Analysis of the membrane binding mechanism of Remorins and their role in beneficial endosymbioses

The plasma membrane is highly organized and within the plasma membrane proteins cluster into so-called membrane domains. Remorins are well-established membrane domain marker proteins. However, the general plasma membrane anchoring mechanism of these proteins was so far unknown. Biochemical approaches and localization studies investigating different remorins from Medicago truncatula and Arabidopsis thaliana enabled us to demonstrate that S-acylation (palmitoylation) within a C-terminal plasma membrane anchoring motif mediates tight plasma membrane attachment of these proteins. However, we could show that S-acylation is not the sole driving force for remorin immobilization in membrane nanodomains. The focus of the second part of this thesis was on the beneficial interaction between plants and symbionts. More than 80% of today´s land plants can undergo an interaction with endosymbiotic fungi that is known as Arbuscular Mycorrhiza (AM). In addition, legume plants have gained the ability to establish a second type of endosymbiosis by interacting with nitrogen-fixing rhizobia: the Root Nodule Symbiosis (RNS). Both interactions are partly controlled by the same pathway, the so-called Common Symbiosis Pathway (CSP) that has evolved through recruitment of signaling components from the evolutionary older AM to the more recently evolved RNS signaling pathway. Depending on the recognition of either fungi or rhizobia downstream of this pathway two morphologically different symbiotic structures are formed within the inner root cortex, either arbuscules or root nodules, respectively. In parallel to the evolution of RNS a local negative regulatory circuit must have evolved to suppress root nodule organogenesis when both interacting symbionts are present and arbuscule formation takes place. In this study first evidence for such a postulated regulatory pathway is presented based on the characterization of the legume-specific remorin MYCREM, which co-evolved with RNS. Phenotypic studies of mutant plants revealed that in the presence of both symbionts MYCREM functions as a negative regulator with respect to root nodule organogenesis events in a CSP-dependent manner. Analyzing the effect of overexpression of auto-active CSP-signaling components, which are known to spontaneously induce root nodule organogenesis, demonstrated a negative regulatory function of MCYREM as well. In summary, this work could serve as basis for further studies to understand the tripartite interaction of legume plants, fungi and rhizobia, as it is found in nature

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

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

Analysis of the membrane binding mechanism of Remorins and their role in beneficial endosymbioses

The plasma membrane is highly organized and within the plasma membrane proteins cluster into so-called membrane domains. Remorins are well-established membrane domain marker proteins. However, the general plasma membrane anchoring mechanism of these proteins was so far unknown. Biochemical approaches and localization studies investigating different remorins from Medicago truncatula and Arabidopsis thaliana enabled us to demonstrate that S-acylation (palmitoylation) within a C-terminal plasma membrane anchoring motif mediates tight plasma membrane attachment of these proteins. However, we could show that S-acylation is not the sole driving force for remorin immobilization in membrane nanodomains. The focus of the second part of this thesis was on the beneficial interaction between plants and symbionts. More than 80% of today´s land plants can undergo an interaction with endosymbiotic fungi that is known as Arbuscular Mycorrhiza (AM). In addition, legume plants have gained the ability to establish a second type of endosymbiosis by interacting with nitrogen-fixing rhizobia: the Root Nodule Symbiosis (RNS). Both interactions are partly controlled by the same pathway, the so-called Common Symbiosis Pathway (CSP) that has evolved through recruitment of signaling components from the evolutionary older AM to the more recently evolved RNS signaling pathway. Depending on the recognition of either fungi or rhizobia downstream of this pathway two morphologically different symbiotic structures are formed within the inner root cortex, either arbuscules or root nodules, respectively. In parallel to the evolution of RNS a local negative regulatory circuit must have evolved to suppress root nodule organogenesis when both interacting symbionts are present and arbuscule formation takes place. In this study first evidence for such a postulated regulatory pathway is presented based on the characterization of the legume-specific remorin MYCREM, which co-evolved with RNS. Phenotypic studies of mutant plants revealed that in the presence of both symbionts MYCREM functions as a negative regulator with respect to root nodule organogenesis events in a CSP-dependent manner. Analyzing the effect of overexpression of auto-active CSP-signaling components, which are known to spontaneously induce root nodule organogenesis, demonstrated a negative regulatory function of MCYREM as well. In summary, this work could serve as basis for further studies to understand the tripartite interaction of legume plants, fungi and rhizobia, as it is found in nature

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin