Directional change during active diffusion of viral ribonucleoprotein particles through cytoplasm.

A mesh of cytoskeletal fibers, consisting of microtubules, intermediate filaments, and fibrous actin, prevents the Brownian diffusion of particles with a diameter larger than 0.10 µm, such as vesicular stomatitis virus ribonucleoprotein (RNP) particles, in mammalian cells. Nevertheless, RNP particles do move in random directions but at a lower rate than Brownian diffusion, which is thermally driven. This nonthermal biological transport process is called "active diffusion" because it is driven by ATP. The ATP powers motor proteins such as myosin II. The motor proteins bend and cross-link actin fibers, causing the mesh to jiggle. Until recently, little was known about how RNP particles get through the mesh. It has been customary to analyze the tracks of particles like RNPs by computing the slope of the ensemble-averaged mean-squared displacement of the particles as a signature of mechanism. Although widely used, this approach "loses information" about the timing of the switches between physical mechanisms. It has been recently shown that machine learning composed of variational Bayesian analysis, Gaussian mixture models, and hidden Markov models can use "all the information" in a single track to reveal that that the positions of RNP particles are spatially clustered. Machine learning assigns a number, called a state, to each cluster. RNP particles remain in one state for 0.2-1.0 s before switching (hopping) to a different state. This earlier work is here extended to analyze the movements of a particle within a state and to determine particle directionality within and between states.

Mechanisms of active diffusion of vesicular stomatitis virus inclusion bodies and cellular early endosomes in the cytoplasm of mammalian cells.

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.

Dynamic Actin Filament Traps Mediate Active Diffusion of Vesicular Stomatitis Virus Ribonucleoproteins.

A recently developed variational Bayesian analysis using pattern recognition and machine learning of single viral ribonucleoprotein (RNP) particle tracks in the cytoplasm of living cells provides a quantitative molecular explanation for active diffusion, a concept previously "explained" largely by hypothetical models based on indirect analyses such as continuum microrheology. Machine learning shows that vesicular stomatitis virus (VSV) RNP particles are temporarily confined to dynamic traps or pores made up of cytoskeletal elements. Active diffusion occurs when the particles escape from one trap to a nearby trap. In this paper, we demonstrate that actin filament disruption increased RNP mobility by increasing trap size. Inhibition of nonmuscle myosin II ATPase decreased mobility by decreasing trap size. Trap sizes were observed to fluctuate with time, dependent on nonmuscle myosin II activity. This model for active diffusion is likely to account for the dominant motion of other viral and cellular elements. IMPORTANCE RNA virus ribonucleoproteins (RNPs) are too large to freely diffuse in the host cytoplasm, yet their dominant motions consist of movements in random directions that resemble diffusion. We show that vesicular stomatitis virus (VSV) RNPs overcome limitations on diffusion in the host cytoplasm by hopping between traps formed in part by actin filaments and that these traps expand and contract by nonmuscle myosin II ATPase activity. ATP-dependent random motion of cellular particles has been termed "active diffusion." Thus, these mechanisms are applicable to active diffusion of other cellular and viral elements.

Vesicular stomatitis virus nucleocapsids diffuse through cytoplasm by hopping from trap to trap in random directions.

Within 2-6 hours after infection by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of infected cells. In that time, viral ribonucleoprotein (RNP) particles (nucleocapsids) travel from their initial sites of synthesis near the nucleus to the edge of the cell, a distance of 5-10 µm. The hydrodynamic radius of RNP particles (86 nm) precludes simple diffusion through the mesh of cytoskeletal fibers. To reveal the relative importance of different transport mechanisms, movement of GFP-labeled RNP particles in live A549 cells was recorded within 3 to 4 h postinfection at 100 frames/s by fluorescence video microscopy. Analysis of more than 200 RNP particle tracks by Bayesian pattern recognition software found that 3% of particles showed rapid, directional motion at about 1 µm/s, as previously reported. 97% of the RNP particles jiggled within a small, approximately circular area with Gaussian width σ = 0.06 µm. Motion within such "traps" was not directional. Particles stayed in traps for approximately 1 s, then hopped to adjacent traps whose centers were displaced by approximately 0.17 µm. Because hopping occurred much more frequently than directional motion, overall transport of RNP particles was dominated by hopping over the time interval of these experiments.

Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells Is Dependent on both Microtubules and Actin Filaments.

UNLABELLED: The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCE: Nucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions.

Mechanical properties of normal versus cancerous breast cells.

A cell's mechanical properties are important in determining its adhesion, migration, and response to the mechanical properties of its microenvironment and may help explain behavioral differences between normal and cancerous cells. Using fluorescently labeled peroxisomes as microrheological probes, the interior mechanical properties of normal breast cells were compared to a metastatic breast cell line, MDA-MB-231. To estimate the mechanical properties of cell cytoplasms from the motions of their peroxisomes, it was necessary to reduce the contribution of active cytoskeletal motions to peroxisome motion. This was done by treating the cells with blebbistatin, to inhibit myosin II, or with sodium azide and 2-deoxy-D-glucose, to reduce intracellular ATP. Using either treatment, the peroxisomes exhibited normal diffusion or subdiffusion, and their mean squared displacements (MSDs) showed that the MDA-MB-231 cells were significantly softer than normal cells. For these two cell types, peroxisome MSDs in treated and untreated cells converged at high frequencies, indicating that cytoskeletal structure was not altered by the drug treatment. The MSDs from ATP-depleted cells were analyzed by the generalized Stokes-Einstein relation to estimate the interior viscoelastic modulus G* and its components, the elastic shear modulus G' and viscous shear modulus G", at angular frequencies between 0.126 and 628 rad/s. These moduli are the material coefficients that enter into stress-strain relations and relaxation times in quantitative mechanical models such as the poroelastic model of the interior regions of cancerous and non-cancerous cells.

Combining capillary electrophoresis and next-generation sequencing for aptamer selection.

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.

Force-velocity relationship for multiple kinesin motors pulling a magnetic bead.

Although the velocity of single kinesin motors against an opposing force F of 0-10 pN is well known, the behavior of multiple kinesin motors working to overcome a larger load is still poorly understood. We have carried out gliding assays in which 3-7 Drosophila kinesin-1 motors moved a microtubule at 200-700 µm/s against a 0-31 pN load at saturating [ATP]. The load F was generated by applying a spatially uniform magnetic field gradient to a superparamagnetic bead attached to the (+) end of the microtubule. When F was scaled by the average number of motors [Symbol: see text]n[Symbol: see text], the force-velocity relationship for multiple motors was similar to the force-velocity relationship for a single motor, supporting a minimal load-sharing model. The velocity distribution at low load has a single mode consistent with rapid fluctuations of n. However, against a load of 2.5-4.7 pN/motor, additional modes appeared at lower velocity. These observations support the Klumpp-Lipowsky model of multimotor transport [Proc Natl Acad Sci USA 102. 17284-17289 (2005)].

Measuring the number and spacing of molecular motors propelling a gliding microtubule.

The molecular motor gliding assay, in which a microtubule or other filament moves across a surface coated with motors, has provided much insight into how molecular motors work. The kinesin-microtubule system is also a strong candidate for the job of nanoparticle transporter in nanotechnology devices. In most cases, several motors transport each filament. Each motor serves both to bind the microtubule to a stationary surface and to propel the microtubule along the surface. By applying a uniform transverse force of 4-19 pN to a superparamagnetic bead attached to the trailing end of the microtubule, we have measured the distance d between binding points (motors). The average value of d was determined as a function of motor surface density σ. The measurements agree well with the scaling model of Duke, Holy, and Liebler, which predicts that (d)~σ(-2/5) if 0.05≤σ≤20 µm(-2) [Phys. Rev. Lett. 74, 330 (1995)]. The distribution of d fits an extension of the model. The radius of curvature of a microtubule bent at a binding point by the force of the magnetic bead was ≈1 µm, 5000-fold smaller than the radius of curvature of microtubules subjected only to thermal forces. This is evidence that at these points of high bending stress, generated by the force on the magnetic bead, the microtubule is in the more flexible state of a two-state model of microtubule bending proposed by Heussinger, Schüller, and Frey [Phys. Rev. E 81, 021904 (2010)].

Magnet polepiece design for uniform magnetic force on superparamagnetic beads.

Here we report construction of a simple electromagnet with novel polepieces which apply a spatially uniform force to superparamagnetic beads in an optical microscope. The wedge-shaped gap was designed to keep partial differential B(x)/ partial differential y constant and B large enough to saturate the bead. We achieved fields of 300-600 mT and constant gradients of 67 T/m over a sample space of 0.5x4 mm(2) in the focal plane of the microscope and 0.05 mm along the microscope optic axis. Within this space the maximum force on a 2.8 microm diameter Dynabead was 12 pN with a spatial variation of approximately 10%. Use of the magnet in a biophysical experiment is illustrated by showing that gliding microtubules propelled by the molecular motor kinesin can be stopped by the force of an attached magnetic bead.

Selection of bead-displayed, PNA-encoded chemicals.

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction--wet or dry--of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.

Kinesin velocity increases with the number of motors pulling against viscoelastic drag.

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 microm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4-15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.

In silico evidence for functional specialization after genome duplication in yeast.

A fairly recent whole-genome duplication (WGD) event in yeast enables the effects of gene duplication and subsequent functional divergence to be characterized. We examined 15 ohnolog pairs (i.e. paralogs from a WGD) out of c. 500 Saccharomyces cerevisiae ohnolog pairs that have persisted over an estimated 100 million years of evolution. These 15 pairs were chosen for their high levels of asymmetry, i.e. within the pair, one ohnolog had evolved much faster than the other. Sequence comparisons of the 15 pairs revealed that the faster evolving duplicated genes typically appear to have experienced partially--but not fully--relaxed negative selection as evidenced by an average nonsynonymous/synonymous substitution ratio (dN/dS(avg)=0.44) that is higher than the slow-evolving genes' ratio (dN/dS(avg)=0.14) but still <1. Increased number of insertions and deletions in the fast-evolving genes also indicated loosened structural constraints. Sequence and structural comparisons indicated that a subset of these pairs had significant differences in their catalytically important residues and active or cofactor-binding sites. A literature survey revealed that several of the fast-evolving genes have gained a specialized function. Our results indicate that subfunctionalization and even neofunctionalization has occurred along with degenerative evolution, in which unneeded functions were destroyed by mutations.

Force-velocity curves of motor proteins cooperating in vivo.

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule "force-velocity curves" have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force-velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37 degrees C, three parameters were measured--velocity (v), radius (a), and effective cytoplasmic viscosity (eta')--as they applied to moving vesicles. These parameters were combined in Stokes' equation, F = 6piaeta'v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force-velocity curve for a single motor agrees with previously reported in vitro single motor force-velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.

Intraneuronal vesicular organelle transport changes with cell population density in vitro.

Primary neuron cultures are widely used in research due to the ease and usefulness of observing individual cells. Therefore, it is vital to understand how variations in culture conditions may affect neuron physiology. One potential variation for cultured neurons is a change in intracellular transport. As transport is necessary for the normal delivery of organelles, proteins, nucleic acids, and lipids, it is a logical indicator of a cell's physiology. We test the hypothesis that organelle transport may change with varying in vitro population densities, thus indicating a change in cellular physiology. Using a novel background subtraction imaging method we show that, at 5 days in vitro (DIV), transport of vesicular organelles in embryonic rat spinal cord neurons is positively correlated with cell density. When density increased 6.5-fold, the number of transported organelles increased 2.2+/-0.3-fold. Intriguingly, this effect was not observable at 3-4 DIV. These results show a significant change in cellular physiology with a relatively small change in plating procedure; this indicates that cells appearing to be morphologically similar, and at the same DIV, may still suffer from a great degree of variability.

Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro.

Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1-2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (<0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~0.6 mum/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 mum/s (taken as the slope of a fit to peak position vs. peak number, R(2)=0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.

Speckled microtubules improve tracking in motor-protein gliding assays.

Gliding assays of motor proteins such as kinesin, dynein and myosin are commonly carried out with fluorescently labeled microtubules or filamentous actin. In this paper, we show that speckled microtubules (MTs), prepared by copolymerizing 98% unlabeled tubulin with 2% rhodamine-labeled tubulin, can be localized to +/-7.4 nm (24 measurements) in images acquired every 125 ms. If the speckled MTs move at about 800 nm s(-1), ten images are sufficient to determine their velocity to a precision of +/-6.8 nm s(-1) (6 microtubules, 24 measurements). This velocity precision is four-fold better than manual methods for measuring the gliding velocity of uniformly labeled MTs by end-point localization. The improved velocity precision will permit the determination of velocity-force curves when one, two and three kinesin motors pull a single load in vitro.

Stepping statistics of single HIV-1 reverse transcriptase molecules during DNA polymerization.

DNA polymerases are protein machines that processively incorporate complimentary nucleotides into a growing double-stranded DNA (ds-DNA). Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity, such as the human immunodeficiency virus type 1 reverse transcriptase (HIV RT) polymerase. Here we find, using a new fluorescence-based single-molecule polymerization assay, that the intrinsic processive DNA-dependent polymerization of HIV RT is approximately Poissionian (i.e. each nucleotide is added sequentially) with a rate of about 100 bases per second at 21 degrees C. From the same experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV RT early termination and final release of completely replicated primer-template DNA. In addition, by measuring the rate of polymerization as a function of temperature, we have estimated the activation energy for processive nucleotide incorporation.

Closing of the fingers domain generates motor forces in the HIV reverse transcriptase.

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (approximately 15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization.

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.