Current Research
Structured illumination to spatially map chromatin motions. Dynamic motions of 0chromatin during interphase are thought to critically influence genomic processes such as gene express and DNA repair. In collaboration with Keith Bonin(Physics) and Pierre Vidi(Cancer Biology), we are developing new optical methods to detect chromatin motion. Our first experiments are using structured illumination in a light microscope to photoactivate a 7×7 array of histone 2A tagged with photoactivatable GFP in the nuclei of live mammalian cells. Movies of the spots are obtained by conventional epifluorescence microscopy of GFP. The 49 spots are tracked to subpixel resolution. From the track, the diffusion rate of the spots can be determined, using Matlab code which we have written.
Transport modes of viral nucleoproteins in live cells. The proteins and nucleic acids which make up a virus are synthesized near the nucleus. A few hours after infection, the complete virus is assembled and then extruded at the surface of the cell. In collaboration with Doug Lyles(Biochemistry), David Ornelles(Microbiology), and Jed Macosko(Physics) we are using high-speed video microscopy of fluorescently tagged ribonucleoprotein VSV particles to observe motion of the particles. After tracking, a 2D variational Bayesian image analysis Matlab program is used to determine the mode of transport of individual particles at specific times. Hopping from trap to trap is the dominant mode of transport.
Undergraduate projects. Undergraduate physics and computer science majors can participate in either of the two ongoing projects described above. Such participation could be for credit (Physics 381) or for pay.
Previous Research Projects:
Mechanical properties of live cells in 2D culture. We tracked the motions of fluorescently tagged peroxisomes in live cells, specifically normal, tumorigenic and metastic human breast cells, in 2D culture. From the tracks, we extracted intracellular mechanical properties such as the elastic and viscous shear moduli of the cytoplasm. These moduli play a role in the dynamics of cell deformation, which occurs during metastasis.
Variational Bayes analysis of particle tracks. A long-standing difficulty afflicting the analysis of particle tracks is their start-stop nature. We developed a hidden Markov, variational Bayes, Gaussian mixture model analysis method which can objectively separate tracks into 2 or more states characterized by whether the vesicle is motor-driven or obeys the laws of diffusion.
Durotaxis and mechanical properties of live cells grown in 3D culture.Together with graduate student Amanda Smelser, we grew cells within collagen gels which had been crosslinked by photogenerated free radicals. Using an optical mask, gels with a spatially varying elastic modulus in a physiologically useful range were generated..
Kinesin-microtoubule interactions during gliding assays under a uniform magnetic force (Todd Fallesen). We attached superparamagnetic beads to the +ends of microtubules, then carried out upside-down motility assays between the polepieces of a novel electromagnet which applied a uniform force to the beads. We were able to measure the number of motors pulling a microtubule. We were also able to dependence of microtubule velocity on the number of attached motors.
Vesicle transport and molecular motors in PC12 cells (David Hill)
We measured the drag force and mechanical work required for fast transport of vesicles in cells and to related this cellular task to the mechanical performance of motor proteins, especially kinesin. In buffer, the maximum steady force which kinesin can exert is 6.5 pN. One ATP is hydrolyzed per 8 nm step, and each step takes 50 microseconds. About 100 such steps occur per second during processive movement. We have tracked large vesicles in the neurites of live PC12 cells at 37C. The tracks suggest that the number of motors pulling an individual vesicle is not constant, but changes roughly once per second between 1, 2, 3, and occasionally 4 motors. The maximum velocity of vesicles in PC12 cells is about 2.5 microns/s, about 4 times the maximum velocity in vitro. We analyyze the Brownian motion of single vesicles in PC12 cells to determine the viscoelastic modulus of PC12 cytoplasm. The viscous drag on the vesicles is 4.6 pN per kinesin, about half the maximum force developed by kinesin against a steady load in a trap. [See Hill, Plaza, Bonin, Holzwarth, 2004 in Publications)]
Our conclusion that vesicle velocity in vivo increases with an increasing number of active motors is no longer as controversial as it was when published; the conventional view was that for processive motors, such as kinesin, the number of motors does not change the speed. To bolster our view, we determinied force-velocity curves for 2, 3, and 4 full-length dimeric kinesin motors during gliding and bead assays in vitro.
Motion-enhanced DIC microscopy (MEDIC)
One of the most widely used methods for observing live cells is differential interference contrast light microscopy. To further improve contrast and thus to better observe small vesicles, one can subtract a background image from the DIC images. Our lab has developed software which continuously updates this background image and displays the background-subtracted image to the microscopist in real time. We do this by constructing the background image from an average of the most recent 8 frames. Non-moving objects disappear in the backgrouond-subtracted image, so the contrast of moving objects such as vesicles can be aggressively enhanced. Our method takes full advantage of the full grayscale of the superior grayscale information available with 12-bit, scientifiic-grade CCD cameras. We are continuing to improve upon this powerful technique. A flow-chart of the software is provided under RESEARCH.
Polarization-modulated DIC microscopy
A second way to improve differential interference contrast light microscopy is to modulate the sign of the offset retardance with a fast, computer-controllable liquid-crystal retardation modulator. Such modulators are easily inserted into a standard microscope. Switching the sign of the offset in alternate frames switches image highlights into shadows and vice versa. By subtracting alternate images(0-1, 2-1, 2-3, 4-3, 4-5, 6-5…) with an image-processing board, the pm-dic difference images are displayed in “real time”, with background automatically subtracted and contrast improved by a factor of two. Because pmdic is a difference method, one can use a modern 12-bit cooled digital CCD cameras to produce images with very flat background, low noise, and excellent spatial resolution. [More details about PM-DIC].