Cell mechanics studied using atomic force microscopy

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 June 17, 2009)Vita.Includes bibliographical references.Thesis (Ph. D.) University of Missouri-Columbia 2008.Dissertations, Academic -- University of Missouri--Columbia -- Physics.Cholesterol plays an indispensable role in regulating the properties of the cell membrane. In particular, lipid rafts, specific membrane domains, which are thought to be required for a number of cell functions, such as receptor mediated signaling and membrane trafficking, are dispersed when cell cholesterol is extracted. There is also evidence showing that cholesterol affects the cells' deformability, an important factor in the development of atherosclerosis. In this study, we investigated the effect of cellular cholesterol on the mechanical properties of bovine aortic endothelial cells (BAECs) and their correlation with the development of atherosclerosis. To compare the mechanical properties of cells with different cholesterol content, we have developed a method to measure the forces needed to extract nanotubes (tethers) from their membranes, using atomic force microscopy (AFM). Our observations show that cholesterol depletion of BAECs resulted in significant increase of membrane-cytoskeleton adhesion. An increase in cellular cholesterol to a level higher than that in normal cells caused decrease of the membrane cytoskeleton adhesion and dramatic decrease of the effective surface viscosity of their membranes. While cholesterol depletion and enrichment had no apparent effect on the intensity of F-actin specific fluorescence, disrupting F-actin with latrunculin A abrogated the observed effects. Fluorescence recovery after photobleaching experiments were also performed to measure the lateral mobility of a lipid probe (DiIC₁₂) at different cholesterol contents. The results are consistent with the AFM measurement.To investigate the molecular bases of the phenomena, we focussed on the regulatory phospholipid, phosphatidylinositol 4,5-biophosphate (PIP2), which is involved in a variety of cell functions, especially the regulation of cytoskeleon, and membrane-cytoskeleton adhesion. In the plasma membrane, PIP2 accumulates in cholesterol-rich domains, and its concentration decreases upon cholesterol depletion.By culturing BAECs with neomycin or by transfecting them to express the GFP-tagged PH domain from phospholipase C [delta], we sequester PIP2 to mimic the effect induced by cholesterol depletion. Interestingly, PIP2 sequestering by either approach decreases cell membrane deformability as cholesterol depletion does. This result suggests that cholesterol depletion affects cell mechanical properties by altering the concentration/distribution of PIP2, which may further change the cortical F-actin network. Furthermore, our studies demonstrate that AFM can be used to relate and correlate biomolecular and biophysical properties

Phase transitions during fruiting body formation in Myxococcus xanthus

The formation of a collectively moving group benefits individuals within a population in a variety of ways such as ultra-sensitivity to perturbation, collective modes of feeding, and protection from environmental stress. While some collective groups use a single organizing principle, others can dynamically shift the behavior of the group by modifying the interaction rules at the individual level. The surface-dwelling bacterium Myxococcus xanthus forms dynamic collective groups both to feed on prey and to aggregate during times of starvation. The latter behavior, termed fruiting-body formation, involves a complex, coordinated series of density changes that ultimately lead to three-dimensional aggregates comprising hundreds of thousands of cells and spores. This multi-step developmental process most likely involves several different single-celled behaviors as the population condenses from a loose, two-dimensional sheet to a three-dimensional mound. Here, we use high-resolution microscopy and computer vision software to spatiotemporally track the motion of thousands of individuals during the initial stages of fruiting body formation. We find that a combination of cell-contact-mediated alignment and internal timing mechanisms drive a phase transition from exploratory flocking, in which cell groups move rapidly and coherently over long distances, to a reversal-mediated localization into streams, which act as slow-spreading, quasi-one-dimensional nematic fluids. These observations lead us to an active liquid crystal description of the myxobacterial development cycle.Comment: 16 pages, 5 figure

The role of the cytoskeleton in the formation and properties of membrane tethers

Abstract only availableMembrane tethers play a critical role in cell adhesion and cell motility. This can be observed in the arrest of neutrophils on the endothelial wall of blood vessels during inflammatory response. Tethers are employed to slow down neutrophils when they are attracted to the periphery of the vessels due to chemotactic gradients set up by cytokines. As a result of slowing down, the neutrophil has time to form specific bonds with endothelial cells and start extravasating from the circulatory system into the surrounding tissue. Metastasizing cancer cells use a similar mechanism. A wide array of factors affects the mechanical characteristics of tethers. A major contribution is provided by the interaction between the cytoskeleton and the membrane. Interbilayer slip and the interaction between the membrane and the glycocalix are additional determinants of tether properties. Previous studies have shown a strong dependence of force needed to extract and pull a tether on the interaction between the membrane and the cytoskeleton. It has also been shown that disrupting the integrity of the cytoskeleton significantly reduces the tether force. The focus of this study was to further elucidate the contribution of the cytoskeleton-lipid bilayer interaction to the tether force, in particular how it affects cell membrane surface viscosity. Atomic Force Microscopy based force spectroscopy was used to determine the tether force and surface viscosity of the membrane prior and after the actin microfilament system had been depolyermerized by latrunculin-A. Two cells lines, Chinese Hamster Ovary (CHO) and Human Brain tumor (HB) cells were investigated. The tether force was determined when the membrane was stretched by a cantilever moving at a constant velocity over a range 3 to 21 micron/s. Surface viscosity was obtained from the slope of the linear force-speed curve. Quantitative information on tether forces and membrane surface viscosities allow for a better understanding of the mechanism responsible for the arrest of neutrophils during their attachment to the endothelial wall.NSF-REU Biosystems Modelin

Myxococcus xanthus gliding motors are elastically coupled to the substrate as predicted by the focal adhesion model of gliding motility

Myxococcus xanthus is a model organism for studying bacterial social behaviors due to its ability to form complex multi-cellular structures. Knowledge of M. xanthus surface gliding motility and the mechanisms that coordinate it are critically important to our understanding of collective cell behaviors. Although the mechanism of gliding motility is still under investigation, recent experiments suggest that there are two possible mechanisms underlying force production for cell motility: the focal adhesion mechanism and the helical rotor mechanism which differ in the biophysics of the cell-substrate interactions. Whereas the focal adhesion model predicts an elastic coupling, the helical rotor model predicts a viscous coupling. Using a combination of computational modeling, imaging, and force microscopy, we find evidence for elastic coupling in support of the focal adhesion model. Using a biophysical model of the M. xanthus cell, we investigated how the mechanical interactions between cells are affected by interactions with the substrate. Comparison of modeling results with experimental data for cell-cell collision events pointed to a strong, elastic attachment between the cell and substrate. These results are robust to variations in the mechanical and geometrical parameters of the model. We then directly measured the motor-substrate coupling by monitoring the motion of optically trapped beads and find that motor velocity decreases exponentially with opposing load. At high loads, motor velocity approaches zero velocity asymptotically and motors remain bound to beads indicating a strong, elastic attachment

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)

Dysfunction in the βII Spectrin-Dependent Cytoskeleton Underlies Human Arrhythmia.

Background: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical activity is not well understood, and often overlooked in the cardiac arrhythmia field. Methods and Results: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the post-translational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electrical and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes that include the Na/Ca exchanger, RyR2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin-deficient mice. Conclusions: Our findings identify βII spectrin as critical for normal myocyte electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology

Direct Measurement of Cell Wall Stress-Stiffening and Turgor Pressure in Live Bacterial Cells

We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the ove rall cell stiffness. We find strong evidence of power–law stress–stiffening in the E. coli cell wall, with an exponent of 1 . 22 ± 0 . 12, such that the wall is significantly stiffer in intact cells ( E = 23 ± 8 MPa and 49 ± 20 MPa in the axial and circumferential directions) than in unpressurized saccul i. These measurements also indicate that the turgor pressure in living cells E. coli is 29 ± 3 kPa