1,039 research outputs found

    In vivo Analysis and Modeling Reveals that Transient Interactions of Myosin XI, its Cargo, and Filamentous Actin Overcome Diffusion Limitations to Sustain Polarized Cell Growth

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    Tip growth is a ubiquitous process throughout the plant kingdom in which a single cell elongates in one direction in a self-similar manner. To sustain tip growth in plants, the cell must regulate the extensibility of the wall to promote growth and avoid turgor-induced rupture. This process is heavily dependent on the cytoskeleton, which is thought to coordinate the delivery and recycling of vesicles containing cell wall materials at the cell tip. Although significant work has been done to elucidate the various molecular players in this process, there remains a need for a more mechanistic understanding of the cytoskeletonÂ’s role in tip growth. For this reason, specific emphasis should be placed on understanding the dynamics of the cytoskeleton, its associated motors, and their cargo. Since the advent of fluorescence fusion technology, various quantitative fluorescence dynamics techniques have emerged. Among the most prominent of these techniques is fluorescence recovery after photobleaching (FRAP). Despite its prominence, it is unclear how to interpret fluorescence recoveries in confined cellular geometries such as tip-growing cells. Here we developed a digital confocal microscope simulation of FRAP in tip-growing cells. With this simulation, we determined that fluorescence recoveries are significantly influenced by cell boundaries. With this FRAP simulation, we then measured the diffusion of VAMP72-labeled vesicles in the moss Physcomitrella patens. Using finite element modeling of polarized cell growth, and the measured VAMP72-labeled vesicle diffusion coefficient, we were able to show that diffusion alone cannot support the required transport of wall materials to the cell tip. This indicates that an actin-based active transport system is necessary for vesicle clustering at the cell tip to support growth. This provides one essential function of the actin cytoskeleton in polarized cell growth. After establishing the requirement for actin-based transport, we then sought to characterize the in vivo binding interactions of myosin XI, vesicles, and filamentous actin. Particle tracking evidence from P. patens protoplasts suggests that myosin XI and VAMP72-labeled vesicles exhibit fast transient interactions. Hidden Markov modeling of particle tracking indicates that myosin XI and VAMP72- labeled vesicles move along actin filaments in short-lived linear trajectories. These fast transient interactions may be necessary to achieve the rapid dynamics of the apical actin, important for growth. This work advances the fieldÂ’s understanding of fluorescence dynamics, elucidates a necessary function of the actin cytoskeleton, and provides insight into how the components of the cytoskeleton interact in vivo

    Persistent Symmetry Frustration in Pollen Tubes

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    Pollen tubes are extremely rapidly growing plant cells whose morphogenesis is determined by spatial gradients in the biochemical composition of the cell wall. We investigate the hypothesis (MP) that the distribution of the local mechanical properties of the wall, corresponding to the change of the radial symmetry along the axial direction, may lead to growth oscillations in pollen tubes. We claim that the experimentally observed oscillations originate from the symmetry change at the transition zone, where both intervening symmetries (cylindrical and spherical) meet. The characteristic oscillations between resonating symmetries at a given (constant) turgor pressure and a gradient of wall material constants may be identified with the observed growth-cycles in pollen tubes

    Reverse Genetics Approach to Examine Myosin XI Functions in Pollen Tube Growth

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    Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which non-motile sperm cells are delivered to ovules thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth depends on actin filaments. Plants encode class VIII and class XI myosins as actin-based motor proteins, of which class XI myosins are required for cell expansion in vegetative tissues. In Arabidopsis thaliana, 6 of 13 myosin XI genes are expressed in pollen: XIA, XIB, XIC, XID, XIE, and XIJ. Initially, two artificial microRNA constructs were designed to target multiple class XI myosins; however, plants expressing the artificial microRNAs had no reduction in overall fertility and only a slight reduction in root hair elongation. Therefore, to explore the functions of individual pollen myosins, homozygous T-DNA insertion mutants were isolated for all six pollen myosin genes. Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, xic xie pollen tubes grew more slowly than WT pollen tubes, which resulted in reduced fitness compared to WT and a drastic reduction in seed set. Organelle motility was significantly reduced in xic xie pollen tubes; however, vesicle accumulation and actin filament dynamics were not altered in the double mutant. Thus, it remains unclear how reduced organelle motility in xic xie pollen tubes leads to a slower growth rate. A novel role of myosin XI in pollen germination was also revealed in this study. Pollen from mutants of XIJ, the only short-tailed myosin XI in Arabidopsis, germinated poorly in vitro. This in vitro pollen germination defect was rescued by the addition of diffusible components from female tissues. Interestingly in pollen grains, YFP-XIJ and YFP-XIA localized to the future site of germination, suggesting a role in pollen germination for multiple myosin XI isoforms. In summary, this study provided the first direct evidence that class XI myosins are involved in pollen tube growth and pollen germination

    EXO70C2 is a key regulatory factor for optimal tip growth of pollen

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    The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes

    Cell wall composition regulates cell shape and growth behaviour in pollen tubes

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    L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style.One of the most important features characterizing plant cells and differentiating them from animal cells is the cell wall that surrounds them. The cell wall plays a critical role in providing protection to the protoplast; it acts as a filtering mechanism and is the location of many biochemical reactions implicated in the regulation of the cell metabolism and the mechanical properties of the cell. The local stiffness, extensibility, plasticity and elasticity of the different cell wall components determine the shape and geometry of the cell during differentiation and morphogenesis. The goal of my thesis is to understand the role played by the different cell wall components in shaping the plant cell and controlling its growth behaviour. To achieve this goal, I studied the pollen tube, or male gametophyte, as a cellular model system. The pollen tube is a cellular protuberance formed by the pollen grain upon its contact with the stigma. Its main purpose is to deliver the sperm cells to the female gametophyte to ensure double fertilization. The pollen tube is a tip-growing cell characterized by its simple cell wall composition and by the fact that it is the fastest growing cell of the plant kingdom. This makes it the ideal model to study the effects of drugs, mutations or stresses on cellular growth behaviour, metabolism and cell wall mechanics. The pollen tube cell wall consists mainly of proteins and three major polysaccharidic components: pectins, cellulose and callose. To understand the role played by these components in regulating pollen tube growth, I investigated the effects of mutations, enzymatic treatments, hyper-gravity and omni-directional gravity on the pollen tube cell wall. Using mathematical modeling combined with molecular biology and high-resolution electron and fluorescent microscopy I was able to show that the regulation of pectin chemistry is required for the regulation of the growth rate and pollen tube shape and that cellulose is crucial for determining the pollen tube diameter in the sup-apical region. Moreover, I investigated the role of the pectate lyases, a group of pectin digesting enzymes expressed during pollen tube development, and I showed that this enzyme activity is required for the initiation of pollen germination. More importantly, I directly showed for the first time that the pollen tube secretes cell wall loosening enzymes to facilitate its penetration through the style

    Imaging vesicle trafficking and organelle dynamics in living fungal hyphae

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    Modélisation par éléments finis de la croissance du tube pollinique.

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    RÉSUMÉ Dans le domaine de la biologie végétale, la reproduction sexuelle des plantes par pollinisation est un sujet d’intérêt présentant plusieurs enjeux humains et économiques. Le tube pollinique est une cellule végétale indispensable à la pollinisation des fleurs. La croissance de la cellule végétale se fait par une déformation de la paroi l’entourant sous l’effet de la pression hydrostatique interne, la turgescence. Simultanément, de nouveaux matériaux doivent être livrés vers la paroi pour empêcher qu’elle n’éclate à cause de l'amincissement causé par l'étirement. Chez le tube pollinique, l'expansion de surface est localisée à l’apex de la cellule et le résultat du processus est une protubérance de forme cylindrique. Ce mode de croissance combiné au système biologique particulier du tube pollinique en fait un modèle privilégié pour les études mécaniques des cellules végétales en croissance. De nombreux modèles portant sur la croissance apicale ont été construits afin d’étudier les paramètres et processus impliqués. Ces modèles présentent différentes manières d’aborder le problème que ce soit des approches géométriques, mathématiques ou mécaniques mais dans chacun des constantes demeurent comme l’axisymétrie du tube. La méthode des éléments finis permet de représenter des formes complexes ou de changer facilement les modèles mathématiques utilisés pour les calculs. Ce potentiel est particulièrement adapté pour l’étude de structures biologiques amenées à changer de forme ou de propriétés mécaniques. Cette approche de modélisation par éléments finis a été privilégiée dans ce projet. Le premier objectif de ce projet de maitrise visait à développer un modèle par éléments finis représentant la croissance du tube pollinique permettant de changer facilement les paramètres mécaniques, géométriques et de chargement du tube pollinique. Le modèle a été construit en se basant sur l’observation expérimentale, soit un long cylindre avec un rayon de 6 μm, terminé par un apex de la forme d’un sphéroïde prolate. La paroi, d’une épaisseur moyenne fixée à 50 nm, a été modélisée par des éléments de type SHELL 181 via un maillage structuré. Ces éléments possèdent un comportement de type «coque» adéquat pour représenter la paroi et pourraient être modifiés dans le futur pour intégrer la viscoplasticité. Le tube a cependant été soumis à un comportement linéaire élastique dans un premier temps. Le coefficient de Poisson utilisé était alors fixé à 0.3. Les principaux paramètres du modèle incluaient entre autres les modules d’Young de la paroi cellulaire ainsi que leur gradient le long du tube, dans le but de tenir compte----------ABSTRACT In the field of plant biology, sexual reproduction through pollination is a topic of interest since it affects human life and economic issues. The pollen tube is a plant cell that is indispensable for the pollinisation of flowers. Plant cell growth occurs by a deformation of the surrounding wall under the effect of an internal hydrostatic pressure, the turgor pressure. At the same time, new material has to be delivered to the wall in order to prevent bursting caused by the thinning of the wall that in turn is a result of the turgor induced stretching. In the pollen tube, surface growth is located at the apex of the cell and it leads to the formation of a cylindrical protuberance. This type of growth, combined to the particular biological system of the pollen tube, provides an excellent model for mechanical studies of growing plant cells. Numerous models for apical growth have been used to study the parameters and processes involved. These models present different methods to approach the problem whether it is geometrical, mathematical or mechanical. However, most of these models depend on the axisymmetry of the tubular structure. The finite element method allows one to represent complex shapes or to easily change the mathematical models used for calculation. This potential is particularly suited for the study of biological structures that are able to change shape or to manipulate their mechanical properties. The finite element modeling approach has been adopted in this project. The first objective of this project was to develop a finite element model representing the growth of the pollen tube that would allow to easily change mechanical parameters, geometry and loading. The model was constructed based on experimental observation. It consisted in a long cylinder with a radius of 6 μm, terminated by an apex shaped as a prolate spheroid. The wall, with an average thickness fixed at 50 nm was modeled by elements of type SHELL 181 via a structured mesh. These elements behave as a thin "shell" and are thus an appropriate choice to represent the relatively thin wall of the pollen tube. Although they offer the possibility to incorporate viscoplastic material properties, we have chosen a linear elastic behavior for this first version of the model. The value for Poisson’s coefficient was fixed at 0.3. The main parameters of the model included the Young’s modulus of the cell wall and its gradient along the tube, in order to take into account the changing biochemical composition of the cell wall between the apex and the cylindrical region. To cover a large number of possible cases, different values fo

    Cell wall mediated regulation of plant cell morphogenesis : pectin esterification and cellulose crystallinity

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    La morphogenèse cellulaire est une composante fondamentale du développement d’un organisme. Toute cellule végétale est entourée de parois régulant sa morphogenèse. Cette matrice extra-cellulaire est principalement composée de polysaccharides. Afin de montrer le lien entre la forme et la fonction d’une cellule il est primordial de comprendre la façon dont ces polysaccharides sont modifiés durant le développement et l’expansion cellulaire. Chez les plantes, la mécanique de l'expansion cellulaire est principalement régulée par la cellulose, le biopolymère le plus abondant sur Terre. Comme les microfibrilles de cellulose présentent une forte résistance à la traction le long de leur axe d’orientation principal, elles réguleraient le processus d'expansion en conférant des propriétés mécaniques aux composants pariétaux qui contrôlent l’ampleur et la directivité de la croissance expansive au niveau subcellulaire. Les homogalacturonanes, type de pectine le plus abondant des parois cellulaires primaires, sont des biopolymères également susceptibles d’agir sur l’expansion cellulaire. La distribution spatiale et le degré d’estérification des pectines homogalacturonanes affectent les propriétés mécaniques de la paroi et par conséquent le pattern d’expansion. J'ai utilisé une approche génétique combinée à des stratégies novatrices de biochimie, de biomécanique et d’imagerie, afin de comprendre comment la dynamique spatio-temporelle de la cellulose et des homogalacturonanes régule l'expansion et la morphogenèse cellulaires. Pour ce faire, je me suis basé sur l’étude de deux types de cellules épidermiques de formes différentes: celles du cotylédon et de l'hypocotyle d'Arabidopsis thaliana. J’ai prouvé que la formation des ondulations des cellules fondamentales du cotylédon nécessite des modifications spatiales et temporelles des microfibrilles de cellulose et des pectines déméthylestérifiées. Ces modifications régulent la rigidité mécanique de la paroi péricline à deux moments distincts : lors de l’initiation de la formation du lobe et lors de son expansion ultérieure. L’initiation de la formation du lobe requiert une augmentation de la rigidité de la paroi péricline au niveau des potentielles saillies de l’ondulation, et ce par une accumulation locale de pectines déméthylestérifiées. L’expansion ultérieure est quant à elle contrôlée par le degré de cristallinité de la cellulose et par l’alignement perpendiculaire des microfibrilles tangentiellement aux saillies de l’ondulation de la paroi péricline. Durant l'élongation et l'expansion anisotrope des cellules épidermiques de l’hypocotyle, la cellulose et les pectines homogalacturonanes jouent des rôles distincts lors de chaque phase d'élongation. Durant la première phase de développement, une réduction du taux de pectines déméthylestérifiées diminue la rigidité de la paroi et accélère l’élongation des cellules. Lors de la seconde phase du développement, une réduction de la cristallinité de la cellulose diminue la vitesse d’élongation de l’hypocotyle. À partir de l’étude des deux systèmes cellulaires, nous pouvons conclure que, contrairement à l’hypothèse acceptée de longue date, la cellulose ne serait pas un élément essentiel au déclenchement d’évènements morphogénétiques mais qu’elle jouerait plutôt un rôle au sein de mécanismes de rétroaction accentuant le processus de morphogenèse. De plus, la morphogenèse induite par des contraintes joue un rôle clé lors des étapes initiales et serait dépendante du degré d’estérification des pectines. Mes expériences permettent de corréler les données de mécanique cellulaire expérimentale à la biologie cellulaire fonctionnelle et à la génétique.Cellular morphogenesis is a fundamental underpinning of development. All cells in the plant kingdom are surrounded by walls that govern shape formation. This extracellular matrix is composed mainly of polysaccharides. How these polysaccharides are modified during cellular development to regulate cell expansion, and thus cell shape, must be understood to link form with function. In plants, the mechanical aspect of cell expansion is known to be mainly influenced by cellulose, the most abundant biopolymer on Earth. Because cellulose microfibrils exhibit a strong tensile strength along their long axis, they may be used to control the expansion process by conferring mechanical properties to the cell wall material that determine the directionality and the magnitude of expansive growth at subcellular level. Another wall polymer that may influence cell expansion is homogalacturonan pectin, the most abundant type of pectin in the primary wall. The spatial distribution and esterification status of homogalacturonan pectin may affect the mechanical aspects of the wall and, therefore, the expansion pattern. I used a genetic approach combined with novel biochemical, biomechanical and imaging strategies to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin during cell expansion and shape formation. I investigated cell shape formation in two differently shaped types of epidermal cells: those of the cotyledon and of the hypocotyl of Arabidopsis thaliana. I show that undulation formation in pavement cells of the cotyledon requires spatial and temporal changes of cellulose microfibrils and demethyl-esterified pectin. These changes regulate the mechanical stiffness of the periclinal wall at two different stages: lobe initiation and subsequent expansion. Lobe initiation involves an increase in the stiffness of the periclinal wall at the prospective neck region of the undulation through a local accumulation of demethyl-esterified pectin. The subsequent expansion is controlled by the degree of cellulose crystallinity and the perpendicular alignment of the microfibrils at the tangent of the neck side of the undulation at the periclinal wall. During the elongation process and the anisotropic expansion of the epidermal hypocotyl cells, cellulose and homogalacturonan pectin make distinct contributions in each developmental phase of the elongation. During the first developmental phase, reduction in the proportion of demethyl-esterified pectin decreases the wall stiffness and accelerates the elongation. A reduction in the cellulose crystallinity decreases the elongation of the hypocotyl at the second developmental phase. It may be concluded from the two cell systems that cellulose, contrary to a long-established hypothesis, may not be essential for the initiation of morphogenetic events and their function may be reassigned to the feedback-mediated augmentation of cell shaping processes. Moreover, stress-induced shape formation plays a key role during the initiating steps and it is likely to be dominated by the degree of pectin esterification. My data link experimental cell mechanics to functional cell biology and genetics
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