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In JoVE (2)

Other Publications (14)

Articles by Ido Braslavsky in JoVE

 JoVE Chemistry

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University


JoVE 4189

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

 JoVE Chemistry

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

1Department of Biomedical and Molecular Sciences, Queen's University, 2National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, 3Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology, 4The Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science, and Nutrition, The Hebrew University of Jerusalem


JoVE 51185

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Other articles by Ido Braslavsky on PubMed

Sequence Information Can Be Obtained from Single DNA Molecules

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

Fluorescence Microscopy Evidence for Quasi-permanent Attachment of Antifreeze Proteins to Ice Surfaces

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.

Direct Visualization of Spruce Budworm Antifreeze Protein Interacting with Ice Crystals: Basal Plane Affinity Confers Hyperactivity

Antifreeze proteins (AFPs) protect certain organisms from freezing by adhering to ice crystals, thereby preventing their growth. All AFPs depress the nonequilibrium freezing temperature below the melting point; however AFPs from overwintering insects, such as the spruce budworm (sbw) are 10-100 times more effective than most fish AFPs. It has been proposed that the exceptional activity of these AFPs depends on their ability to prevent ice growth at the basal plane. To test the hypothesis that the hyperactivity of sbwAFP results from direct affinity to the basal plane, we fluorescently tagged sbwAFP and visualized it on the surface of ice crystals using fluorescence microscopy. SbwAFP accumulated at the six prism plane corners and the two basal planes of hexagonal ice crystals. In contrast, fluorescently tagged fish type III AFP did not adhere to the basal planes of a single-crystal ice hemisphere. When ice crystals were grown in the presence of a mixture of type III AFP and sbwAFP, a hybrid crystal shape was produced with sbwAFP bound to the basal planes of truncated bipyramidal crystals. These observations are consistent with the blockage of c-axial growth of ice as a result of direct interaction of sbwAFP with the basal planes.

Single-molecule DNA Sequencing of a Viral Genome

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

Universality of Persistence Exponents in Two-dimensional Ostwald Ripening

We measured persistence exponents theta(phi) of Ostwald ripening in two dimensions, as a function of the area fraction phi occupied by coarsening domains. The values of theta(phi) in two systems, succinonitrile and brine, quenched to their liquid-solid coexistence region, compare well with one another, providing compelling evidence for the universality of the one-parameter family of exponents. For small phi, theta(phi) approximately = 0.39phi, as predicted by a model that assumes no correlations between evolving domains. These constitute the first measurements of persistence exponents in the case of phase transitions with a conserved order parameter.

Caught in the Act: the Lifetime of Synaptic Intermediates During the Search for Homology on DNA

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell's biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be approximately 10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3'- or 5'-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.

Structural Basis for the Superior Activity of the Large Isoform of Snow Flea Antifreeze Protein

The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP.

Superheating of Ice Crystals in Antifreeze Protein Solutions

It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

Compound Ice-binding Site of an Antifreeze Protein Revealed by Mutagenesis and Fluorescent Tagging

By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent protein to different mutants and isoforms and by examining the binding of these fusion proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make the SP isoforms more QAE-like slow down the rate of ice growth. On the basis of these observations we postulate that other types of AFP also have compound ice-binding sites that enable them to bind to multiple planes of ice.

Antifreeze Protein from Freeze-tolerant Grass Has a Beta-roll Fold with an Irregularly Structured Ice-binding Site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

New Insights into Ice Growth and Melting Modifications by Antifreeze Proteins

Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs.

Microfluidic Experiments Reveal That Antifreeze Proteins Bound to Ice Crystals Suffice to Prevent Their Growth

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules.

Inhibition of Ice Growth and Recrystallization by Zirconium Acetate and Zirconium Acetate Hydroxide

The control over ice crystal growth, melting, and shaping is important in a variety of fields, including cell and food preservation and ice templating for the production of composite materials. Control over ice growth remains a challenge in industry, and the demand for new cryoprotectants is high. Naturally occurring cryoprotectants, such as antifreeze proteins (AFPs), present one solution for modulating ice crystal growth; however, the production of AFPs is expensive and inefficient. These obstacles can be overcome by identifying synthetic substitutes with similar AFP properties. Zirconium acetate (ZRA) was recently found to induce the formation of hexagonal cavities in materials prepared by ice templating. Here, we continue this line of study and examine the effects of ZRA and a related compound, zirconium acetate hydroxide (ZRAH), on ice growth, shaping, and recrystallization. We found that the growth rate of ice crystals was significantly reduced in the presence of ZRA and ZRAH, and that solutions containing these compounds display a small degree of thermal hysteresis, depending on the solution pH. The compounds were found to inhibit recrystallization in a manner similar to that observed in the presence of AFPs. The favorable properties of ZRA and ZRAH suggest tremendous potential utility in industrial applications.

Model of Pore Formation in a Single Cell in a Flow-through Channel with Micro-electrodes

Microfluidic channels with embedded micro-electrodes are of growing use in devices that aim to electroporate single cells. In this article we present an analysis of pore evolution in a single cell passing by two planar electrodes that are separated by a nano-gap. The cell experiences an electric field that changes in time, as it goes over the electrodes in the channel. The nano-gap between the electrodes enhances the electric field's strength in the micro-channel, thus enabling the use of low potential difference between the electrodes. By computing the electric field on the surface of the cell we can calculate the pore density, as predicted by the model described by Krassowska and Filev (Biophys. J. 92(2):404-417, 2007). The simulation presented in this article is a useful tool for planning and executing experiments of single-cell electroporation in flow-through devices. We demonstrate how different parameters, such as cell size and the size of the gap between the electrodes, change the pore density and show how electroporation between micro-electrodes on the same plane is different from conventional electroporation between facing electrodes.

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