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 0.10µm, such as vesicular stomatitis virus ribonucleoprotein particles (RNP), 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 crosslink actin fibers, causing the mesh to jiggle. Until recently, little was known about how RNP particles get through the mesh.

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.

DNA damage reduces heterogeneity and coherence of chromatin motions.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate.

Significance: Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth ( z ) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field. Aim: To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system. Approach: We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker. Results: Bead localization precision was < 25 nm in the transverse dimensions and ≤ 70 nm along the axial dimension over an axial range of 6 µ m . Higher axial precision ( ≤ 50 nm ) was achieved over a shallower focal depth of 2.9 µ m . 3D diffusion constants of chromatin matched expected values. Conclusions: Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.

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.

Structured illumination to spatially map chromatin motions.

We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340 ± 30 nm, which simultaneously photoactivate a 7 × 7 matrix pattern of GFP-labeled histones, with spots 1.70 µm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

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.

Dividing organelle tracks into Brownian and motor-driven intervals by variational maximization of the Bayesian evidence.

Many organelles and vesicles in live cells move in a start-stop manner when observed for ~10 s by optical microscopy. Changes in velocity and directional persistence of such particles are a potentially rich source of insight into the mechanisms leading to the start and stop states. Unbiased assessment of the most probable number of states, the properties of each state, and the most probable state for the particle at each moment can be accomplished by variational Bayesian methods combined with a hidden Markov model and a Gaussian mixture model. Our track analysis method, "vbTRACK", applied this combination of methods to particle velocity v or changes in the direction of travel evaluated from simulated tracks and from tracks of peroxisomes in live cells. When tested with numerical data, vbTRACK reliably determined the number of states, the mean and variance of the velocity or the direction of travel for each state, and the most probable state during each frame. When applied to the tracks of peroxisomes in live cells, some tracks separated into two states, one with high velocity and directionality, the other approximately Brownian. Other tracks of particles in live cells separated into several diffusive states with distinct diffusion constants.

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.

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.

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.

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.