Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.
19 Related JoVE Articles!
One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles
Institutions: University of Toronto, University of Toronto, Campbell Family Institute For Cancer Research and Techna Institute, University at Buffalo, The State University of New York.
We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide α, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 °C to 62 °C rapidly upon one minute exposure of 750 mW (1.18 W/cm2
), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.
Bioengineering, Issue 79, Nanoparticles, Porphysome, photothermal therapy, nanoparticle, porphyrin
Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration
Institutions: Harvard Medical School, University of Pennsylvania.
Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 μm, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.
Bioengineering, Issue 87, oxygen, microparticles, microbubbles, homogenization, encapsulation, lipid monolayer, drug delivery
Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer
Institutions: Southern Illinois University.
FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1
. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1
) after an analyte interacts with receptors attached to PDA2,3,4,7
. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J
) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8
. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.
Biochemistry, Issue 66, Molecular Biology, Chemistry, Physics, Fluorescence Resonance Energy Transfer (FRET), Polydiacetylene (PDA), Biosensor, Liposome, Sensing
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
Institutions: Harvard Medical School, Harvard Medical School.
Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro,
two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 μm2
area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.
Biomedical Engineering, Issue 31, Viral fusion, membrane fusion, supported lipid bilayer, biophysics, single molecule
Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles
Institutions: University of Colorado Boulder, University of Colorado Boulder.
Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3
, monitor drug delivery4,5
, and encapsulation4
. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6
Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7
. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8
. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions3
. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9
. Drug encapsulation can be achieved as reported during liposome formation9
. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10
, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency10,5
, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5
is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10
, electron microscopy11
and nanoparticle tracking analysis (NTA)12
were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.
Bioengineering, Issue 64, Biomedical Engineering, Liposomes, particle extrusion, nano-sized vesicles, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA)
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Institutions: Weill Cornell Medical College.
Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12
. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+
that has been successfully used in other fluorescence quenching assays 5,13
permeates the lipid bilayer slowly 8
but passes readily through conducting gramicidin channels 1,14
. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12
Microbiology, Issue 44, membrane properties, bilayer properties, gramicidin, fluorescence quenching, high throughput drug screening
Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy
Institutions: Rush University, Rush University.
Previous studies showed that knockdown of ITSN-1s (KDITSN
), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1
. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN
). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2
. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3
Here, efficient and specific KDITSN
in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN
/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN
throughout the study4
. Mouse tissues collected at several time points post-siRNAITSN
were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN
, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e.
disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo
Bioengineering, Issue 76, Biomedical Engineering, Biochemistry, Genetics, Molecular Biology, Cellular Biology, Anatomy, Physiology, Medicine, Immunology, Pharmacology, animal models, Cardiovascular Diseases, intersectin-1s, siRNA, liposomes, retro-orbital injection, acute and chronic ITSN-1s knockdown, transgenic mice, liposome, endothelial cells, tissue, lung, perfusion, electron microscopy, animal model
Postproduction Processing of Electrospun Fibres for Tissue Engineering
Institutions: University of Sheffield , University of Sheffield , University of Sheffield .
Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3
Many tissues in vivo
undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro
can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4
An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.
Bioengineering, Issue 66, Materials Science, Biomedical Engineering, Tissue Engineering, Medicine, Chemistry, Electrospinning, bilayer, biaxial distension, heat and vapour annealing, mechanical testing, fibres
Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique
Institutions: University of North Carolina School of Medicine, University of North Carolina .
Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4
. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications.
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5
(Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10
Bioengineering, Issue 55, Nanoparticles, nanomedicine, drug delivery, polymeric micelles, polymeric nanoparticles, diblock co-polymers, nanoplatform, nanoparticle molecular imaging, polymer conjugation.
Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation
Institutions: Boston University .
High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro
experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.
Mechanical Engineering, Issue 67, Physics, Materials Science, Cancer Biology, Phase-shift nanoemulsions, narrow size distribution, acoustic droplet vaporization, bubble-enhanced heating, HIFU ablation, polyacrylamide hydrogel
Intra-lymph Node Injection of Biodegradable Polymer Particles
Institutions: University of Maryland, College Park.
Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.
size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.
Bioengineering, Issue 83, biomaterial, immunology, microparticle, nanoparticle, vaccine, adjuvant, lymph node, targeting, polymer
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
Institutions: Scuola Normale Superiore, Instituto Italiano di Tecnologia, University of California, Irvine.
It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn’t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn
-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.
Bioengineering, Issue 92, fluorescence, protein dynamics, lipid dynamics, membrane heterogeneity, transient confinement, single molecule, GFP
Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method
Institutions: University of Minnesota, University of Minnesota, Mayo Clinic College of Medicine, Mayo Clinic College of Medicine.
Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2
beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.
Bioengineering, Issue 87, supported lipid bilayer, beads, microarray, fluorescence, microfabrication, nanofabrication, atomic layer deposition, myelin, lipid rafts
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology
Institutions: University of Washington.
The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.
Physiology, Issue 76, Biophysics, Molecular Biology, Biochemistry, Genetics, Cellular Biology, Proteins, Membranes, Artificial, Lipid Bilayers, Liposomes, Phospholipids, biochemistry, Lipids, Giant Unilamellar Vesicles, liposome, electrophysiology, electroformation, reconstitution, patch clamp
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans
has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans
triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans
. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
Institutions: AntiCancer, Inc..
There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.
Cellular Biology, Issue 13, Springer Protocols, hair follicles, liposomes, adenovirus, genes, stem cells