pH-Controlled Coacervate-Membrane Interactions within Liposomes
Last, Mart G.F. ; Deshpande, Siddharth ; Dekker, Cees - \ 2020
ACS Nano 14 (2020)4. - ISSN 1936-0851 - p. 4487 - 4498.
coacervates - liposomes - liquid−liquid phase separation - membranes - microfluidics
Membraneless organelles formed by liquid-liquid phase separation are dynamic structures that are employed by cells to spatiotemporally regulate their interior. Indeed, complex coacervation-based phase separation is involved in a multitude of biological tasks ranging from photosynthesis to cell division to chromatin organization, and more. Here, we use an on-chip microfluidic method to control and study the formation of membraneless organelles within liposomes, using pH as the main control parameter. We show that a transmembrane proton flux that is created by a stepwise change in the external pH can readily bring about the coacervation of encapsulated components in a controlled manner. We employ this strategy to induce and study electrostatic as well as hydrophobic interactions between the coacervate and the lipid membrane. Electrostatic interactions using charged lipids efficiently recruit coacervates to the membrane and restrict their movement along the inner leaflet. Hydrophobic interactions via cholesterol-tagged RNA molecules provide even stronger interactions, causing coacervates to wet the membrane and affect the local lipid-membrane structure, reminiscent of coacervate-membrane interactions in cells. The presented technique of pH-triggered coacervation within cell-sized liposomes may find applications in synthetic cells and in studying biologically relevant phase separation reactions in a bottom-up manner.
Melamine-Based Microporous Organic Framework Thin Films on an Alumina Membrane for High-Flux Organic Solvent Nanofiltration
Amirilargani, Mohammad ; Yokota, Giovana N. ; Vermeij, Gijs H. ; Merlet, Renaud B. ; Delen, Guusje ; Mandemaker, Laurens D.B. ; Weckhuysen, Bert M. ; Winnubst, Louis ; Nijmeijer, Arian ; Smet, Louis C.P.M. de; Sudhölter, Ernst J.R. - \ 2020
ChemSusChem 13 (2020)1. - ISSN 1864-5631 - p. 136 - 140.
membranes - microporous materials - organic solvent nanofiltration - polymers - porous organic frameworks
Microporous polymer frameworks have attracted considerable attention to make novel separation layers owing to their highly porous structure, high permeability, and excellent molecular separation. This study concerns the fabrication and properties of thin melamine-based microporous polymer networks with a layer thickness of around 400 nm, supported on an α-alumina support and their potential use in organic solvent nanofiltration. The modified membranes show excellent solvent purification performances, such as n-heptane permeability as high as 9.2 L m−2 h−1 bar −1 in combination with a very high rejection of approximately 99 % for organic dyes with molecular weight of ≥457 Da. These values are higher than for the majority of the state-of-the-art membranes. The membranes further exhibit outstanding long-term operation stability. This work significantly expands the possibilities of using ceramic membranes in organic solvent nanofiltration.
In vivo 1H NMR methods to study dynamics of chloroplast water and thylakoid membrane lipids in leaves and in photosynthetic microorganisms
Pagadala, Shanthi - \ 2017
Wageningen University. Promotor(en): H. van Amerongen, co-promotor(en): H. van As. - Wageningen : Wageningen University - ISBN 9789463431569 - 130
cell membranes - membranes - chloroplasts - thylakoids - photosynthesis - in vivo experimentation - stress conditions - stress - proteins - lipids - mobility - dynamics - celmembranen - membranen - chloroplasten - thylakoïden - fotosynthese - in vivo experimenten - stress omstandigheden - stress - eiwitten - lipiden - mobiliteit - dynamica
Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for in vitro studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete in vivo conditions in leaf disks of F. benjamina and A. platanoides and in suspensions of the green alga Chlamydomonas reinhardtii and blue-green alga Synechocystissp.PCC 6803.
In leaf disks of Ficus benjamina and Acer platanoides, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in F. benjamina but not in A. platanoides) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of Synechocystissp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different.
Thermo-responsive block copolymers : synthesis, self-assembly and membrane development
Mocan Cetintas, Merve - \ 2017
Wageningen University. Promotor(en): F.A.M. Leermakers, co-promotor(en): M.M.G. Kamperman. - Wageningen : Wageningen University - ISBN 9789463431583 - 177
polymer chemistry - polymers - membranes - synthesis - self assembly - thermal properties - polymeerchemie - polymeren - membranen - synthese - zelf-assemblage - thermische eigenschappen
Block copolymers (BCPs) are remarkable materials because of their self-assembly behavior into nano-sized regular structures and high tunable properties. BCPs are in used various applications such as surfactants, nanolithography, biomedicine and nanoporous membranes. In these thesis, we aimed to fabricate thermo-responsive iso- and nanoporous membranes from BCPs.
First, we optimized the synthesis of a thermo-responsive BCP, i.e. polystyrene-poly(N-isopropyl acrylamide) (PS-PNIPAM) with desired properties using controlled/living polymerization methods. We fabricated membranes using self-assembly and non-solvent induced phase separation (SNIPS) method. The membranes were nanoporous, thermo-responsive and exhibited an interconnected worm-like surface.
We investigated the self-assembly behavior of BCPs using both theoretical and experimental approaches. The theoretical investigation involves self-consistent field modelling of Scheutjens and Fleer (SF-SCF) which is used for the first time for BCP self-assembly phenomena. Using SF-SCF, first, we found a chain length dependence on the critical point of BCP phase diagram which confirms well with the reported literature. Second, we worked on the stability of the common mesophases (e.g. single and double gyroids, double diamond, hexagonally perforated lamellae) that is observed between hexagonally ordered cylindrical (HEX) and lamellar (LAM) phases; at chain length, =300 and at intermediate segregation regime, =30. Among the mentioned mesophases double gyroid was the only phase dominant over HEX and LAM phases. At strong segregation regime of =120 with the same chain length, double gyroid was found as a metastable phase.
The experimental approach of the BCP self-assembly was performed by solvent annealing of BCP thin films. For annealing, common laboratory solvents e.g. methanol, tetrahydrofuran, toluene were used with various ratios to tune the selectivity of the solvent mixtures to the blocks in the copolymer. A lamellar forming triblock copolymer using the solvent mixtures methanol: THF (v:v) 1:2 or methanol: toluene (v:v) 1:1 resulted in HEX phase. In contrast, no sustained long-range order was found when only one type of solvent was used.
Next, we optimized the membrane fabrication parameters to obtain membranes with an isoporous surface. We investigated the effect of solvent selectivity, evaporation time and polymer concentration. For PS selective solvents, membranes exhibited a disordered surface whereas PNIPAM selective solvents resulted in membranes with an isoporous surface. For a large parameter space, isoporous membranes were attained which is not common for SNIPS method. Permeability tests at various temperatures proved fully reversible thermo-responsive behavior of these membranes.
Finally, we concluded our work with future recommendations to obtain block copolymer membranes that have improved properties and suggested tests that will prove membranes’ suitability for industrial applications.
Tuning for light and more : engineering phototrophy and membrane proteins in Escherichia coli
Claassens, Nicolaas J.H.P. - \ 2017
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Vitor Martins dos Santos. - Wageningen : Wageningen University - ISBN 9789463430920 - 328
escherichia coli - phototropism - membranes - proteins - light - photosystem i - gene expression - escherichia coli - fototropie - membranen - eiwitten - licht - fotosysteem i - genexpressie
The application of microbial and plant photosynthesis for biobased production on the one hand has a huge potential but on the other hand photosynthesis has serious limitations regarding its efficiency. Hence, studying both fundamental features of photosynthetic processes and engineering of photosystems is of paramount interest, exploring the engineering of photosystems is the overarching aim of this thesis. As described in Chapter 1, natural photosystems may be modified or transplanted to allow for more efficient conversion of solar light energy into biochemical energy. Hereto ambitious proposals to engineer photosystems have been made, and to realize those endeavors the disciplines of synthetic and systems biology are required. To explore how to apply and improve those disciplines hereto, the work described in this thesis has focused on the transplantation of simple photosystems (proton-pumping rhodopsins; PPRs) into the cell membrane of the heterotrophic model bacterium Escherichia coli. Both in silico analyses, including metabolic and thermodynamic modeling (Chapter 3) and a series of experimental studies on transplanting PPR photosystems (Chapters 4,6 and 7) were performed, which identified several challenges, limitations and most importantly opportunities. This thesis also describes the application of novel tools to substantially improve the functional production of PPRs and a variety of other membrane proteins in E. coli.
Chapter 2 provides more details on previously reported examples of heterologous expression of PPRs in several hosts, and on the physiological impact of these transplanted photosystems. Based on this evaluation, some suggestions are made to improve and further exploit the transplantation of these photosystems.
In Chapter 3 a systematic, integrated in silico analysis is made of anaerobic, photo-electro-autotrophic synthetic metabolism in E. coli, consisting of (i) a PPR photosystem for ATP regeneration, (ii) an electron uptake pathway, and (iii) a natural or synthetic carbon fixation pathway. Constraint-based metabolic modelling of E. coli central metabolism is used, in combination with kinetic and thermodynamic pathway analyses. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the PPR photosystems, and the relatively high ATP consumption due to maintenance. In general these analyses illustrate the potential of in silico analyses to identify potential bottlenecks and solutions in complex designs for autotrophic and photosynthetic metabolism, as a basis for subsequent experimental implementation.
To tackle a main bottleneck of PPR systems: their functional membrane-embedded production level, the heterologous production in E. coli of the proton-pumping rhodopsins from Gloeobacter violaceus (GR) and from Thermus thermophilus JL18 (TR) is quantified and experimentally optimized in Chapter 4. High constitutive production of both rhodopsin proteins is achieved by fine-tuning transcription and translation. Besides the canonical retinal pigment, the GR system has the ability to bind a light-harvesting antennae pigment, echinenone. After optimization of the heterologous pigment biosynthesis pathways for either retinal or echinenone production, appropriate quantities of retinal or echinenone for PPR reconstitution were detected in E. coli. Association of echinenone with GR broadens its absorption spectrum in E. coli, broadening the potential for light-harvesting also to blue light. Optimization of the branched pathway for simultaneous biosynthesis of both retinal and echinenone has been attempted by using a smart library of variable Ribosome Binding Sites (RBSs) with varying strengths (RedLibs). In general, the here described approaches towards improved functional production of rhodopsin photosystems in E. coli and their pigments may prove more widely applicable for heterologous production of more complex photosystems and other systems.
In Chapter 5 an up-to-date overview is provided on how codon usage can influence functional protein production. The fact that all known organisms have an incomplete set of tRNAs, indicates that biased codon usage could act as a general mechanism that allows for fine-tuning the translation speed. Although translation initiation is the key control step in protein production, it is broadly accepted that codon bias, especially in regions further downstream of the start codon, can contribute to the translation efficiency by tuning the translation elongation rate. Modulation of the translation speed depends on a combination of factors, including the secondary structure of the transcript (more or less RNA hairpins), the codon usage landscape (frequent and more rare codons) and for bacteria also RBS-like sequences at which ribosomes can pause. The complex combination of interdependent factors related to codon usage that can influence translation initiation and elongation. This complexity makes that the design of synthetic genes for heterologous expression is still in its infancy, and despite the availability of some codon usage algorithms, it is often as well a matter of trial and error.
In Chapter 6 the effect of different codon usage algorithms (optimization and harmonization) has been experimentally tested for heterologous production of membrane proteins. Apart from the codon usage algorithms also the combined effect of transcriptional fine-tuning in E. coli LEMO21(DE3) was assessed. The overproduction of 6 different membrane-embedded proteins, including 4 PPR variants (from bacteria, archaea and eukaryotes), was tested. For production of tested PPR variants, the different codon usage algorithms hardly influenced production, while transcriptional tuning had a large impact on production levels. Interestingly, for the other two tested non-PPR membrane proteins, some codon usage variants significantly improved production on top of transcriptional tuning. For both these proteins the codon-optimization algorithm reduced functional production below that of the wild-type codon variant, while the harmonization algorithm gave significantly higher production, equal or even higher than for the wild-type variant.
In Chapter 7 it is demonstrated that a translational-tuning system can be used to successfully optimize the expression of several membrane proteins, based on initial findings presented in Chapter 4. The employed, recently developed Bicistronic Design (BCD) system is based on translational coupling of a gene encoding a short leader peptide and the gene of interest that is under control of a variable ribosome binding site. A standardized library of 22 RBSs allows for precise, gene context-independent, fine-tuning of expression of this second gene, here encoding a membrane protein. For all four membrane proteins tested in this study the BCD approach resulted in 3 to 7-fold higher protein levels than those obtained by two other recently developed methods for optimizing membrane protein production. The presented approach allows for inducer-free, constitutive, high-level production of membrane proteins in E. coli, which can be widely applicable for both membrane protein purification studies as well as for synthetic biology projects involving membrane proteins.
In Chapter 8 a broad review and perspectives are provided on the potential of microbial autotrophs for the production of value-added compounds from CO2. Both photoautotrophic and chemolithoautotrophic production platforms are discussed, and recent progress in improving their efficiency and production potential is highlighted. Transplantation efforts for photosystems, but also for CO2 fixation pathways and electron uptake systems are discussed. An overview is provided on novel in silico and experimental approaches to engineer components related to autotrophy in heterotrophic and autotrophic model hosts, including approaches applied in this thesis. Future avenues are discussed for realizing more efficient autotrophic production platforms.
Finally, in Chapter 9 and 10 the work in this thesis is summarized and a general discussion is provided on future avenues for engineering of PPR photosystems, photosystems in general and on the optimization of membrane protein production.
Breaking down barriers: construction of a hybrid heterochiral membrane
Siliakus, Melvin - \ 2016
Wageningen University. Promotor(en): John van der Oost, co-promotor(en): Servé Kengen. - Wageningen : Wageningen University - ISBN 9789462579293 - 237
membranes - engineering - escherichia coli - fatty acids - isoprenoids - archaea - thermococcus kodakarensis - polymerase chain reaction - gene knock-out - dna modification - membranen - engineering - escherichia coli - vetzuren - isoprenoïden - archaea - thermococcus kodakarensis - polymerase-kettingreactie - inactivering van genen - dna-modificatie
Because of a chemical disparity between Archaeal and Bacterial membrane-lipids, these organisms thrive under distinct environmental conditions. Archaea are generally more resistant to extreme habitats like low pH, high temperature or presence of solvents. It has therefore long been hypothesized that the archaeal lipids provide archaeal cells with a higher robustness than bacterial lipids do for Bacteria. A recent study in which bacterial and archaeal lipids were mixed to form hybrid vesicles “lipid enclosed round structures”, for instance showed a higher temperature dependent stability than either the bacterial or archaeal lipid vesicles separately. In the present study, we therefore introduced the enzymatic machinery for assembly of archaeal lipids into the bacterium Escherichia coli. This engineering led to cells with a mixed membrane at a surprisingly high amount of 28% archaeal lipids. Although the intervention led to severe morphological malformations, the cells indeed showed an increased robustness to extreme cold and butanol.
Preparation of polylactide microcapsules at a high throughput with a packed-bed premix emulsification system
Sawalha, Hassan ; Sahin, Sami ; Schroën, Karin - \ 2016
Journal of Applied Polymer Science 133 (2016)24. - ISSN 0021-8995
biomedical applications - colloids - drug-delivery systems - membranes
Core-shell polymer microcapsules are well known for their biomedical applications as drug carriers when they are filled with drugs and gas-filled microcapsules that can be used as ultrasound contrast agents. The properties of microcapsules are strongly dependent on their size (distribution); therefore, equipment that allows the preparation of small and well-defined microcapsules is of great practical relevance. In this study, we made polylactide microcapsules with a packed-bed premix emulsification system that previously gave good results for regular emulsions. Here, we tested it for applicability to a system in which droplets shrank and solidified to obtain capsules. The packed-bed column was loaded with glass beads of different sizes (30-90 μm) at various bed heights (2-20 mm), and coarse emulsions consisting of the polymer, a solvent, and a nonsolvent were pushed repeatedly through this system at selected applied pressures (1-4 bar). The obtained transmembrane fluxes (100-1000 m3 m-2 h-1) were much higher than those recorded for other membrane emulsification techniques. The average size of the obtained microcapsules ranged between 2 and 8 μm, with an average span of about 1; interestingly, the capsules were 2-10 times smaller than the interstitial voids of the beds. The droplets were larger when we used thicker beds and larger glass beads, and these effect correlated with the pore Reynolds number (Rep). Two breakup mechanisms were identified: spontaneous droplet snap-off dominated the system at low Reps, and localized shear forces dominated the system at higher Rep.
Sensory quality of drinking water produced by reverse osmosis membrane filtration followed by remineralisation
Vingerhoeds, M.H. ; Nijenhuis, M.A. ; Ruepert, N. ; Bredie, W.L.P. ; Kremer, S. - \ 2016
Water Research 94 (2016). - ISSN 0043-1354 - p. 42 - 51.
drinking water - water quality - sensory evaluation - taste research - reverse osmosis - membranes - filtration - drinkwater - waterkwaliteit - sensorische evaluatie - smaakonderzoek - omgekeerde osmose - membranen - filtratie
Membrane filtration of ground, surface, or sea water by reverse osmosis results in permeate, which is almost free from minerals. Minerals may be added afterwards, not only to comply with (legal) standards and to enhance chemical stability, but also to improve the taste of drinking water made from permeate. Both the nature and the concentrations of added minerals affect the taste of the water and in turn its acceptance by consumers. The aim of this study was to examine differences in taste between various remineralised drinking waters. Samples selected varied in mineral composition, i.e. tap water, permeate, and permeate with added minerals (40 or 120 mg Ca/L, added as CaCO3, and 4 or 24 mg Mg/L added as MgCl2), as well as commercially available bottled drinking waters, to span a relevant product space in which the remineralised samples could be compared. All samples were analysed with respect to their physical–chemical properties. Sensory profiling was done by descriptive analysis using a trained panel. Significant attributes included taste intensity, the tastes bitter, sweet, salt, metal, fresh and dry mouthfeel, bitter and metal aftertaste, and rough afterfeel. Total dissolved solids (TDS) was a major determinant of the taste perception of water. In general, lowering mineral content in drinking water in the range examined (from <5 to 440 mg/L) shifted the sensory perception of water from fresh towards bitter, dry, and rough sensations. In addition, perceived freshness of the waters correlated positively with calcium concentration. The greatest fresh taste was found for water with a TDS between 190 and 350 mg/L. Remineralisation of water after reverse osmosis can improve drinking quality significantly.
Lipid bilayer stability in relation to oxide nanoparticles
Pera, H. - \ 2015
Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Mieke Kleijn. - Wageningen : Wageningen University - ISBN 9789462574670 - 144
lipids - membranes - stability - nanotechnology - particles - analytical methods - models - modeling - lipiden - membranen - stabiliteit - nanotechnologie - deeltjes - analytische methoden - modellen - modelleren
Lipid bilayer stability in relation to oxide nanoparticles
All living organisms are composed of cells that are filled with a thick molecular soup. These molecules constitute a complex machinery that brings these cells to life. To contain these molecules, and to protect them from the hostile outer environment, a phospholipid bilayer envelopes the cell. It is essential that this lipid bilayer, also known as the cell membrane, should remain intact and form a perfect barrier at all times. Industrially manufactured nanoparticles are suspect to be able to penetrate this barrier, and thus endanger living organisms in the environment. This thesis deals with some aspects of the structural integrity of lipid bilayers, and especially how this integrity is affected by the interaction with nanoparticles.
Experiments were performed with silica and titanium dioxide nanoparticles, interacting with lipid bilayers, using a variety of experimental techniques. In addition, a theoretical model was applied that is based on the Scheutjens-Fleer Self Consistent Field (SCF) theory. This model delivered detailed structural and thermodynamic information about the lipid bilayer. The modelling work helped us to improve our understanding of lipid bilayer stability, and showed the effect of the interaction with the nanoparticles on the phospholipid bilayer. These latter results could be related directly to our experiments.
Let us first experimentally regard the interaction of lipid bilayers with synthetic oxide nanoparticles. We developed a protocol for high-throughput screening of the nanoparticle-bilayer interaction using a fluorescence technique. Results from this method were combined with reflectometry measurements and atomic force microscopy (AFM). The combination of these methods allowed us to relate lipid bilayer integrity to its interaction with nanoparticles and their adsorption onto the bilayer. In addition, the AFM results yielded detailed structural information at the nano-scale. We found that the interaction strongly depends on both lipid bilayer and nanoparticle charge. However, the specific interaction that depends on the nanoparticle type, starts to play a role when the charges are low. When the total interaction strength is regarded, a regime was found at which interaction is strong enough for the nanoparticles to adsorb onto the bilayer, but too weak to disrupt the bilayer. If, however, the bilayer is disrupted by the nanoparticles, the particle may steal away some lipid molecules from the bilayer, and leave again to disrupt the bilayer elsewhere.
Let us now go into more detail on the SCF modelling. Bilayers are composed of phospholipids, which consist of a hydrophilic head group, and a hydrophobic tail. These bilayers were modelled using a single lipid molecule type, of which the head group structure and lipid tail length could be varied. We thus obtained bilayers that varied in their thickness, and the space that a single lipid takes within the bilayer. Changes in bilayer composition affect the bilayer mechanical properties, such as those constants that describe bilayer stretching or bending. This thesis shows how vesicles, which are bilayers in a globular shape, may become unstable if the bilayer lipid composition is changed. Under certain conditions, a vesicle would prefer to fall apart into many smaller vesicles, which is when highly charged head groups start to repel each other. Or the bilayer may form continuous cubic phases, which might occur if lipids with uncharged head groups but with very long tails are used to form the bilayer. Under very specific and finely tuned conditions, a lipid bilayer may become unstable to form stable pores in the membrane, or to fall apart into tiny lipid discs.
Intracellular accommodation of rhizobia in legume host cell: the fine-tuning of the endomembrane system
Gavrin, A.Y. - \ 2015
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): E. Federova. - Wageningen : Wageningen University - ISBN 9789462574182 - 160
peulgewassen - rhizobium - bodembacteriën - endosymbiose - wortelknolletjes - membranen - waardplanten - legumes - rhizobium - soil bacteria - endosymbiosis - root nodules - membranes - host plants
The symbiosis of legumes with rhizobia leads to the formation of root nodules. Rhizobia which are hosted inside specialized infected cells are surrounded by hostderived membranes, forming symbiosomes. Although it is known that symbiosome formation involves proliferation of membranes and changing of host cell architecture the mechanisms involved in these processes remain largely uncovered.
In this thesis, I studied in more detail the adaptation of the endomembrane system of infected cells to intracellular rhizobia. I have shown that in the first cell layer of the nitrogen-fixing zone, the vacuole of the infected cells shrinks, creating space for the expanding symbiosomes. Here the expression of homotypic fusion and vacuole protein sorting complex (HOPS) genes VPS11 and VPS39 are switched off, whereas tonoplast proteins, like the vacuolar aquaporin TIP1g, are targeted to the symbiosome membrane. These observations suggest that tonoplast-targeted traffic in infected cells is altered. This retargeting is essential for the maturation of symbiosomes.
Accommodation of intracellular rhizobia requires also the reorganization of the actin cytoskeleton. I have shown that during symbiosome development the symbiosomes become surrounded by a dense actin network and in this way, the actin configuration in infected cells is changed markedly. The actin nucleating factor ARP3 is operational in the rearrangement of actin around the symbiosome.
It is known that the plasma membrane is inelastic; its capacity to stretch is only around 1-3%. Exocytosis of new membrane material is therefore involved in changes in the size of the membrane surface and in repair of damaged membrane loci. Membrane tension may create a vector for the fusion of membrane vesicles. To test this, the localization of proteins from the group of synaptotamin calcium sensors involved in membrane fusion, was studied. I have shown that the Medicago synaptotamins, MtSyt2 and MtSyt3, are localised on protrusions of the host plasma membrane created by expanding rhizobia (infection threads, cell wall-free unwalled droplets). Hence, at these sites of contact between symbionts membrane tension may create a vector for exocytosis.
It is known that the host cell wall is modified during the development of infected cells. This process is mediated by the exocytotic pathway employing vesicle-associated membrane proteins (VAMPs) from the VAMP721 family. Previously it was shown in Medicago nodules, that cell-wall free interface membrane formation during bacterial release is dependent on these proteins. I have shown that the pectin modifying enzyme pectate lyase is delivered to the site of bacterial release in soybean nodules by VAMP721-positive vesicles.
My study uncovered new mechanisms involved in the adaptation of host cells to intracellular rhizobia: defunctionalization of the vacuole, actin cytoskeleton rearrangement and the retargeting of host cell proteins to the interface membrane.
Ion adsorption-induced wetting transition in oil-water-mineral systems
Mugele, F. ; Bera, B. ; Cavalli, A. ; Siretanu, I. ; Maestro, A. ; Duits, M. ; Cohen Stuart, M.A. ; Ende, D. van den - \ 2015
Scientific Reports 5 (2015). - ISSN 2045-2322 - 8 p.
hydration forces - surfaces - recovery - wettability - interfaces - membranes - charge - layer
The relative wettability of oil and water on solid surfaces is generally governed by a complex competition of molecular interaction forces acting in such three-phase systems. Herein, we experimentally demonstrate how the adsorption of in nature abundant divalent Ca2+ cations to solid-liquid interfaces induces a macroscopic wetting transition from finite contact angles (˜10°) with to near-zero contact angles without divalent cations. We developed a quantitative model based on DLVO theory to demonstrate that this transition, which is observed on model clay surfaces, mica, but not on silica surfaces nor for monovalent K+ and Na+ cations is driven by charge reversal of the solid-liquid interface. Small amounts of a polar hydrocarbon, stearic acid, added to the ambient decane synergistically enhance the effect and lead to water contact angles up to 70° in the presence of Ca2+. Our results imply that it is the removal of divalent cations that makes reservoir rocks more hydrophilic, suggesting a generalizable strategy to control wettability and an explanation for the success of so-called low salinity water flooding, a recent enhanced oil recovery technology.
Listeria monocytogenes repellence by enzymatically modified PES surfaces
Veen, S. van der; Nady, N. ; Franssen, M.C.R. ; Zuilhof, H. ; Boom, R.M. ; Abee, T. ; Schroën, C.G.P.H. - \ 2015
Journal of Applied Polymer Science 132 (2015)10. - ISSN 0021-8995 - 6 p.
stainless-steel - catalyzed modification - biofilm formation - attachment - growth - membranes - water - acid - functionalization - hydrophobicity
: The effect of enzyme-catalyzed modification of poly(ethersulfone) (PES) on the adhesion and biofilm formation of two Listeria monocytogenes strains is evaluated under static and dynamic flow conditions. PES has been modified with gallic acid, ferulic acid and 4-hydroxybenzoic acid. The surfaces modified with any of these compounds show up to 70% reduced adhesion of L. mono-cytogenes under static conditions and up to 95% under dynamic flow conditions compared with unmodified surfaces. Also, under static conditions the formation of biofilms is reduced by 70%. These results indicate that the brush structures that are formed by the polymers on the PES surface directly influence the ability of microorganisms to interact with the surface, thereby reducing attachment and biofilm formation of L. monocytogenes. Based on these results, it is expected that enzyme-catalyzed surface modification is a promising tool to reduce microbial adhesion and biofilm formation
Fluidized Capacitive Bioanode As a Novel Reactor Concept for the Microbial Fuel Cell
Deeke, A. ; Sleutels, T.H.J.A. ; Donkers, T.F.W. ; Hamelers, B. ; Buisman, C.J.N. ; Heijne, A. ter - \ 2015
Environmental Science and Technology 49 (2015)3. - ISSN 0013-936X - p. 1929 - 1935.
waste-water treatment - electricity-generation - power-generation - iron reduction - scaled-up - performance - carbon - resistance - membranes - biofilms
The use of granular electrodes in Microbial Fuel Cells (MFCs) is attractive because granules provide a cost-effective way to create a high electrode surface area, which is essential to achieve high current and power densities. Here, we show a novel reactor design based on capacitive granules: the fluidized capacitive bioanode. Activated carbon (AC) granules are colonized by electrochemically active microorganisms, which extract electrons from acetate and store the electrons in the granule. Electricity is harvested from the AC granules in an external discharge cell. We show a proof-of-principle of the fluidized capacitive system with a total anode volume of 2 L. After a start-up period of 100 days, the current increased from 0.56 A/m2 with 100 g AC granules, to 0.99 A/m2 with 150 g AC granules, to 1.3 A/m2 with 200 g AC granules. Contact between moving AC granules and current collector was confirmed in a control experiment without biofilm. Contribution of an electro-active biofilm to the current density with recirculation of AC granules was limited. SEM images confirmed that a biofilm was present on the AC granules after operation in the fluidized capacitive system. Although current densities reported here need further improvement, the high surface area of the AC granules in combination with external discharge offers new and promising opportunities for scaling up MFCs.
Spatial constraints and the organization of the cytoskeleton
Ga^rlea, I.C. - \ 2015
Wageningen University. Promotor(en): Bela Mulder. - Wageningen : Wageningen University - ISBN 9789462572126 - 175
celskelet - microtubuli - actine - eiwitten - celdeling - microvezels - membranen - cytoskeleton - microtubules - actin - proteins - cell division - microfilaments - membranes
The shape of animal cells is in controlled by a network of filamentous polymers called the cytoskeleton. The two main components of the cytoskeleton are actin filaments and microtubules. These polymers continuously reorganize in order to performed their diverse cellular functions. For example, in processes such as cell migration actin filaments grow against the membrane, creating flat protrusions called lamellipodia. The lamellipodia enable the cells to move over surfaces. Microtubules are a key player in the cell division mechanism. There, the proper separation of the genetic material between the two daughter cells is controlled by two microtubule asters. The positioning of these two asters also determines the location where the cells will physically separate. Both migration and division are crucial processes for the cell, however the mechanisms underlying these processes are still poorly understood. The organization of the cytoskeleton in cells, and thus their functioning as cell shapers, is an interplay between mutual interaction, confinement and protein mediated interactions. Since cells are exquisitely complex systems, experimentally, the bottom-up approach proves useful in understanding the contribution of each of these interactions on the cytoskeleton organization. This approach is based on the idea of reconstructing a minimal system and adding more complexity to it as our understanding of this system increases.
Starting by a bottom-up approach, as it is done in experimental systems, we study various aspects of confinement and mutual interactions on cytoskeleton organization. The simplest system in which these two interactions are expected to compete is when dense enough rigid cytoskeletal polymers are confined. Experimentally, this question is addressed by confining these polymers in microchambers which are small compared to the persistence length of the enclosed polymers. In Chapter 2, using Monte Carlo simulations, we investigate the organization of rigid polymers confined in shallow square containers, this geometry being simplified model of a lamellipodium. We find that, in the regime where the confinement effect, which causes wall alignment of the polymers, competes with the self-aligning tendency of the polymers, the organization is characterized by a nematic droplet aligned along a diagonal and wall aligned polymers. The pattern is stabilized by linear defect structures.
By the same methods, in Chapter 3, we study rigid polymers in curved wall confinement, finding that the bipolar structure appearing in the disk geometry is drastically modified by the opening of a hole in the middle of the container. Unexpectedly, in this annular geometry, the organization is characterized by highly aligned domains separated by radial defect walls. The patterns observed are the result of the finite size of the particles.
When the rigid polymers are small compared to the confining volume, their orientation is expected to vary over lengths which are much larger than the length of the polymer. In this regime the system is well described by continuum theories. Since currently employed continuum models either exclude the emergence of singularities by the way they are constructed (Oseen-Frank model) or are valid only in for a limited density range around the transition from an unordered to an ordered system (Landau-De Gennes model), in Chapter 4, we construct a mean-field model combining the virtues of these two models. We apply this model to a system of rigid small polymers enclosed in rectangular shallow container (geometry similar to the one in Chapter 2), finding that patterns which are minimizing the energy of the system are characterized by continuum variation of the orientation. However, our model also yields patterns containing point defects which have slightly higher energy.
So far we have considered only rigid cytoskeletal polymers, however at the length scale of the cell the polymers are better described by an elastic rod. In Chapter 5 we study the configurations adopted by a cytoskeletal polymers when enclosed by a rigid ellipsoidal membrane. We find that, compared to the spherical confinement, the change in shape of the confining membrane leads to non-trivial organization of the enclosed polymers. Among the patters observed are single bundles, planar asters, circular and elliptical rings. In reconstructed systems such as emulsion droplets the cytoskeletal polymers push against the membrane, deforming it but, since the membrane is under tension, it also constrains them to bend. Determining the polymeric configurations as a function of the confining surface is the first step towards understanding this mechanical interplay between the cytoskeleton and the membrane.
For proper cell division, a precise positioning of the two microtubule asters involved is required. The positioning of the two asters is based on pushing and pulling forces generated by the microtubule-membrane interaction. Experimental evidence shows that, in reconstructed systems, a spatial separation between the two asters in always present. Therefore, in Chapter 6, we investigate the steric repulsion between two asters finding that it indeed leads to a spatial separation.
The models that we developed in this thesis are a starting point for understanding the cytoskeletal organization and its role in the cell. In the last Chapter of this thesis we give some directions that the present work opens.
Effect of antimicrobial compounds on cut Gerbera flowers: Poor relation between stem bending and numbers of bacteria in the vase water
Witte, Y. van de; Harkema, H. ; Doorn, W.G. van - \ 2014
Postharvest Biology and Technology 91 (2014). - ISSN 0925-5214 - p. 78 - 83.
jamesonii flowers - essential oils - rose flowers - membranes - longevity - stress - sugars - plants - life - acid
Gerbera flowers (Gerbera jamesonii) often show stem bending. In four cultivars (Tamara, Liesbeth, Cora, and Mickey), we tested the effects on bending of antimicrobial compounds (chlorine bleach, a slow release chlorine compound, 8-hydroxyquinoline citrate [HQC], silver nitrate, carvacrol and thymol), some combined with sugars. At concentrations used for other cut flowers, inclusion in the vase solution of several of the antimicrobial compounds delayed bending, had no effect, or hastened bending. Hastening of bending was found at higher concentrations. It was accompanied with visible damage on the stem ends. Results with HQC indicated high toxicity as it did not delay bending at any of the concentration tested (100-400 mg L-1). At 200 mg L-1 HQC induced growth of bacteria that were not found in the controls. The number of bacteria in the vase water showed a low correlation with bending. Visible toxicity on the stem surface was often associated with a high bacteria count. However, at relatively high concentrations of the antimicrobial compounds stem bending was associated with a low count. This indicated an effect other than bacteria. Water uptake was low in stems that bent early. It is hypothesized that material from dead stem cells resulted in a xylem blockage which led to early bending. Sucrose at 15 g L-1 in combination with an antimicrobial compound (slow release chlorine, HQC) resulted in the absence of stem damage and produced much less bending than the same concentration of the antimicrobial compounds alone. Sucrose apparently counteracted the toxic effects of the antimicrobial chemicals. (C) 2014 Published by Elsevier B.V.
KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery
Mansoori Zangir, N. ; Timmers, J.F.P. ; Desprez, T. ; Lessa Alvim Kamei, C. ; Dees, D.C.T. ; Vincken, J.P. ; Visser, R.G.F. ; Höfte, H. ; Vernhettes, S. ; Trindade, L.M. - \ 2014
PLoS ONE 9 (2014)11. - ISSN 1932-6203
secondary cell-wall - arabidopsis-thaliana - endo-1,4-beta-glucanase - expression - membranes - protein - system - plants - gene - endo-1,4-beta-d-glucanase
Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane–cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.
Linking lipid architecture to bilayer structure and mechanics using self-consistent field modelling
Pera, H. ; Kleijn, J.M. ; Leermakers, F.A.M. - \ 2014
Journal of Chemical Physics 140 (2014). - ISSN 0021-9606 - 23 p.
interacting chain molecules - statistical thermodynamics - spontaneous curvature - bending moduli - association colloids - membranes - elasticity - adsorption - monolayers - vesicles
To understand how lipid architecture determines the lipid bilayer structure and its mechanics, we implement a molecularly detailed model that uses the self-consistent field theory. This numerical model accurately predicts parameters such as Helfrichs mean and Gaussian bending modulus k c and k ¯ and the preferred monolayer curvature J m 0 , and also delivers structural membrane properties like the core thickness, and head group position and orientation. We studied how these mechanical parameters vary with system variations, such as lipid tail length, membrane composition, and those parameters that control the lipid tail and head group solvent quality. For the membrane composition, negatively charged phosphatidylglycerol (PG) or zwitterionic, phosphatidylcholine (PC), and -ethanolamine (PE) lipids were used. In line with experimental findings, we find that the values of k c and the area compression modulus k A are always positive. They respond similarly to parameters that affect the core thickness, but differently to parameters that affect the head group properties. We found that the trends for k ¯ and J m 0 can be rationalised by the concept of Israelachivili's surfactant packing parameter, and that both k ¯ and J m 0 change sign with relevant parameter changes. Although typically k ¯
High loaded MBRs for organic matter recovery from sewage: Effect of solids retention time on bioflocculation and on the role of extracellular polymers
Faust, L. ; Temmink, B.G. ; Zwijnenburg, A. ; Kemperman, A.J.B. ; Rijnaarts, H. - \ 2014
Water Research 56 (2014). - ISSN 0043-1354 - p. 258 - 266.
waterzuivering - membranen - biofilms - organische stof - water treatment - membranes - biofilms - organic matter - municipal waste-water - submerged membrane bioreactor - improved energy recovery - activated-sludge process - microbial community - surface-properties - substances eps - performance - extraction - constituents
High loaded MBRs (HL-MBR) can concentrate sewage organic matter by aerobic bioflocculation for subsequent anaerobic conversion to methane or volatile fatty acids. In the range of very short solid retention times (SRT), the effect of SRT on bioflocculation and EPS production in HL-MBR was investigated. This short SRT range was selected to find an optimum SRT maximising recovery of organics by aerobic bioflocculation and minimizing losses of organics by aerobic mineralization. Bioflocculation was studied in five HL-MBRs operated at SRTs of 0.125, 0.25, 0.5, 1 and 5 d. The extent of flocculation, defined as the fraction of suspended COD in the concentrate, increased from 59% at an SRT of 0.125 d to 98% at an SRT of 5 d. The loss of sewage organic matter by biological oxidation was 1, 2, 4, 11 and 32% at SRT of 0.125–5 d. An SRT of 0.5–1 d gave best combination of bioflocculation and organic matter recovery. Bound extracellular polymeric substances (EPS) concentrations, in particular EPS-protein concentrations, increased when the SRT was prolonged from 0.125 to 1 d. This suggests that these EPS-proteins govern the bioflocculation process. A redistribution took place from free (supernatant) EPS to bound (floc associated) EPS when the SRT was prolonged from 0.125 to 1 d, further supporting the fact that the EPS play a dominant role in the flocculation process. Membrane fouling was most severe at the shortest SRTs of 0.125 d. No positive correlation was detected between the concentration of free EPS and membrane fouling, but the concentration of submicron (45–450 nm) particles proved to be a good indicator for this fouling.
Charge-driven co-assembly of polyelectrolytes across oil-water interfaces
Monteillet, H. ; Hagemans, F. ; Sprakel, J.H.B. - \ 2013
Soft Matter 9 (2013)47. - ISSN 1744-683X - p. 11270 - 11275.
controlled flocculation - opposite charge - small particles - emulsions - membranes
We report a simple strategy to co-assemble oppositely charged polyelectrolytes across oil–water interfaces; this allows the accumulation of an electrostatic complex at the interface of species that are not surface active by themselves. To this end, we use a new, oil-soluble anionic polymer, poly-(fluorene-co-benzothiadiazole-co-benzoic acid), in combination with a cationic polyelectrolyte that is dissolved in the aqueous phase. When only one of the two charged components is present, no positive adsorption is observed in interfacial tension measurements; by contrast, when both polyelectrolytes are present, in the oil and water phases respectively, a rapid decrease of the interfacial tension is observed, indicating co-adsorption of the cationic and anionic polyelectrolytes. The complexation strength can be tuned through changes in both ionic strength and pH. Confocal microscopy and co-localization analysis further verifies the presence of both polyelectrolytes at the interface. With this approach, emulsions can be stabilized for several weeks; moreover, using the sensitivity of the complex to changes in pH, we are able to reversibly break and make the emulsions on demand.
Bending rigidities of surfactant bilayers using self-consistent field theory
Leermakers, F.A.M. - \ 2013
Journal of Chemical Physics 138 (2013). - ISSN 0021-9606 - 11 p.
interacting chain molecules - lipid-bilayers - curvature elasticity - statistical-theory - adsorption - membranes - size - thermodynamics - interface - relevance
Self-consistent field (SCF) theory is used to find bending moduli of surfactant and lipid bilayers. Recently, we successfully applied low-memory search methods to solve the SCF equations. Using these we are now able to directly evaluate the Gaussian bending modulus for molecularly detailed models of bilayers by evaluating the excess Helmholtz energy of tensionless bilayers in a (part of the) Im3m cubic phase. The result prompted us to reconsider the protocol that has been used thus far to find the mean bending modulus k c and Gaussian bending modulus k¯. With respect to previous predictions, the value of k c is reduced by a factor of two and the Gaussian bending modulus is less negative and much closer to zero. In line with experimental data we now find that k¯can also become positive. In this paper we use the non-ionic surfactants series of the type C n E m for illustration.