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

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

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)