Protuberance Formation Research Articles

A dynamic WUSCHEL/Layer 1 interplay directs shoot apical meristem formation during regeneration in tobacco.

De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early-stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues and is characterized by autonomous initiation of cellular patterning. Here, we report on the initial stages of SAM organogenesis and on the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli starts with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. We also exposed a complex interdependent relationship between L1 and WUS expression and revealed that any disruption in this interplay compromises shoot formation. Silencing WUS in nascent protuberances prevented L1 formation and caused the disorganization of the outer cell layers exhibiting both anticlinal and periclinal divisions, suggesting WUS plays a critical role in the proper establishment and organization of L1 during SAM organogenesis. We further discovered that silencing TONNEAU1 prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity and L1 formation, ultimately impeding SAM formation and regeneration. This study provides a novel molecular framework for the characterization of a WUS/L1 interplay that mediates SAM formation during regeneration.

Stability of stationary points for one-dimensional Willmore energy with spatially heterogeneous term

We consider a variational problem that consists of the Willmore energy of planar curves and a linear term in the curvature with a spatially heterogeneous coefficient. This coefficient is assumed to be a piecewise constant function. The variational problem arises from the modeling of the epidermal membrane in human skin. It has been predicted that the spatial heterogeneity of cell adhesion is one of the important factors in the protuberance formation of the membrane. We investigate the existence and stability of stationary points represented by symmetric graphs and find a condition for the existence of such stationary points by using the methods developed in the study of the Navier problem for Willmore energy. Especially, we give a necessary and sufficient condition for the existence of symmetric stationary points in terms of the gap size of the spatial heterogeneity. Moreover, we show some results on the stability of these stationary points.

Cooperation between β‐ and γ‐cytoplasmic actins in the mechanical regulation of endothelial microparticle formation

Elevated endothelial microparticle (MP) levels are observed in numerous diseases, increasingly supporting roles as effectors and valuable markers of vascular dysfunction. While a contractile role for the actin cytoskeleton has been implicated in vesiculation, i.e., MP production, the precise interactions and mechanisms of its constituents, β- and γ-cytoplasmic actins, is unknown. Human cerebral microvascular endothelial cells were stimulated with known agonists, and vesiculation development was monitored by scanning electron microscopy (SEM) and flow cytometry. These data in combination provide new insight into the kinetics, patterns of vesiculating cell recruitment, and degrees of response specific to stimuli. Reorganization of β- and γ-actins, F-actin, vinculin, and talin accompanied significant MP release. β-Actin redistribution into basal stress fibers following stimulation was associated with increased apically situated actin-rich particulate structures, which in turn directly correlated with electron-lucent membrane protrusions observed by SEM. Y-27632 Rho-kinase inhibition abolished basal β-actin fiber formation, minimizing apically associated actin-rich structures, significantly reducing membrane protrusions and MP release to near basal levels. Cytoskeletal protein expression and distribution varied between MPs and mother cells, as determined by Western blot. These data strongly suggest that β-actin plays an active facilitative role in agonist-induced protuberance formation, through mechanical interactions with newly described actin-rich structures.

Finite Element Modeling of Polar Growth in Walled Cells

Walled cells such as those of plants and fungi grow by expanding their cellular surface driven by the intracellular turgor pressure. The generation of a particular cellular shape necessitates the precise spatial control of mechanical properties in the polymer-network forming the cell wall. To model protuberance formation in walled cells we established a finite element model. We aimed to identify the requirements for spatial distribution of mechanical properties in the cell wall that would allow the generation of tubular shapes that agree with experimental observations on the pollen tube, a rapidly elongating plant cell. We based our structural model on the parameterized description of a tip growing cell that allows the manipulation of cell size, shape, cell wall thickness and local mechanical properties. The mechanical load was applied in the form of hydrostatic pressure. We used two validation methods to compare different simulations based on cellular shape and the displacement of surface markers. We compared the resulting optimal distribution of cell mechanical properties with the spatial distribution of biochemical cell wall components in pollen tubes and found remarkable agreement between the gradient in mechanical properties and the distribution of de-esterified pectin.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Finite Element Model of Polar Growth in Pollen Tubes

Cellular protuberance formation in walled cells requires the local deformation of the wall and its polar expansion. In many cells, protuberance elongation proceeds by tip growth, a growth mechanism shared by pollen tubes, root hairs, and fungal hyphae. We established a biomechanical model of tip growth in walled cells using the finite element technique. We aimed to identify the requirements for spatial distribution of mechanical properties in the cell wall that would allow the generation of cellular shapes that agree with experimental observations. We based our structural model on the parameterized description of a tip-growing cell that allows the manipulation of cell size, shape, cell wall thickness, and local mechanical properties. The mechanical load was applied in the form of hydrostatic pressure. We used two validation methods to compare different simulations based on cellular shape and the displacement of surface markers. We compared the resulting optimal distribution of cell mechanical properties with the spatial distribution of biochemical cell wall components in pollen tubes and found remarkable agreement between the gradient in mechanical properties and the distribution of deesterified pectin. Use of the finite element method for the modeling of nonuniform growth events in walled cells opens future perspectives for its application to complex cellular morphogenesis in plants.

A NIMA‐related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules

To study cellular morphogenesis genetically, we isolated loss-of-function mutants of Arabidopsis thaliana, designated ibo1. The ibo1 mutations cause local outgrowth in the middle of epidermal cells of the hypocotyls and petioles, resulting in the formation of a protuberance. In Arabidopsis, the hypocotyl epidermis differentiates into two alternate cell files, the stoma cell file and the non-stoma cell file, by a mechanism involving TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA2 (GL2). The ectopic protuberances of the ibo1 mutants were preferentially induced in the non-stoma cell files, which express GL2. TTG1-dependent epidermal patterning is required for protuberance formation in ibo1, suggesting that IBO1 functions downstream from epidermal cell specification. Pharmacological and genetic analyses demonstrated that ethylene promotes protuberance formation in ibo1, implying that IBO1 acts antagonistically to ethylene to suppress radial outgrowth. IBO1 is identical to NEK6, which encodes a Never In Mitosis A (NIMA)-related protein kinase (Nek) with sequence similarity to Neks involved in microtubule organization in fungi, algae, and animals. The ibo1-1 mutation, in which a conserved Glu residue in the activation loop is substituted by Arg, completely abolishes its kinase activity. The intracellular localization of GFP-tagged NEK6 showed that NEK6 mainly accumulates in cytoplasmic spots associated with cortical microtubules and with a putative component of the gamma-tubulin complex. The localization of NEK6 is regulated by the C-terminal domain, which is truncated in the ibo1-2 allele. These results suggest that the role of NEK6 in the control of cellular morphogenesis is dependent on its kinase action and association with the cortical microtubules.

A procedure for shoot organogenesis in vitro from leaves and nodes of an elite Eucalyptus gunnii clone: comparative histology

In Eucalyptus, shoot regeneration has mainly been achieved from juvenile material (i.e. seedlings). Here, we present a procedure for shoot regeneration from different organs (leaves, internodes, nodes) of micropropagated E. gunnii clones derived from selected trees using a basal regeneration medium that included 0.04 μM picloram and 2.25 μM N6-benzyladenine (BA). About 10% of the leaves or internodes regenerated an average of four buds after 10–12 weeks of culture and more than 50% of the nodes regenerated an average of 20 buds after 6–8 weeks of culture. Explant browning was reduced by increasing the BA concentration in a two-step procedure. The ability to regenerate buds was strictly correlated with the formation of protuberances of chlorophyll-containing cells from the different organs tested. Node-derived protuberances originated from pre-existing meristematic areas, whereas leaf-derived protuberance formation involved the leafs vascular system. Histological studies revealed that adventitious buds originated from the peripheral layers of both leaf-derived and node-derived protuberances.

Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn(II)-phthalocyanine

The destructive process of mouse MS-2 fibrosarcoma induced by photodynamic therapy (PDT) with liposome-administered Zn(II)-phthalocyanine (ZnPc) was studied by electron microscopy. Pronounced ultrastructural changes characteristic of apoptosis were observed for several tumour cells, including early occurrence of condensation and margination of chromatin, disappearance of nuclear pores, karyopyknosis, karyorrhexis, protuberance formation at the cell surface and cell fragmentation. The findings indicate that apoptosis was involved in the process of tumour cell death induced by ZnPc-PDT. The detailed mechanism and pathways controlling this phenomenon need to be elucidated further.

Morphological responses of dissociated sponge cells to different organic substrata

To study interactions between sponge cells and components of the extracellular matrix (ECM), cells of the calcareous sponge Clathrina cerebrum were investigated in vitro by scanning electron microscopy. Cells were settled on glass coverslips, used as controls, and on coverslips coated with various ECM components (laminin, collagens and fibronectin), and with an adhesive substance (polylysine). Cells tended to conserve a rounded shape, producing thin, stiff processes (scleropodia) and lamellipodia, whose shape and extension varied according to the substrata. Spreading was observed only on polylysine, inducing cells to assume a fibroblast-like aspect. On laminin, cell adhesion was assured only by scleropodia. On fibronectin, scleropodia and lamellipodia were present, but reduced in size and length. On collagens, laminar processes occurred among prevailing scleropodia. Measurements of cell area and perimeter allowed statistical comparison of substrata, on the basis of their induction of cell flattening and protuberance formation. In summary, sponge cells were found to modulate their morphology in response to the external environment, expressing features for dynamic activities most fully in the presence of substances close to their natural ECM constituents. These results are discussed in the context of tissue rearrangement as a basic adaptation occurring throughout the life span of these organisms.

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Experimental analysis of polar body formation in starfish eggs

AbstractIn polar body formation, a protuberance develops on the oocyte surface and subsequently separates from it by a process resembling normal cytokinesis. Protuberance formation has usually been attributed to controlled cytoplasmic outflow associated with local surface weakening. This investigation was designed to ascertain by physical manipulation more information concerning the physical mechanisms that accomplish the form changes. In unoperated Pisaster giganteus and Asterias vulgaris oocytes, unsymmetrical polar body formation was associated with failure of the mitotic apparatus to assume its normal orientation perpendicular to the oocyte surface. Needles inserted perpendicular to the plane of division straddling the mitotic apparatus can block separation of the polar body depending on the distance between them. Consequences of moving the mitotic apparatus with a needle were consistent with those in oocytes in which the mitotic apparatus was spontaneously maloriented. Shifting all or part of the mitotic apparatus resulted in polar body formation activity if the apparatus was close enough to the surface. Shifting at interphase did not affect the surface until the next meiotic cycle. Shifting during meiotic activity resulted in rapid local protuberance formation at the new site (av. 1.74 min, n=21). Increasing internal hydrostatic pressure by flattening the oocyte did not enhance cytoplasmic flow into the normal protuberance and usually caused it to recede rapidly. When oocytes were immersed in 50% seawater, the osmotic swelling did not enhance the flow of cytoplasm into the protuberance and the point of rupture showed no tendency to be localized in the polar body area. There did not appear to be a cytochalasin B resistant form change during starfish polar body formation. Extensive tears in the oocyte surface, which prompted massive cytoplasmic outflow, had no effect on the form of the nearby protuberance. Experiments indicated clear differences in response to hydrostatic pressure between normal early stages of polar body formation and similarly sized excrescences formed on the oocyte by mechanical or chemical weakening of a restricted part of the surface. They suggest that the protuberance of normal polar body formation may not result from local surface weakening.

Ultrastructural Development of Mesophyll Cells in Rice Seedlings

Ultrastructural observations were made on the mesophyll cells in leaves at different stages of development. In the ground meristem of the leaf primordium, the plastids and mitochondria, both are approximately the same in dimention, locate around the large nucleus (Fig. 1). The plastids contain several lamellae and small starch grains (Fig. 4). The plastids in the intercalary meristem have more developed lamellae and larger starch grains than those in the leaf primordium. The protuberance formation of the arm-palisade cell progresses in parallel with the cell enlargement, and has completed just before emerging from sheath (Fig. 18). At the stage of cell enlargement, the number of lamellae, the size of starch grains and the rudimentary grana increase in the plastids (Fig. 6, 10). The dividing plastids are frequently observed (Fig. 6). The plasma membrane of the vacuolated cells is observed to possess numerous invaginations (Fig. 7). These invaginations continue to increase in size and project into the large central vacuole (Fig. 8, 9). These structures are bounded by two membranes, an outer tonoplast and inner invaginated plasma membranes. Some invaginations may separate from the peripheral cytoplasm and subsequently attach to the plastids (Fig. 10, 11, 12). The inner membranes of the invagination attached to the plastid, are connected with the plastid envelope. It is assumed that the invaginations may possibly be related to the formation of plastid envelope. In the leaf portion emerged out from sheath, all of the plastids take up a peripheral position in the cell and increase in size (Fig. 14). Another prominent features of the plastids are the increase in both the number of grana and thylakoids per granum (Fig. 16). The microbodies of 0.5-1.0 μm in diameter, are found in the mesophyll cells of expanded leaves (Fig. 17), but they are not found at the stage of cell division and cell enlargement.