Kinesin and dynein mechanics: measurement methods and research applications

Zachary Abraham, Emma Hawley, Daniel Hayosh, Victoria Webster-Wood and Ozan Akkus

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical for researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research towards novel treatment and nanoengineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will: a) discuss such techniques, specifically microscopy, atomic force microscopy, optical trapping, and magnetic tweezers, and, b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity and dependence on ATP concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

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Volumetric Stress-Strain Analysis of Optohydrodynamically Suspended Biological Cells

Sean S. Kohles, Yu Liang and Asit K. Saha

Ongoing investigations are exploring the biomechanical properties of isolated and suspended biological cells in pursuit of understanding single-cell mechanobiology. An optical tweezer with minimal applied laser power has positioned biologic cells at the geometric center of a microfluidic cross-junction, creating a novel optohydrodynamic trap. The resulting fluid flow environment facilitates unique multiaxial loading of single cells with site-specific normal and shear stresses resulting in a physical albeit extensional state. A recent two-dimensional analysis has explored the cytoskeletal strain response due to these fluid-induced stresses [Wilson and Kohles, 2010, “Two-Dimensional Modeling of Nanomechanical Stresses-Strains in Healthy and Diseased Single-Cells During Microfluidic Manipulation,” J Nanotechnol Eng Med, 1(2), p. 021005]. Results described a microfluidic environment having controlled nanometer and piconewton resolution. In thispresent study, computational fluid dynamics combined with multiphysics modeling has further characterized the applied fluid stress environment and the solid cellular strain response in three dimensions to accompany experimental cell stimulation. A volumetric stress-strain analysis was applied to representative living cell biomechanical data. The presented normal and shear stress surface maps will guide future microfluidic experiments as well as provide a framework for characterizing cytoskeletal structure influencing the stress to strain response.

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Axisymmetric Optical-Trap Measurement of Red Blood Cell Membrane Elasticity

Alexandre Lewalle and Kim H. Parker

The elastic properties of the cell membrane play a crucial role in determining the equilibrium shape of the cell, as well as its response to the external forces it experiences in its physiological environment. Red blood cells are a favored system for studying membrane properties because of their simple structure: a lipid bilayer coupled to a membrane cytoskeleton and no cytoplasmic cytoskeleton. An optical trap is used to stretch a red blood cell, fixed to a glass surface, along its symmetry axis by pulling on a micron-sized latex bead that is bound at the center of the exposed cell dimple. Thesystem, at equilibrium, shows Hookean behavior with a spring constant of 1.5×10−6 N/m over a 1–2 µm range of extension. This choice of simple experimental geometry preserves the axial symmetry of the native cell throughout the stretch, probes membrane deformations in the small-extension regime, and facilitates theoretical analysis. The axisymmetry makes the experiment amenable to simulation using a simple model that makes no a priori assumption on the relative importance of shear and bending in membrane deformations. We use an iterative relaxation algorithm to solve for the geometrical configuration of the membrane at mechanical equilibrium for a range of applied forces. We obtain estimates for the out-of-plane membrane bending modulus B1×10−19 Nm and an upper limit to the in-plane shear modulus H<2×10−6 N/m. The partial agreement of these results with other published values may serve to highlight the dependence of the cell's resistance to deformation on the scale and geometry of the deformation.

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Mechanical Stress Analysis of Microfluidic Environments Designed for Isolated Biological Cell Investigations

Sean S. Kohles, Nathalie Nève, Jeremiah D. Zimmerman, and Derek C. Tretheway

Advancements in technologies for assessing biomechanics at the cellular level have led to discoveries in mechanotransduction and the investigation of cell mechanics as a biomarker for disease. With the recent development of an integrated optical tweezer with micron resolution particle imagevelocimetry, the opportunity to apply controlled multiaxial stresses to suspended single cells is available (Nève, N., Lingwood, J. K., Zimmerman,J., Kohles, S. S., and Tretheway, D. C., 2008, “The µPIVOT: An Integrated Particle Image Velocimetry and Optical Tweezers Instrument for Microenvironment Investigations,” Meas. Sci. Technol., 19(9), pp. 095403). A stress analysis was applied to experimental and theoretical flow velocitygradients of suspended cell-sized polystyrene microspheres demonstrating the relevant geometry of nonadhered spherical cells, as observed for osteoblasts, chondrocytes, and fibroblasts. Three flow conditions were assessed: a uniform flow field generated by moving the fluid sample with an automated translation stage, a gravity driven flow through a straight microchannel, and a gravity driven flow through a microchannel cross junction. The analysis showed that fluid-induced stresses on suspended cells (hydrodynamic shear, normal, and principal stresses in the range of 0.02–0.04 Pa) are generally at least an order of magnitude lower thanadhered single cell studies for uniform and straight microchannel flows (0.5–1.0 Pa). In addition, hydrostatic pressures dominate (1–100 Pa) overhydrodynamic stresses. However, in a cross junction configuration, orders ofmagnitude larger hydrodynamic stresses are possible without the influence of physical contact and with minimal laser trapping power.

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