Quantitative biological studies at cellular and sub-cellular level

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on March 23, 2009)Vita.Includes bibliographical references.Thesis (Ph.D.) University of Missouri-Columbia 2007.Dissertations, Academic -- University of Missouri--Columbia -- Biological sciences.We developed a translational magnetic tweezers for quantitative measurements at cellular and sub-cellular level. In the cellular studies, multiple membrane tethers were extracted simultaneously under constant force transduced to eukaryotic cells through magnetic beads attached to their membranes. The tethers were characterized in terms of viscoelastic parameters. The contribution of the actin cytoskeleton in the process of tether formation was investigated. The membrane tether system was used to test the applicability of the Crooks fluctuation theorem (a recent finding in non-equilibrium thermodynamics) at the mesoscopic level. In the sub-cellular studies, the cytoplasmic viscoelastic coefficients of mouse oocytes were determined using magnetic beads trapped into their cytoskeletal mesh. We found that cryopreservation altered all the viscoelastic parameters. We demonstrated that the reversible disassembly of the actin cytoskeleton with latrunculin A before cryopreservation increased the number of survivors and preserved their viscoelastic parameters. This finding promoted latrunculin as a candidate cryoprotective agent

Dynamics of Mechanosensing in the Bacterial Flagellar Motor

Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.Molecular and Cellular BiologyPhysic

Adaptation at the output of the chemotaxis signalling pathway

In the bacterial chemotaxis network, receptor clusters process input1–3, and flagellar motors generate output4. Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell5. However, the motor is ultrasensitive, with a narrow [CheY-P] operating range6. How might the match between receptor output and motor input be optimized? Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase7–8 and why motors exhibit signal-dependent FliM turnover9. Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.Molecular and Cellular Biolog

Membrane tether formation studied with magnetic tweezers

Abstract only availableMembrane tethers are ubiquitous nanometer-diameter cylindrical extensions of biological membranes. They form through either active or passive processes, by locally acting tensional forces. The physical properties of membrane tethers depend on the viscoelastic properties of the biological membrane and its immediate surroundings, such as the cortical cytoskeleton in the case of the cell membrane. Tether formation is integral to such physiological processes as extravasation from the circulatory system of leukocytes as part of the inflammatory response or malignant cells during metastasis, as well as cell-to-cell communication. Quantifying the viscoelasticity of the membrane with characteristic biophysical parameters provides insight into these physiological processes. Using magnetic tweezers, we applied constant tensional forces to the plasma membrane through non-specifically attached magnetic beads. The physical response of the resulting tethers was analyzed in terms of standard viscoelastic models. This provided the characteristic biophysical parameters of the tethers. In order to identify the contribution of the cytoskeleton to tether formation, tethers were pulled before and after disrupting the cytoskeleton with Latrunculin A, an actin depolymerizing agent. Measurements were performed using Chinese Hamster Ovary (CHO) and Human Brain Tumor (HB) cells. The membrane of HB cells was systematically found to be less rigid and viscous than that of CHO cells, possibly reflecting the invasive potential of the cancerous cells.NSF-REU Biosystems Modelin

Eukaryotic membrane tethers revisited using magnetic tweezers

doi: 10.1088/1478-3975/4/2/001 http://iopscience.iop.org/1478-3975/4/2/001/Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever- force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.This study was partially supported by grants from NSF and NASA (to GF) and NSERC (MG)

Magnetic tweezers for intracellular applications

doi:10.1063/1.1599066 http://rsi.aip.org/rsinak/v74/i9/p4158_s1We have designed and constructed a versatile magnetic tweezer primarily for intracellular investigations. The micromanipulator uses only two coils to simultaneously magnetize to saturation micron-size superparamagnetic particles and generate high magnitude constant field gradients over cellular dimensions. The apparatus resembles a miniaturized Faraday balance, an industrial device used to measure magnetic susceptibility. The device operates in both continuous and pulse modes. Due to its compact size, the tweezers can conveniently be mounted on the stage of an inverted microscope and used for intracellular manipulations. A built-in temperature control unit maintains the sample at physiological temperatures. The operation of the tweezers was tested by moving 1.28 μm diameter magnetic beads inside macrophages with forces near 500 pN.This work was partially supported by the NSF ~Grant No. DBI-9730999!

Bioprinting: Development of a novel approach for engineering three-dimensional tissue structures [abstract]

Abstract only availableBioprinting is a tissue engineering technique in which spherical cell aggregates, the "bio-ink", are deposited into biocompatible hydrogels, the "bio-paper", by a 3-axis "bio-printer". The aggregates can be deposited into essentially any 3D configuration, and when comprised of adhesive and motile cells aggregate fusion occurs. This self-organizing, liquid-like nature of these tissues is described on a molecular basis by The Differential Adhesion Hypothesis (DAH). The techniques we have developed are quite unique because of the high degree of automation that has been incorporated into our processes and the variety of engineered tissues that we are capable of creating. Despite automation, the creation of aggregates remains a nontrivial and time intensive process. The entire process of aggregate formation from initial cell culture to mature aggregate ready to be loaded into the printer takes approximately five days. This time is a limiting factor in the potential use of bio-printing as a source of on-demand tissues for clinical applications. A solution to this potential problem lies in the cryopreservation of aggregates. Freezing mediums and freezing protocols were tested and the effect of the freezing process on aggregate fusion was determined. An alternate solution to expedite the bioprinting process could lie in the printing of cell 'sausages', tightly packed cylinders of cells. In this method aggregate preparation is forgone. Elimination of this step could allow for increased time in tissue creation. Cell sausage printing provides another technique that could be incorporated into the fabrication of complex tissues. Our experiments in this novel and developing technology of bioprinting represent steps towards building complex tissues via self-assembly.McNair Scholars Progra

Osmotic Pressure in a Bacterial Swarm

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ~30 mm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ~120 mm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (~30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.Molecular and Cellular BiologyPhysic