critical role in the microtubule-dependent mechanism for specifying axial cell growth in the expanding hypocotyl, independent of any mechanical role in microtubule array organization.

SummaryThe role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1–3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays—termed an amphiaster (“a star on both sides” [4])—that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ∼35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP—the small GTPase Ran in its GTP bound state—could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.

We generalize the Dogterom–Leibler model for microtubule dynamics (Dogterom and Leibler in Phys Rev Lett 70(9):1347–1350, 1993) to the case where the rates of elongation as well as the lifetimes of the elongating shortening phases are a function of GTP-tubulin concentration. We analyze also the effect of nucleation rate in the form of a damping term which leads to new steady-states. For this model, we study existence and stability of steady states satisfying the boundary conditions at x = 0. Our stability analysis introduces numerical and analytical Evans function computations as a new mathematical tool in the study of microtubule dynamics.

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.

SummaryRan is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2–5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7–9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10–12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14 XCKT2 is important for spindle assembly and pole organization [15–20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/β from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here, we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP gradient. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/β. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation.

Plant cell morphogenesis depends critically on two processes: the deposition of new wall material at the cell surface and the mechanical deformation of this material by the stresses resulting from the cell's turgor pressure. We developed a model of plant cell morphogenesis that is a first attempt at integrating these two processes. The model is based on the theories of thin shells and anisotropic viscoplasticity. It includes three sets of equations that give the connection between wall stresses, wall strains and cell geometry. We present an algorithm to solve these equations numerically. Application of this simulation approach to the morphogenesis of tip-growing cells illustrates how the viscoplastic properties of the cell wall affect the shape of the cell at steady state. The same simulation approach was also used to reproduce morphogenetic transients such as the initiation of tip growth and other non-steady changes in cell shape. Finally, we show that the mechanical anisotropy built into the model is required to account for observed patterns of wall expansion in plant cells.

Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level.

As the legume-rhizobia symbiosis is established, the plant recognizes bacterial-signaling molecules, Nod factors (NFs), and initiates transcriptional and developmental changes within the root to allow bacterial invasion and the construction of a novel organ, the nodule. Plant mutants defective in nodule initiation (Nod - ) are thought to have defects in NF-signal transduction. However, it is unknown whether WT plants respond to NF-independent bacterial-derived signals or whether Nod - plant mutants show defects in global symbiosis-associated gene expression. To characterize plant gene expression in the establishment of the symbiosis, we used an Affymetrix oligonucleotide microarray representing 9,935 Medicago truncatula expressed sequences. We identified 46 sequences that are differentially expressed in plants exposed for 24 h to WT Sinorhizobium meliloti or to the invasion defective S. meliloti mutant, exoA . Eight of these genes encode nucleolar proteins, which are implicated in ribosome biogenesis. We also identified differentially expressed transcription factors, signaling components, defense response proteins, stress response proteins, and several previously uncharacterized genes. NF appears both necessary and sufficient to induce most changes. Six of seven Nod - M. truncatula mutants ( nfp , dmi1 , dmi2 , dmi3 , nsp1 , and nsp2 ) showed no transcriptional response to S. meliloti , suggesting that the encoded proteins are required for initiating new transcription. The Nod - mutant hcl , however, exhibits a reduced transcriptional response to S. meliloti , indicating that the machinery responsible for initiating new transcription is at least partially functional in this mutant.

Calcineurin is a Ca 2+ - and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae , calcineurin is required for survival during several environmental stresses, including high concentrations of Na + , Li + , and Mn 2+ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor. We have identified Hph1p as a novel substrate of calcineurin. HPH1 ( YOR324C ) and its homolog HPH2 ( YAL028W ) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum. Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress. Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo. Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN. This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin. In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin. Ca 2+ -induced Crz1p-mediated transcription is unaffected in hph1 Δ hph2 Δ mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin. Thus, Hph1p and Hph2p are components of a novel Ca 2+ - and calcineurin-regulated response required to promote growth under conditions of high Na + , alkaline pH, and cell wall stress.

Plant cells create highly structured microtubule arrays at the cell cortex without a central organizing center to anchor the microtubule ends. In vivo imaging of individual microtubules in Arabidopsis plants revealed that new microtubules are initiated at the cell cortex and exhibit dynamics at both ends. Polymerization-biased dynamic instability at one end and slow depolymerization at the other end result in sustained microtubule migration across the cell cortex by a hybrid treadmilling mechanism. This motility causes widespread microtubule repositioning and contributes to changes in array organization through microtubule reorientation and bundling.

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.

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.

The bipolar budding pattern of a/α Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2-μm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 μm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.

The wealth of information from genetic and more recently genomic studies of the budding yeast has been staggering. However, the small size of the organism has limited traditional cytological approaches. Thus there is a disparity in our detailed understanding of the genetic control of mitosis, for instance, relative to the morphology of mitosis. The application of time-lapse high-resolution digital-enhanced differential interference contrast (DE-DIC) and multimode fluoresence microscopy to studying yeast cell division has revealed a very dynamic process that reflects the interplay among microtubule dynamics, microtubule-based motor proteins, and positional determinants in the mother and bud (Salmon et al., 1998a ,b ; Shaw et al., 1997a ,b ).

The interphase microtubule arrays in flowering plant cells assemble at the cell cortex into patterns that affect cellular morphogenesis. A decade of live cell imaging studies has provided significant information about the in vivo properties of the microtubule polymers. Efforts to extrapolate individual properties to larger roles in organizing or patterning the microtubule array have produced models focused on self-organization and local levels of biological control. Recent studies looking at cortical microtubule arrays as they transition from an existing pattern to a new pattern have re-emerged as a testbed for examining these models and the molecular hypotheses underpinning them. The evidence suggests that microtubule patterning is locally controlled on the scale of a cell face, using or circumventing self-organizating properties as necessary.

222, 84-94 Over the past five years, photographic and video-tape documentation of microscopic data has gradually be- come outmoded and has been super- seded by digital image acquisition systems. There are many advantages of digital images over film or video. Acquisition is convenient and fast, and images can be duplicated without loss of information. Furthermore, digital images provide quantitative and pos- itional data maps of specimens, mak- ing them amenable to digital process- ing to enhance features of interest and extract information that is not readily apparent to the eye. With the advent of the digital-imaging age has come the problem of how to present digital movies in a video world. Televisions, monitors and projectors in the USA adhere to the National Televisions Systems Committee (NTSC) standard signal with 525 horizontal lines of resolution scanned at 30 times a second; most of Western Europe uses the phase-alternating line system (PAL) standard of 625 lines at 25 Hz, while in France and Eastern Europe 'Sequential Couleur ~ Memoire' (SECAM, sequen- tial colour with memory), which is similar in resolution to PAL, is standard video format. By contrast, modern computer displays typically offer res- olutions nearly twice as good as video (e.g. 1280) 150 Hz. In preparing for presentations at major scientific meetings, we were recently confronted with the task of presenting our time-lapse series of digi- tal fluorescence images of living cells using the audio visual (A/V) equipment available, without degrading the qual- ity of our data. We produced 20-min movies on super-video (S-VHS) tape, including timed text, graphics and movie clips. This article outlines the equipment requirements for this type of small-scale scientific digital movie production (see Table 1 ).

Recent studies using Fucus embryos indicate that the cell wall relays positional information to the underlying plasma membrane and cytosol to establish a polar axis, initiate polar growth and orient the first cell division plane of the zygote. It is the formation and stabilization of the polar axis in the zygote — not the position of the first division plane — which is critical for the formation of the initial embryonic pattern in Fucus . The pattern of asymmetry in the cell wall set at the time of polar axis fixation may also be the basis for the determination of rhizoid and thallus characteristics in the embryo. The cell division in early Arabidopsis embryos is very similar to that in Fucus embryos, and thus these findings may be pertinent to higher plants.

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.

Computers dominate image capture and analysis in modern light microscopy. The output of an imaging experiment is a binary coded file, called an image file, which contains the spatial, temporal and intensity information present in the sample. Understanding what comprises an image file, and how these files are generated is necessary in order to optimize the use of the digital light microscope. In this chapter, we discuss image file formats, and the various components of these files, such as bit-depth, sampling rate, color theory, and compression, from the perspective of the non-computer scientist. We also discuss the problem of proprietary file formats, and how these often are incompatible with certain types of imaging software. We present several solutions to this issue. Finally, we present the use of digital movie formats, compression routines, and provide some real world examples for optimizing the generation of digital movies.

The advent of fluorescent proteins and access to modern imaging technologies have dramatically accelerated the pace of discovery in plant cell biology. Remarkable new insights into such diverse areas as plant pathogenesis, cytoskeletal dynamics, sugar transport, cell wall synthesis, secretory control, and hormone signaling have come from careful examination of living cells using advanced optical probes. New technologies, both commercially available and on the horizon, promise a continued march toward more quantitative methods for imaging and for extending the optical exploration of biological structure and activity to molecular scales. In this review, we lay out fundamental issues in imaging plant specimens and look ahead to several technological innovations in molecular tools, instrumentation, imaging methods, and specimen handling that show promise for shaping the coming era of plant cell biology.

The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell's midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces.