Crowding Induces Complex Ergodic Diffusion and Dynamic Elongation of Large DNA Molecules

Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0–40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA.     

Arbovirus Identification by an Agar-Gel Diffusion Technique

A double diffusion-in-agar test was used to investigate precipitation reactions of 75 arboviruses. Specific reactions were regularly observed with members of arbovirus groups B, California, Simbu, Turlock, Hart Park, vesicular stomatitis, and several other arboviruses as well as with a member of the Tacaribe group and a herpesvirus. The results demonstrated the feasibility of applying this technique to the identification of arboviruses.     

Collective Bacterial Dynamics Revealed Using a Three-Dimensional Population-Scale Defocused Particle Tracking Technique

An ability to monitor bacterial locomotion and collective dynamics is crucial to our understanding of a number of well-characterized phenotypes including biofilm formation, chemotaxis, and virulence. Here, we report the tracking of multiple swimming Escherichia coli cells in three spatial dimensions and at single-cell resolution using a novel three-dimensional (3D) defocused particle tracking (DPT) method. The 3D trajectories were generated for wild-type Escherichia coli strain RP437 as well as for isogenic derivatives that display smooth swimming due to a cheA deletion (strain RP9535) or incessant tumbling behavior due to a cheZ deletion (strain RP1616). The 3D DPT method successfully differentiated these three modes of locomotion and allowed direct calculation of the diffusion coefficient for each strain. As expected, we found that the smooth swimmer diffused more readily than the wild type, and both the smooth swimmer and the wild-type cells exhibited diffusion coefficients that were at least two orders of magnitude larger than that of the tumbler. Finally, we found that the diffusion coefficient increased with increasing cell density, a phenomenon that can be attributed to the hydrodynamic disturbances caused by neighboring bacteria.     

Introduction of Impermeant Molecules into Synaptosomes Using Freeze/Thaw Permeabilization

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.     

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.     

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.Download video file.^{(127M, mp4)}     

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.     

Diffusion of DNA Molecules in Gel at High Electric Fields

Problems related to the gel electrophoretic migration of polymers can be investigated by models based on a Brownian-type ratchet where a particle can undergo a net transport on a potential energy surface that is externally driven to fluctuate between several distinct states. Here we describe the method of polymer transport and separation by means of the cellular automata technique. Numerical simulations of the polymer reptation in the model system allow us to understand the band-broadening processes in the gel electrophoresis experiments. They indicate also possible ways of fine-tuning the parameters in designing desired resolution of the experiments.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope''s objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.Download video file.^{(65M, mp4)}     

New Insights into the Developing Rabbit Brain Using Diffusion Tensor Tractography and Generalized q-Sampling MRI

The use of modern neuroimaging methods to characterize the complex anatomy of brain development at different stages reveals an enormous wealth of information in understanding this highly ordered process and provides clues to detect neurological and neurobehavioral disorders that have their origin in early structural and functional cerebral maturation. Non-invasive diffusion tensor magnetic resonance imaging (DTI) is able to distinguish cerebral microscopic structures, especially in the white matter regions. However, DTI is unable to resolve the complicated neural structure, i.e., the fiber crossing that is frequently observed during the maturation process. To overcome this limitation, several methods have been proposed. One such method, generalized q-sampling imaging (GQI), can be applied to a variety of datasets, including the single shell, multi-shell or grid sampling schemes that are believed to be able to resolve the complicated crossing fibers. Rabbits have been widely used for neurodevelopment research because they exhibit human-like timing of perinatal brain white matter maturation. Here, we present a longitudinal study using both DTI and GQI to demonstrate the changes in cerebral maturation of in vivo developing rabbit brains over a period of 40 weeks. Fractional anisotropy (FA) of DTI and generalized fractional anisotropy (GFA) of GQI indices demonstrated that the white matter anisotropy increased with age, with GFA exhibiting an increase in the hippocampus as well. Normalized quantitative anisotropy (NQA) of GQI also revealed an increase in the hippocampus, allowing us to observe the changes in gray matter as well. Regional and whole brain DTI tractography also demonstrated refinement in fiber pathway architecture with maturation. We concluded that DTI and GQI results were able to characterize the white matter anisotropy changes, whereas GQI provided further information about the gray matter hippocampus area. This developing rabbit brain DTI and GQI database could also be used for educational purposes and neuroscience investigations.     

Transplantation of Nuclei from Lymphocytes of Adult Frogs into Enucleated Eggs

The technique of transplantation of nuclei from adult lymphocytes into enucleated eggs from Xenopus laevis (South African clawed toad) is described. The splenic lymphocytes from the one nucleolus mutant were bound via their immunoglobulin receptors to nylon fibers, derivatized with the antigen used for immunization. A technique for coupling other cell types with Woodward reagent is also described. The cells were broken by aspiration into a micropipette and injected into enucleated eggs. The egg pronucleus was eliminated by UV treatment followed by surgical removal. The origin of the genome of developing embryos was determined on karyotype preparations by looking for the nucleolar organizer on the chromosome pair No. 12. Participation of egg pronucleus in development was frequent as judged by the incidence of gynogenetic diploid individuals and of tetraploid animals exhibiting characteristics of both recipient egg and somatic cell donor karyotypes. Apparently normal, lymphocyte-derived tadpoles were obtained, but they died at 12 days. This shows that differentiated cells from adults, such as lymphocytes, can re-express the genes necessary for ontogeny.     

Detection and Measurement of Staphylococcus epidermidis Slime Using an ELISA Technique

A polyclonal rabbit anti-serum against the strong slime-producing Staphylococcus epidermidis strain RP62A was absorbed with the slime-negative phase variant of this strain PV1 in order to remove not slime-specific antibodies. Using this antiserum we established an ELISA which enables detection of slime production in S. epidermidis extracts. The ELISA showed high absorbance when extracts from slime-positive strains (confirmed in the tissue culture tube test) were used as antigens. The high absorbance of slime-positive strains was greatly reduced by periodate oxidation of the extracts and was resistant to proteinase digestion suggesting that the detected antigen is composed of polysaccharides. In contrast to other rapid and simple laboratory detection methods for S. epidermidis slime, the slime-specific ELISA gave positive results in the presence of human serum.