Tradeoff between enzyme and metabolite efficiency maintains metabolic homeostasis upon perturbations in enzyme capacity

Substrate metabolite concentrations are inversely related to the in vivo capacity of their converting enzymes.Local metabolite responses represent a passive mechanism to achieve metabolic homeostasis upon perturbations in enzyme capacity.Enzyme capacity and metabolite concentration control the metabolic reaction rate

Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system

Physical principles underlying structure, mechanics and dynamic re-organization of hyaluronan-rich matrices - from tissues to supramolecular models in experiment and theory

154 p.The goal of this research project is to understand the physical principles that underlie the structure, mechanics and dynamic reorganization of hyaluronan-rich pericellular and extracellular matrices. Many cells produce a carbohydrate-rich cellular coat that plays a crucial role in the protection of the cell and which is also vital in structuring and communicating with the cell¿s environment. Important examples of such self-organizing supramolecular structures are the hyaluronan-rich pericellular matrices that are found for example around oocytes or endothelial cells. An outstanding feature is their dynamic self-organization into large, hydrated matrices. The supra-molecular level of organization that results from the assembly of glycans and proteins into soft, hydrated networks gives rise to new qualities and functions, which differ from those characterizing the individual constituents. The work involved in this project, based on a state of the art biophysical charactization tool box and polymer theory, contributed to understanding the relation between the organizational and dynamic features of such supramolecular assemblies, their physicochemical (in particular mechanical) properties, and the resulting biological functions.CIC biomaGUNE: biomaterialetako Ikerkuntza Kooperatibiko Zentroa; Centro de Investigación Cooperativa en Biomateriales. Max-Planck-Institute for Intelligence System

Nutrient availability modulating physiology and pathogenicity of <i>Legionella pneumophila</i>

A virulent strain of Legionella pneumophila serogroup 1 was established in continuous culture under defined iron-replete conditions at pH 6.9. Iron-limitation and extremes of pH (6.0 and 7.8) influenced the growth and metabolism of L. pneumophila, as manifested by increased metabolic activity, impaired energy coupling, and altered metabolic fluxes. In particular, the physiological versatility of L. pneumophila was demonstrated by a significant decrease in the iron content of biomass (6-fold increase in Yiron), coupled with reduced metabolic efficiency (Ycarbon), in response to iron-limited growth. Iron limitation promoted the accumulation of significant intracellular reserves of poly- ß-hydroxybutyrate (16 % cell dry wt.), which supported long-term survival of L. pneumophila under starvation conditions. Expression of the important pathogenicity factor, the zinc metalloprotease, was regulated by iron availability. Common iron acquisition mechanisms, such as siderophores and transferrin receptors, were not elaborated by iron-limited cells. However, human transferrin was identified as a potential iron source for L. pneumophila, with the zinc metalloprotease mediating transferrin digestion and possibly iron acquisition. Iron-limitation and extremes of pH also influenced cellular morphology and the surface properties of L. pneumophila, promoting the formation of uniform cultures of short rod-shaped bacteria, with altered fatty acid, phospholipid and protein composition. In addition to morphological and physiological adaptation, iron limitation had a significant effect on the virulence of L. pneumophila. Iron-replete cells grown at pH 6.9 and 6.0 were highly virulent in a guinea pig model. However, the virulence of L. pneumophila was significantly attenuated (P L. pneumophila, inducing the expression of distinct phenotypes differing in their ability to persist in nature and cause infection

Nutrient availability modulating physiology and pathogenicity of <i>Legionella pneumophila</i>

A virulent strain of Legionella pneumophila serogroup 1 was established in continuous culture under defined iron-replete conditions at pH 6.9. Iron-limitation and extremes of pH (6.0 and 7.8) influenced the growth and metabolism of L. pneumophila, as manifested by increased metabolic activity, impaired energy coupling, and altered metabolic fluxes. In particular, the physiological versatility of L. pneumophila was demonstrated by a significant decrease in the iron content of biomass (6-fold increase in Yiron), coupled with reduced metabolic efficiency (Ycarbon), in response to iron-limited growth. Iron limitation promoted the accumulation of significant intracellular reserves of poly- ß-hydroxybutyrate (16 % cell dry wt.), which supported long-term survival of L. pneumophila under starvation conditions. Expression of the important pathogenicity factor, the zinc metalloprotease, was regulated by iron availability. Common iron acquisition mechanisms, such as siderophores and transferrin receptors, were not elaborated by iron-limited cells. However, human transferrin was identified as a potential iron source for L. pneumophila, with the zinc metalloprotease mediating transferrin digestion and possibly iron acquisition. Iron-limitation and extremes of pH also influenced cellular morphology and the surface properties of L. pneumophila, promoting the formation of uniform cultures of short rod-shaped bacteria, with altered fatty acid, phospholipid and protein composition. In addition to morphological and physiological adaptation, iron limitation had a significant effect on the virulence of L. pneumophila. Iron-replete cells grown at pH 6.9 and 6.0 were highly virulent in a guinea pig model. However, the virulence of L. pneumophila was significantly attenuated (P L. pneumophila, inducing the expression of distinct phenotypes differing in their ability to persist in nature and cause infection

Evaluation of spore wall chemistry as a novel biochemical proxy for UV·B radiation

The stratospheric ozone layer provides protection for Earth's land-based organisms against harmful ultraviolet (UV) radiation, but the past century has seen the ozone layer compromised as a result of human activity, resulting in commensurate variations in surface UV -B flux. Despite the importance of UV radiation to the well-being of life, reliable records of surface UV-B flux only exist for a short period of time (20-30 years). In order to gain a deeper understanding of the behaviour of ozone and UV-B flux in the past, an alternative method of determining UV-B is required. Changes in spore chemistry have been proposed as a palaeo-monitor of UV-B flux, which can then be related to stratospheric ozone abundance. By employing the rapid and inexpensive technique of FTIR microspectroscopy to investigate changes in spore chemistry, a large dataset spanning seven different spatial and temporal UV regimes has been generated in order to evaluate the feasibility of routine usage of a spore-based UV-B proxy. Exploring contemporary samples grown under controlled UV conditions and spores preserved in historical archives reveals that modem-day and recent spore chemistry show a positive relationship with known and calculated UV-B flux, with additional environmental factors such as cloudiness and vegetation canopy cover superimposed on these results. Examination of fossil spores obtained from sediments and experimentally matured specimens provides an insight into the chemical changes that occur in organic matter after incorporation into sediments, even under mild burial conditions. This thesis explores the potential of spore chemistry to act as a proxy monitor of past UV-B flux and is the first concerted attempt at applying FTIR spectroscopy to the investigation of spore chemistry in this way

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

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