George Holzwarth

Forces Required of Kinesin during Processive Transport through Cytoplasm

The purpose of this paper is to deduce whether the maximum force, steplike movement, and rate of ATP consumption of kinesin, as measured in buffer, are sufficient for the task of fast transport of vesicles in cells. Our results show that moving a 200-nm vesicle in viscoelastic COS7 cytoplasm, with the same steps as observed for kinesin-driven beads in buffer, required a maximum force of 16 pN and work per step of 1 +/- 0.7 ATP, if the drag force was assumed to decrease to zero between steps. In buffer, kinesin can develop a force of 6-7 pN while consuming 1 ATP/step, comparable to the required values. As an alternative to assuming that the force vanishes between steps, the measured COS7 viscoelasticity was extrapolated to zero frequency by a numerical fit. The force required to move the bead then exceeded 75 pN at all times and peaked briefly to 92 pN, well beyond the measured capabilities of a single kinesin in buffer. The work per step increased to 7 +/- 5 ATP, greatly exceeding the energy available to a single motor.

Fluctuations in the velocity of individual DNA Molecules during agarose gel electrophoresis.

The velocity of the center of mass of individual T4 DNA molecules during agarose gel electrophoresis, computed from digitized video-microscopic images, fluctuated between 0 and 4.5 mum/s after a field E = 5 V/cm was applied; the amplitude of the velocity peaks was twice the averaged steady-state velocity. The velocity fluctuations correlated with changes in molecular configuration. The mean velocity (10 molecules) showed a sharp rise in less than 0.2 s, followed by a shallow minimum and a broad peak, before reaching a plateau. The much smaller amplitude of these oscillatory features demonstrated that the velocity fluctuations of individual molecules were largely, but not entirely, uncorrelated with the onset of the field. The components of the shape tensor S of individual chains, which are a measure of the extension of the chains, were also determined for each image sequence. Only the principal component in the direction of E,S(xx), increased.

Polarization-modulated differential-interference contrast microscopy with a variable retarder.

A liquid-crystal variable retarder inserted into a differential-interference contrast video microscope switches image highlights into shadows and vice versa in alternate frames. Synchronous computation and display of the difference between alternate frames yield a stream of images with doubled contrast and reduced fixed-position noise because of the automatic background subtraction. The measured signal-to-noise ratio (SNR) peaks when the modulation +/- Gamma of the retarder equals the phase shift delta of the sample. A Jones calculus model of the central ray in the polarization-modulated differential-interference contrast microscope yields SNR = (sin Gamma sin delta)/((1 - cos Gamma cos delta)N), where N is the rms time-dependent photon noise. This expression fits the experiments closely for 1.8 degrees < or = Gamma < or = 115 degrees.

Improving DIC microscopy with polarization modulation

It is demonstrated experimentally, as well as analytically, that when the polarization of the light incident upon the first Nomarski–Wollaston prism in a differential interference contrast (DIC) light microscope is switched by 90°, image highlights are changed into shadows and vice versa. Using an inexpensive ferroelectric liquid‐crystal modulator, which is easily installed in the microscope, this switching can be done at 30 frames s−1, synchronized to the camera. Subtraction of alternate digitized frames generates a stream of images in which contrast is doubled, compared with conventional video‐enhanced DIC, while image defects and noise tend to cancel. Subtraction of alternate images is carried out efficiently by frame buffer operations and amounts to massively parallel synchronous detection. The new method eliminates the problems inherent in obtaining a separate background image, as required by current video‐enhanced DIC practice, without loss of resolution.

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.

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated

Video‐enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video‐enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video‐enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12‐bit background image from the incoming 12‐bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion‐enhanced differential interference contrast. By carrying out background subtraction with 12‐bit images, the number of grey levels in the moving vesicles can be maximized and a single look‐up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion‐enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.

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.

Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells is Dependent on Both Microtubules and Actin Filaments

ABSTRACT 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.

Distinguishing Brownian Motion from Motor-Driven Transport within Organelle Trajectories by Bayesian Analysis

Many organelles and vesicles move in an intermittent (start-stop) manner in live cells when observed by video microscopy. We propose that such vesicles travel in one of two states, a driven state in which they are being actively pulled along a cytoskeletal fiber by motor proteins, and a Brownian state in which they are detached from the fiber and obey the laws of diffusion in the cytoplasm. Using variational Bayesian analysis, we analyze the tracks of peroxisomes, lysosomes, and other organelles in PC12 and HME cells to reveal the probability that the vesicle is in the driven or the Brownian state at each time (frame). Instantaneous vesicle velocity and directional persistence are the input data for our hidden Markov, Gaussian mixture model Bayesian analysis. This analysis evaluates the most probable velocity as well the variance of the velocity of each state. It also determines the most probable times at which the state changes. Further analysis of the motions of the vesicles during the Brownian episodes may permit a cleaner assessment of the viscoelastic properties of cytoplasm. This research is supported by the National Science Foundation under Grant No. CMMI-1106105. AMS is supported by the NIGMS of the National Institutes of Health under award number T32GM095440.