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1.  Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains 
Nature communications  2015;6:5984.
The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here, using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the transmembrane domains differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo- and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.
doi:10.1038/ncomms6984
PMCID: PMC4313621  PMID: 25635869
2.  Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains 
Nature Communications  2015;6:5984.
The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.
Clustering of proteins in the plasma membrane plays an important role in the regulation of both cellular signalling and membrane remodelling. Milovanovic et al. demonstrate that mismatch between transmembrane domain length and the lipid bilayer thickness is sufficient to drive clustering of SNARE proteins.
doi:10.1038/ncomms6984
PMCID: PMC4313621  PMID: 25635869
3.  The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots 
Summary
In this work, cytotoxicity and cellular impedance response was compared for CdSe/ZnS core/shell quantum dots (QDs) with positively charged cysteamine–QDs, negatively charged dihydrolipoic acid–QDs and zwitterionic D-penicillamine–QDs exposed to canine kidney MDCKII cells. Pretreatment of cells with pharmacological inhibitors suggested that the uptake of nanoparticles was largely due to receptor-independent pathways or spontaneous entry for carboxylated and zwitterionic QDs, while for amine-functionalized particles involvement of cholesterol-enriched membrane domains is conceivable. Cysteamine–QDs were found to be the least cytotoxic, while D-penicillamine–QDs reduced the mitochondrial activity of MDCKII by 20–25%. Although the cell vitality appeared unaffected (assessed from the changes in mitochondrial activity using a classical MTS assay after 24 h of exposure), the binding of QDs to the cellular interior and their movement across cytoskeletal filaments (captured and characterized by single-particle tracking), was shown to compromise the integrity of the cytoskeletal and plasma membrane dynamics, as evidenced by electric cell–substrate impedance sensing.
doi:10.3762/bjnano.6.26
PMCID: PMC4362492  PMID: 25821666
biocompatibility; CdSe/ZnS; cytotoxicity; ECIS; quantum dots; single-particle tracking
4.  Mechanical properties of MDCK II cells exposed to gold nanorods 
Summary
Background: The impact of gold nanoparticles on cell viability has been extensively studied in the past. Size, shape and surface functionalization including opsonization of gold particles ranging from a few nanometers to hundreds of nanometers are among the most crucial parameters that have been focussed on. Cytoxicity of nanomaterial has been assessed by common cytotoxicity assays targeting enzymatic activity such as LDH, MTT and ECIS. So far, however, less attention has been paid to the mechanical parameters of cells exposed to gold particles, which is an important reporter on the cellular response to external stimuli.
Results: Mechanical properties of confluent MDCK II cells exposed to gold nanorods as a function of surface functionalization and concentration have been explored by atomic force microscopy and quartz crystal microbalance measurements in combination with fluorescence and dark-field microscopy.
Conclusion: We found that cells exposed to CTAB coated gold nanorods display a concentration-dependent stiffening that cannot be explained by the presence of CTAB alone. The stiffening results presumably from endocytosis of particles removing excess membrane area from the cell’s surface. Another aspect could be the collapse of the plasma membrane on the actin cortex. Particles coated with PEG do not show a significant change in elastic properties. This observation is consistent with QCM measurements that show a considerable drop in frequency upon administration of CTAB coated rods suggesting an increase in acoustic load corresponding to a larger stiffness (storage modulus).
doi:10.3762/bjnano.6.21
PMCID: PMC4311714  PMID: 25671166
atomic force microscopy; CTAB; gold nanorods; membrane tension; MDCK II cells; QCM
5.  Mammalian cell growth on gold nanoparticle-decorated substrates is influenced by the nanoparticle coating 
Summary
In this work, we study epithelial cell growth on substrates decorated with gold nanorods that are functionalized either with a positively charged cytotoxic surfactant or with a biocompatible polymer exhibiting one of two different end groups, resulting in a neutral or negative surface charge of the particle. Upon observation of cell growth for three days by live cell imaging using optical dark field microscopy, it was found that all particles supported cell adhesion while no directed cell migration and no significant particle internalization occurred. Concerning cell adhesion and spreading as compared to cell growth on bare substrates after 3 days of incubation, a reduction by 45% and 95%, respectively, for the surfactant particle coating was observed, whereas the amino-terminated polymer induced a reduction by 30% and 40%, respectively, which is absent for the carboxy-terminated polymer. Furthermore, interface-sensitive impedance spectroscopy (electric cell–substrate impedance sensing, ECIS) was employed in order to investigate the micromotility of cells added to substrates decorated with various amounts of surfactant-coated particles. A surface density of 65 particles/µm2 (which corresponds to 0.5% of surface coverage with nanoparticles) diminishes micromotion by 25% as compared to bare substrates after 35 hours of incubation. We conclude that the surface coating of the gold nanorods, which were applied to the basolateral side of the cells, has a recognizable influence on the growth behavior and thus the coating should be carefully selected for biomedical applications of nanoparticles.
doi:10.3762/bjnano.5.257
PMCID: PMC4311695  PMID: 25671143
basolateral application; cytotoxicity; electric cell–substrate impedance sensing; gold; nanoparticles
6.  Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines 
Open Biology  2014;4(5):140046.
Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique, we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent (G″/G′) increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells.
doi:10.1098/rsob.140046
PMCID: PMC4042852  PMID: 24850913
microrheology; atomic force microscopy; cancer; viscoelasticity
7.  Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods 
Autophagy  2013;9(5):770-777.
We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.
doi:10.4161/auto.23978
PMCID: PMC3669185  PMID: 23445924
isothermal titration calorimetry; liposome flotation assays; multi-angle laser light scattering; PROPPIN; small unilamellar vesicle
8.  Co-localization of the Ganglioside GM1 and Cholesterol Detected by Secondary Ion Mass Spectrometry 
The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remains a major challenge. The concept of lipid rafts is widely invoked, however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of Secondary Ion Mass Spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano and micro-scale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the co-localization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d>3 μm); and, (3) nanometer-scale domains (d=100 nm − 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.
doi:10.1021/ja310831m
PMCID: PMC3639293  PMID: 23514537
9.  Covalent and Density-Controlled Surface Immobilization of E-Cadherin for Adhesion Force Spectroscopy 
PLoS ONE  2014;9(3):e93123.
E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5–11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.
doi:10.1371/journal.pone.0093123
PMCID: PMC3968077  PMID: 24675966
10.  WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane 
PLoS ONE  2014;9(1):e85033.
Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.
doi:10.1371/journal.pone.0085033
PMCID: PMC3879356  PMID: 24392163
11.  It takes two to tango 
Autophagy  2013;9(1):106-107.
PROPPINs are a family of PtdIns3P and PtdIns(3,5)P2-binding proteins. The crystal structure now unravels the presence of two distinct phosphoinositide-binding sites at the circumference of the seven bladed β-propeller. Mutagenesis analysis of the binding sites shows that both are required for normal membrane association and autophagic activities. We identified a set of evolutionarily conserved basic and polar residues within both binding pockets, which are crucial for phosphoinositide binding. We expect that membrane association of PROPPINs is further stabilized by membrane insertions and interactions with other proteins.
doi:10.4161/auto.22400
PMCID: PMC3542210  PMID: 23069643
autophagy; protein-lipid interactions; X-ray crystallography; S. cerevisiae
12.  Tension Monitoring during Epithelial-to-Mesenchymal Transition Links the Switch of Phenotype to Expression of Moesin and Cadherins in NMuMG Cells 
PLoS ONE  2013;8(12):e80068.
Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.
doi:10.1371/journal.pone.0080068
PMCID: PMC3855076  PMID: 24339870
13.  Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro 
Biology Open  2013;2(12):1279-1287.
Summary
The directional migration of primordial germ cells (PGCs) to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS) as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro.
doi:10.1242/bio.20135140
PMCID: PMC3863412  PMID: 24285703
Primordial germ cells; Cell migration; Cell adhesion; Cellular dynamics; Xenopus
14.  Mechanics of spreading cells probed by atomic force microscopy 
Open Biology  2013;3(7):130084.
Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.
doi:10.1098/rsob.130084
PMCID: PMC3728925  PMID: 23864554
membrane tension; cell mechanics; adhesion; spreading
15.  Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork 
PLoS Biology  2013;11(6):e1001577.
Myelin basic protein undergoes a phase transition from a cytoplasmic soluble pool into a cohesive functional amyloid-like assembly; this may be one mechanism of myelin membrane biogenesis.
Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.
Author Summary
Myelin is a specialized membrane that covers axons and serves as an insulator to enable the fast conduction of the action potentials. The importance of myelin membrane is highlighted in demyelinating diseases such as multiple sclerosis, which lead to severe neurological disability. Here, we describe a physicochemical mechanism of how myelin is generated and assembled. We find that myelin basic protein (MBP) molecules undergo a phase transition into a cohesive meshwork at the membrane interface, which drives structural changes in the membranes. We provide evidence that the interaction of myelin basic proteins with the inner leaflet of the myelin bilayer results in charge neutralization and triggers self-association of the protein into larger polymers. Interactions between MBP molecules are mediated by hydrophobic phenylalanine residues and amyloid-like association. We propose that phase transition of MBP from a cytoplasmic soluble pool into a cohesive functional amyloid-like assembly is one of the key mechanisms in myelin membrane biogenesis.
doi:10.1371/journal.pbio.1001577
PMCID: PMC3676292  PMID: 23762018
16.  Collective behavior of Dictyostelium discoideum monitored by impedance analysis 
Dictyostelium discoideum cells respond to periodic signals of extracellular cAMP by collective changes of cell-cell and cell-substrate contacts. This was confirmed by dielectric analysis employing electric cell-substrate impedance sensing (ECIS) and impedance measurements involving cell-filled micro channels in conjunction with optical microscopy providing a comprehensive picture of chemotaxis under conditions of starvation.
doi:10.4161/cib.23894
PMCID: PMC3656020  PMID: 23713138
Dictyostelium discoideum; cAMP; chemotaxis; impedance analysis; oscillation; starvation
17.  A model system for cell adhesion mediated by weak carbohydrate-carbohydrate interactions 
The multivalent carbohydrate-carbohydrate interaction between membrane anchored epitopes derived from the marine sponge Microciona prolifera (M. prolifera) has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane coated surfaces was employed to mimic native cell-cell contacts. The dynamic strength of the homomeric self-association was measured as a function of calcium ion concentration and loading rate. A deterministic model was used to estimate the number of participating bonds in the contact zone.
doi:10.1021/ja210304j
PMCID: PMC3288207  PMID: 22296574
Colloidal probe microscopy; Carbohydrates; Parallel bonds; Calcium mediated interaction; Microciona prolifera
18.  Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts 
PLoS ONE  2013;8(1):e54172.
Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.
doi:10.1371/journal.pone.0054172
PMCID: PMC3547869  PMID: 23349816
19.  Macromolecular shape and interactions in layer-by-layer assemblies within cylindrical nanopores 
Summary
Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d 0 and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d 0 = 25–80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n max). The value of n max was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n max is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d 0 = 25–30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d 0 > 25–30 nm) became inhibited when approaching a pore diameter of d eff,n_max = 25–35 nm, a similar size to that of native AAO pores, with d 0 = 25–30 nm. For a reasonable estimation of d eff,n_max, the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers.
doi:10.3762/bjnano.3.54
PMCID: PMC3458591  PMID: 23019541
avidin-biotin; dendrimers; nanoporous substrates; optical lightmode waveguide spectroscopy; polyelectrolytes
20.  Structure and Thermotropic phase Behavior of Fluorinated Phospholipid Bilayers: A combined Attenuated Total Reflection FTIR Spectroscopy and Imaging Ellipsometry Study 
The journal of physical chemistry. B  2008;112(28):8250-8256.
Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39° as compared to 21° for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon–hydrocarbon interactions in the center of the bilayer due to chain staggering.
doi:10.1021/jp800711j
PMCID: PMC2518959  PMID: 18563929
21.  Nanoscale Patterning in Mixed Fluorocarbon-Hydrocarbon Phospholipid Bilayers 
A growing body of literature suggests that fluorocarbons can direct self-assembly within hydrocarbon environments. We report here the fabrication and characterization of supported lipid bilayers (SLBs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a synthetic, fluorocarbon-functionalized analog, 1. AFM investigation of these model membranes reveals an intricate, composition-dependent domain structure consisting of ~50 nm stripes interspersed between ~1 µm sized domains. Although DSC of 1 showed a phase transition near room temperature, DSC of DPPC:1 mixtures exhibited complex phase behavior suggesting domain segregation. Finally, temperature-dependent AFM of DPPC:1 bilayers shows that, while the stripe structures can be melted above the Tm of 1, the stripes and domains result from immiscibility of the hydrocarbon and fluorocarbon lipid gel phases. Fluorination appears to be a promising strategy for chemical self-assembly in two dimensions. In particular, because no modification is made to the lipid headgroups, it may be useful for nanopatterning biologically relevant ligands on bilayers in vitro or in living cells.
doi:10.1021/ja070950l
PMCID: PMC2507729  PMID: 17602478
Lipid bilayers; self-assembly; fluorine; atomic force microscopy

Results 1-21 (21)