Fabienne Furt

Characterization of Lipid Rafts from Medicago truncatula Root Plasma Membranes: A Proteomic Study Reveals the Presence of a Raft-Associated Redox System

Several studies have provided new insights into the role of sphingolipid/sterol-rich domains so-called lipid rafts of the plasma membrane (PM) from mammalian cells, and more recently from leaves, cell cultures, and seedlings of higher plants. Here we show that lipid raft domains, defined as Triton X-100-insoluble membranes, can also be prepared from Medicago truncatula root PMs. These domains have been extensively characterized by ultrastructural studies as well as by analysis of their content in lipids and proteins. M. truncatula lipid domains are shown to be enriched in sphingolipids and Δ7-sterols, with spinasterol as the major compound, but also in steryl glycosides and acyl-steryl glycosides. A large number of proteins (i.e. 270) have been identified. Among them, receptor kinases and proteins related to signaling, cellular trafficking, and cell wall functioning were well represented whereas those involved in transport and metabolism were poorly represented. Evidence is also given for the presence of a complete PM redox system in the lipid rafts.

Proteomics of Plant Detergent-resistant Membranes

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.

Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells.

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F-actin-associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F-actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F-actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F-actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F-actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI-associated structures at the cells apex. In addition, this approach uncovered a mechanism for actin polymerization-dependent motility in the moss cytoplasm, where myosin XI-associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI-associated vesicular structures and F-actin polymerization-driven motility function at the cells apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F-actin in tip growing cells.

Biochemical survey of the polar head of plant glycosylinositolphosphoceramides unravels broad diversity

Although Glycosyl-Inositol-Phospho-Ceramides (GIPCs) are the main sphingolipids of plant tissues, they remain poorly characterized in term of structures. This lack of information, notably with regard to polar heads, currently hampers the understanding of GIPC functions in biological systems. This situation prompted us to undertake a large scale-analysis of plant GIPCs: 23 plant species chosen in various phylogenetic groups were surveyed for their total GIPC content. GIPCs were extracted and their polar heads were characterized by negative ion MALDI and ESI mass spectrometry. Our data shed light on an unexpected broad diversity of GIPC distributions within Plantae, and the occurrence of yet-unreported GIPC structures in green and red algae. In monocots, GIPCs with three saccharides were apparently found to be major, whereas a series with two saccharides was dominant in Eudicots within a few notable exceptions. In plant cell cultures, GIPC polar heads appeared to bear a higher number of glycan units than in the tissue from which they originate. Perspectives are discussed in term of GIPC metabolism diversity and function of these lipids.

Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells

BackgroundIn the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells.ResultsWe used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles.ConclusionsTo our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles from different tip growing plant cells, thus enhancing our understanding of the mechanisms of plant polarized cell growth.

Phylogenetic analysis of the Kinesin superfamily from physcomitrella.

Kinesins are an ancient superfamily of microtubule dependent motors. They participate in an extensive and diverse list of essential cellular functions, including mitosis, cytokinesis, cell polarization, cell elongation, flagellar development, and intracellular transport. Based on phylogenetic relationships, the kinesin superfamily has been subdivided into 14 families, which are represented in most eukaryotic phyla. The functions of these families are sometimes conserved between species, but important variations in function across species have been observed. Plants possess most kinesin families including a few plant specific families. With the availability of an ever increasing number of genome sequences from plants, it is important to document the complete complement of kinesins present in a given organism. This will help develop a molecular framework to explore the function of each family using genetics, biochemistry, and cell biology. The moss Physcomitrella patens has emerged as a powerful model organism to study gene function in plants, which makes it a key candidate to explore complex gene families, such as the kinesin superfamily. Here we report a detailed phylogenetic characterization of the 71 kinesins of the kinesin superfamily in Physcomitrella. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analysis of function. Some of the families, such as kinesins 14s are composed of fewer members in moss, while other families, such as the kinesin 12s are greatly expanded. To improve the comparison between species, and to simplify communication between research groups, we propose a classification of subfamilies based on our phylogenetic analysis.

Plant lipid rafts: fluctuat nec mergitur.

Lipid rafts in plasma membranes are hypothesized to play key roles in many cellular processes including signal transduction, membrane trafficking and entry of pathogens. We recently documented the biochemical characterization of lipid rafts, isolated as detergent-insoluble membranes, from Medicago truncatula root plasma membranes. We evidenced that the plant-specific lipid steryl-conjugates are among the main lipids of rafts together with free sterols and sphingolipids. An extensive proteomic analysis showed the presence of a specific set of proteins common to other lipid rafts, plus the presence of a redox system around a cytochrome b561 not previously identified in lipid rafts of either plants or animals. Here, we discuss the similarities and differences between the lipids and proteins of plant and animal lipid rafts. Moreover we describe the potential biochemical functioning of the M. truncatula root lipid raft redox proteins and question whether they may play a physiological role in legume-symbiont interactions.

Myosin XI localizes at the mitotic spindle and along the cell plate during plant cell division in Physcomitrella patens

Cell division is a fundamental biological process that has been extensively investigated in different systems. Similar to most eukaryotic cells, plant cells assemble a mitotic spindle to separate replicated chromosomes. In contrast, to complete cell division, plant cells assemble a phragmoplast, which is composed of aligned microtubules and actin filaments. This structure helps transport vesicles containing new cell wall material, which then fuse to form the cell plate; the cell plate will expand to create the new dividing cell wall. Because vesicles are known to be transported by myosin motors during interphase, we hypothesized this could also be the case during cell division and we investigated the localization of the plant homologue of myosin V - myosin XI, in cell division. In this work, we used the protonemal cells of the moss Physcomitrella patens as a model, because of its simple cellular morphology and ease to generate transgenic cell lines expressing fluorescent tagged proteins. Using a fluorescent protein fusion of myosin XI, we found that, during mitosis, this molecule appears to associate with the kinetochores immediately after nuclear envelope breakdown. Following metaphase, myosin XI stays associated with the spindles midzone during the rest of mitosis, and when the phragmoplast is formed, it concentrates at the cell plate. Using an actin polymerization inhibitor, latrunculin B, we found that the association of myosin XI with the mitotic spindle and the phragmoplast are only partially dependent on the presence of filamentous actin. We also showed that myosin XI on the spindle partially overlaps with a v-SNARE vesicle marker but is not co-localized with the endoplasmic reticulum and a RabA vesicle marker. These observations suggest an actin-dependent and an actin-independent behavior of myosin XI during cell division, and provide novel insights to our understanding of the function of myosin XI during plant cell division.