Jan H. Hoh

Relative Microelastic Mapping of Living Cells by Atomic Force Microscopy

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.

Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope

The spatial distribution of intermolecular forces governs macromolecular interactions. The atomic force microscope, a relatively new tool for investigating interaction forces between nanometer-scale objects, can be used to produce spatially resolved maps of the surface or material properties of a sample; these include charge density, adhesion and stiffness, as well as the force required to break specific ligand-receptor bonds. Maps such as these will provide fundamental insights into biological structure and will become an important tool for characterizing technologically important biological systems.

Reduced amino acid alphabet is sufficient to accurately recognize intrinsically disordered protein

Intrinsically disordered proteins are an important class of proteins with unique functions and properties. Here, we have applied a support vector machine (SVM) trained on naturally occurring disordered and ordered proteins to examine the contribution of various parameters (vectors) to recognizing proteins that contain disordered regions. We find that a SVM that incorporates only amino acid composition has a recognition accuracy of 87 ± 2%. This result suggests that composition alone is sufficient to accurately recognize disorder. Interestingly, SVMs using reduced sets of amino acids based on chemical similarity preserve high recognition accuracy. A set as small as four retains an accuracy of 84 ± 2%; this suggests that general physicochemical properties rather than specific amino acids are important factors contributing to protein disorder.

Relative Surface Charge Density Mapping with the Atomic Force Microscope

An experimental approach for producing relative charge density maps of biological surfaces using the atomic force microscope is presented. This approach, called D minus D (D-D) mapping, uses isoforce surfaces collected at different salt concentrations to remove topography and isolate electrostatic contributions to the tip-sample interaction force. This approach is quantitative for surface potentials below 25 mV, and does not require prior knowledge of the cantilever spring constant, tip radius, or tip charge. In addition, D-D mapping does not require tip-sample contact. The performance of D-D mapping is demonstrated on surfaces of constant charge and varying topography (mechanically roughened mica and stacked bilayers of dipalmitolphosphatidylserine), a surface of varying charge and varying topography (patches of dipalmitolphosphatidylcholine on mica), and bacteriorhopsin membranes adsorbed to mica.

AFM force measurements on microtubule-associated proteins: the projection domain exerts a long-range repulsive force

Microtubule‐associated proteins (MAPs) are thought to control spacing between microtubules. We propose that the projection domain is largely unstructured and exerts a long‐range repulsive force that is predominantly entropic in origin, providing a physical mechanism for maintaining spacing. To test this hypothesis, we developed an experimental system where MAPs are electrostatically end‐attached to a flat surface, such that the projection domains extend away from the surface. Atomic force microscopy force measurements on this system show that projection domains exert a long‐range (>100 nm) repulsive force. This force depends on the ionic strength of the solution in a way that is consistent with a polyelectrolyte polymer brush.

Microelastic properties of lung cell-derived extracellular matrix

The mechanical properties of the extracellular microenvironment regulate cell behavior, including migration, proliferation and morphogenesis. Although the elastic moduli of synthetic materials have been studied, little is known about the properties of naturally produced extracellular matrix. Here we have utilized atomic force microscopy to characterize the microelastic properties of decellularized cell-derived matrix from human pulmonary fibroblasts. This heterogeneous three-dimensional matrix had an average thickness of 5 ± 0.4 μm and a Youngs modulus of 105 ± 14 Pa. Ascorbate treatment of the lung fibroblasts prior to extraction produced a twofold increase in collagen I content, but did not affect the stiffness of the matrices compared with matrices produced in standard medium. However, fibroblast-derived matrices that were crosslinked with glutaraldehyde demonstrated a 67% increase in stiffness. This work provides a microscale characterization of fibroblast-derived matrix mechanical properties. An accurate understanding of native three-dimensional extracellular microenvironments will be essential for controlling cell responses in tissue engineering applications.

Splaying of Aliphatic Tails Plays a Central Role in Barrier Crossing During Liposome Fusion

The fusion between two lipid bilayers involves crossing a complicated energy landscape. The limiting barrier in the process appears to be between two closely opposed bilayers and the intermediate state where the outer leaflets are fused. We have performed molecular dynamics simulations to characterize the free energy barrier for the fusion of two liposomes and to examine the molecular details of barrier crossing. To capture the slow dynamics of fusion, a model using coarse-grained representations of lipids was used. The fusion between pairs of liposomes was simulated for four systems: DPPC, DOPC, a 3:1 mixture of DPPC/DPPE, and an asymmetric lipid tail system in which one tail of DPPC was reduced to half the length (ASTail). The weighted histogram method was used to compute the free energy as a function of separation distance. The relative barrier heights for these systems was found to be ASTail >> DPPC > DPPC/DPPE > DOPC, in agreement with experimental observations. Further, the free energy curves for all four can be overlaid on a single curve by plotting the free energy versus the surface separation (differing only in the point of fusion). These simulations also confirm that the two main contributions to the free energy barrier are the removal of water between the vesicles and the deformation of the vesicle. The most prominent molecular detail of barrier crossing in all cases examined was the splaying of lipid tails, where initially a single splayed lipid formed a bridge between the two outer leaflets that promotes additional lipid mixing between the vesicles and eventually leads to fusion. The tail splay appears to be closely connected to the energetics of the process. For example, the high barrier for the ASTail is the result of a smaller distance between terminal methyl groups in the splayed molecule. The shortening of this distance requires the liposomes to be closer together, which significantly increases the cost of water removal and bilayer deformation. Before tail splay can initiate fusion, contact must occur between a tail end and the external water. In isolated vesicles, the contact fraction is correlated to the fusogenicity difference between DPPC and DOPC. Moreover, for planar bilayers, the contact fraction is much lower for DPPC, which is consistent with its lack of fusion in giant vesicles. The simulation results show the key roles of lipid tail dynamics in governing the fusion energy landscape.

Role of long-range repulsive forces in organizing axonal neurofilament distributions: Evidence from mice deficient in myelin-associated glycoprotein

When the axon of a motor neuron is sectioned and visualized by electron microscopy, a two‐dimensional distribution of neurofilaments (NFs) with nonrandom spacing is revealed; this ordered arrangement implies the presence of physical interactions between the NFs. To gain insight into the molecular basis of this organization, we characterized NF distributions from mouse sciatic nerve cross sections using two statistical mechanical measures: radial distribution functions and occupancy probability distributions. Our analysis shows that NF organization may be described in terms of effective pairwise interactions. In addition, we show that these statistical mechanical measures can detect differences in NF architecture between wild‐type and myelin‐associated glycoprotein null mutant mice. These differences are age dependent, with marked contrast between the NF distributions by 9 months of age. Finally, using Monte Carlo simulations, we compare the experimental results with predictions for models in which adjacent NFs interact through rigid cross bridges, deformable cross bridges, and long‐range repulsive forces. Among the models tested, a model in which the filaments interact through a long‐range repulsive force is most consistent with the results of our analysis.

Probing the Machinery of Intracellular Trafficking with the Atomic Force Microscope

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular‐scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor–ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.

Modes of remodeling in the cortical cytoskeleton of vascular endothelial cells

The cortical cytoskeleton of vascular endothelial cells plays an important role in responding to mechanical stimuli and controlling the distribution of cell surface proteins. Here, we have used atomic force microscopy to visualize the dynamics of cortical cytoskeleton in living bovine pulmonary artery endothelial cells. We demonstrate that the cortical cytoskeleton, organized as a complex polygonal mesh, is highly dynamic and shows two modes of remodeling: intact‐boundary‐mode where mesh element boundaries remain intact but move at ∼0.08 μm/min allowing the mesh element to change shape, and altered‐boundary‐mode where new mesh boundaries form and existing ones disappear.