Study of lipid bilayer behaviour modified by substrate interactions

Biological membranes rarely exist as free-floating structures but are often confined and supported by various cellular assemblies such as the cytoskeleton and the extracellular matrix. It has already been shown that biological and polymeric substrates can modulate the morphology and response to various stimuli of supported lipid bilayers significantly. The interaction between such structures and the membrane are obviously important yet remain poorly understood even in minimal or synthetic systems. The work of this thesis utilises a variety of fluorescence microscopy and atomic force microscopy (AFM) techniques to investigate the behaviour and structure of supported lipid bilayers, in particular how interfacial features of their support substrate influence and modulate their morphology and biophysical properties. First, surface modification of polydimethylsiloxane is systematically explored, in particular how the interfacial properties of such a polymer substrate can be modified to create fully and partially plasma-treated interfaces that stably support lipid bilayers. Lipid patch formation on such substrates is then investigated, revealing that the membrane undergoes significant morphological reorganisation after vesicle fusion has completed forming a lipid patch. The underlying mechanisms can be altered by substrate interactions following different pathways for fully and partially plasma-treated PDMS substrates. Furthermore, partially plasma-treated substrates are demonstrated to be capable of specifically depleting cholesterol from supported lipid membranes, while stably supporting the other remaining phospholipid species. Studies of cholesterol depletion of lipid patches possessing liquid-ordered and disordered domains reveal a disruption in domains structure, with the partitioning of fluorescent dyes into regions from which they were previously excluded. This structure perturbation was found to be reversible upon the reinsertion of cholesterol into the bilayer. Many of the discussed mechanisms are only observed in the presence of a substrate, emphasising the importance of substrate interactions in both functional biomembranes and the development of supported membrane technologie

Investigating the Role of Microtubules in Glut4 Vesicle Trafficking and the Kinetics of Membrane Attachment by the Myosin Myo1c

The myosin myo1c dynamically localizes to cellular membranes through high affinity phosphoinositide binding and links them to the actin cytoskeleton. Determining the kinetics of membrane attachment will provide insight into the relationship between membrane-attachment and actin-attachment lifetimes, and will also provide details about the regulation of membrane attachment. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1cIQ-tail) to and from 100 nm diameter large unilamellar vesicles (LUVs). The apparent second-order rate constant for association of myo1cIQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was approximately diffusion-limited. Myo1cIQ-tail dissociated from PtdIns(4,5)P2 at a slower rate (2.0 s-1) than the pleckstrin homology domain of phospholipase C-δ (PLCδ-PH) (13 s-1). The presence of additional anionic phospholipid reduced the myo1cIQ-tail dissociation rate constant \u3e 50-fold, but marginally changed the dissociation rate of PLCδ-PH, suggesting that additional electrostatic interactions in myo1cIQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1cIQ-tail from LUVs, suggesting that phosphoinositides are able to bind and dissociate from myo1cIQ-tail as it remains bound to the membrane. In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the molecular role of microtubules, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The time course of the density increase precedes that of the increase in intensity of HA-GLUT4-eGFP in this same region. Microtubule disruption delayed and modestly reduced the accumulation of GLUT4 at the plasma membrane. Interestingly, fusion of GLUT4-containing vesicles with the plasma membrane preferentially occur near microtubules, and long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for fusion events

Efectos de las proteínas SP-B y SP-C del surfactante pulmonar en las propiedades físicas de membranas biológicas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Físicas, Departamento de Física Aplicada III, leída el 10/07/2013Depto. de Estructura de la Materia, Física Térmica y ElectrónicaFac. de Ciencias FísicasTRUEunpu

The effects of membrane physicochemical properties on huntingtin membrane association and downstream aggregation

Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by an expanded glutamine repeat region (polyQ) within the huntingtin protein (htt). As a result of the expanded polyQ domain, htt associates into a variety of toxic aggregate species. The polyQ domain of htt is flanked at the N-terminal end by 17 amino acids (Nt17) that adopt an amphipathic α-helical structure in the presence of binding partners such as lipid membranes. In addition to comprising a lipid binding domain, the Nt17 amphipathic α -helix has been directly implicated in htt aggregation initiation via self-association with other Nt17 α -helices. Due to this, htt/lipid interaction likely has a large impact on the rate and extent of htt aggregate formation, with potential implications for HD pathogenesis. The studies presented here focus on elucidating the effect of membrane physicochemical properties on htt membrane association and downstream htt aggregation. In order to measure membrane association, a method of normalizing polydiacetylene (PDA) lipid binding assays was developed to enable the direct comparison of various molecules’ binding affinity for different lipid systems. Then, the normalized PDA assay was utilized to determine if small molecule aggregation inhibitors influence the interaction of htt with pure and physiologically relevant lipid systems, and thioflavin-T (ThT) assays and atomic force microscopy (AFM) were used to monitor if the interaction altered the ability of the small molecules to inhibit htt aggregation. While both small molecules altered htt-membrane association, EGCG remained an effective aggregation inhibitor while curcumin no longer inhibited htt fibrillization in the presence of either lipid system. These results highlight the complex relationship between htt-membrane association, downstream aggregation, and the ability of small molecules to inhibit htt aggregation in a cellular environment. Subsequent studies utilized ThT, AFM, polydiacetylene (PDA) assays, and native MS to determine how altering membrane physicochemical properties by changing the headgroup or the tail of lipids influences htt aggregation and membrane affinity. Our results indicate that increasing the negative charge of lipid headgroups disrupts the insertion of Nt17 into the bilayer, which in turn decreases membrane disruption and results in a localization effect that increases htt fibrillization. Also, when varying the degree of unsaturation in the lipid tail, the trend of htt aggregation in the presence of each lipid system is different than the trend of association between htt and the vesicles, indicating that membrane properties alter the mechanism of downstream htt aggregation. Further investigation of the interaction between Nt17 and lipid membranes with molecular dynamics (MD) simulations reveal that a combination of the hydrophobic amino acid and available membrane defect sizes influence the orientation of Nt17 on bilayers during the initial stages of interaction. Collectively, these results highlight how the properties of lipid membranes modulate htt-membrane interactions and htt aggregation mechanisms

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase

ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature−protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes

New algorithms for the analysis of live-cell images acquired in phase contrast microscopy

La détection et la caractérisation automatisée des cellules constituent un enjeu important dans de nombreux domaines de recherche tels que la cicatrisation, le développement de l'embryon et des cellules souches, l’immunologie, l’oncologie, l'ingénierie tissulaire et la découverte de nouveaux médicaments. Étudier le comportement cellulaire in vitro par imagerie des cellules vivantes et par le criblage à haut débit implique des milliers d'images et de vastes quantités de données. Des outils d'analyse automatisés reposant sur la vision numérique et les méthodes non-intrusives telles que la microscopie à contraste de phase (PCM) sont nécessaires. Comme les images PCM sont difficiles à analyser en raison du halo lumineux entourant les cellules et de la difficulté à distinguer les cellules individuelles, le but de ce projet était de développer des algorithmes de traitement d'image PCM dans Matlab® afin d’en tirer de l’information reliée à la morphologie cellulaire de manière automatisée. Pour développer ces algorithmes, des séries d’images de myoblastes acquises en PCM ont été générées, en faisant croître les cellules dans un milieu avec sérum bovin (SSM) ou dans un milieu sans sérum (SFM) sur plusieurs passages. La surface recouverte par les cellules a été estimée en utilisant un filtre de plage de valeurs, un seuil et une taille minimale de coupe afin d'examiner la cinétique de croissance cellulaire. Les résultats ont montré que les cellules avaient des taux de croissance similaires pour les deux milieux de culture, mais que celui-ci diminue de façon linéaire avec le nombre de passages. La méthode de transformée par ondelette continue combinée à l’analyse d'image multivariée (UWT-MIA) a été élaborée afin d’estimer la distribution de caractéristiques morphologiques des cellules (axe majeur, axe mineur, orientation et rondeur). Une analyse multivariée réalisée sur l’ensemble de la base de données (environ 1 million d’images PCM) a montré d'une manière quantitative que les myoblastes cultivés dans le milieu SFM étaient plus allongés et plus petits que ceux cultivés dans le milieu SSM. Les algorithmes développés grâce à ce projet pourraient être utilisés sur d'autres phénotypes cellulaires pour des applications de criblage à haut débit et de contrôle de cultures cellulaires.Automated cell detection and characterization is important in many research fields such as wound healing, embryo development, immune system studies, cancer research, parasite spreading, tissue engineering, stem cell research and drug research and testing. Studying in vitro cellular behavior via live-cell imaging and high-throughput screening involves thousands of images and vast amounts of data, and automated analysis tools relying on machine vision methods and non-intrusive methods such as phase contrast microscopy (PCM) are a necessity. However, there are still some challenges to overcome, since PCM images are difficult to analyze because of the bright halo surrounding the cells and blurry cell-cell boundaries when they are touching. The goal of this project was to develop image processing algorithms to analyze PCM images in an automated fashion, capable of processing large datasets of images to extract information related to cellular viability and morphology. To develop these algorithms, a large dataset of myoblasts images acquired in live-cell imaging (in PCM) was created, growing the cells in either a serum-supplemented (SSM) or a serum-free (SFM) medium over several passages. As a result, algorithms capable of computing the cell-covered surface and cellular morphological features were programmed in Matlab®. The cell-covered surface was estimated using a range filter, a threshold and a minimum cut size in order to look at the cellular growth kinetics. Results showed that the cells were growing at similar paces for both media, but their growth rate was decreasing linearly with passage number. The undecimated wavelet transform multivariate image analysis (UWT-MIA) method was developed, and was used to estimate cellular morphological features distributions (major axis, minor axis, orientation and roundness distributions) on a very large PCM image dataset using the Gabor continuous wavelet transform. Multivariate data analysis performed on the whole database (around 1 million PCM images) showed in a quantitative manner that myoblasts grown in SFM were more elongated and smaller than cells grown in SSM. The algorithms developed through this project could be used in the future on other cellular phenotypes for high-throughput screening and cell culture control applications

Elucidating membrane disruption mechanisms of peptide antibiotics

Antimicrobial peptides and proteins hold promise as a next generation of antibiotics. Whilst conventional antibiotics must cross the microbial membrane to target intracellular processes, many of these agents act by disrupting microbial lipid bilayers. This alternative mode of action attacks a conserved structural component of the cell and offers a promising alternative for treating infections caused by multi-resistant pathogens. To facilitate the translational development of antimicrobial peptides, it is beneficial to develop a fundamental understanding of their behaviour. However, resolving the interactions between antimicrobial peptides and lipid bilayers is challenging. Disruption to the lipid packing occurs at the nanoscale, is often dynamic and adapts under different environment conditions. Consequently, the rules linking peptide sequence to mechanisms of membrane disruption and biological activity remain largely unknown. The work presented here directly addresses this challenge. AFM imaging of model membrane systems is used to visualise disruption mechanisms with high temporal and spatial resolution. The findings are then compared to biological assays, and reveal new sequence-function relationships. Starting with a simplified α-helical template, we demonstrate that both the mode of lipid disruption and the biological selectivity of a sequence can be controlled at the single amino acid level. Next, we demonstrate that incorporating motifs from membrane-active but non-lytic peptide sequences into an α-helical design can confer auxiliary lipid interactions. Moving on from single α-helices, we provide the first evidence of membrane disruption by multi-helix bacteriocins, resolving a hitherto unknown multimodal mechanism. In addition we resolve the membrane interactions of supramolecular peptide structures, confirming that self-assembly can offer mechanistic advantage. Finally, preliminary data is presented that improves on the chemical and structural specificity of AFM, and therefore the insights into peptide-lipid interactions that it can provide

Factors Influencing Huntingtin Aggregation at Surfaces: Implications for Huntington’s Disease

Huntington’s Disease (HD) is a genetic, neurodegenerative disease characterized by an abnormal polyglutamine (polyQ) expansion in the first exon of the huntingtin protein (htt). The polyQ domain facilitates aggregation and initiates the formation of a diverse collection of aggregate species, including fibrils, oligomers and annular aggregates. The first 17 amino acids of htt (Nt17) directly flank the polyQ domain and is a key factor in htt’s association to membranous structures. In addition to Nt17 being an amphipathic αhelix, it also promotes aggregation through self-association and contains numerous posttranslational modifications (PTMs) that can modulate toxicity and subcellular localization. For in depth understanding of these mechanisms, particularly in the presence of lipid membrane surfaces, the PTM phosphorylation and macromolecular crowders found in subcellular environments were explored. Through the application of phosphomimetic mutations of htt to a variety of lipid systems, lipid-specific impacts of electrostatic interactions involved in htt/lipid interactions were elucidated. Cytosolic conditions mimicked through the addition of macromolecular crowders and htt were evaluated at both solid/liquid and membrane/liquid interfaces, with each crowder having a distinct effect on htt aggregation. The results presented here aid in the understanding of the multi-faceted nature of htt aggregation in the presence of cellular and subcellular surfaces

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research