Kristi L. Hanson

Polymer surface properties control the function of heavy meromyosin in dynamic nanodevices.

The actin-myosin system, responsible for muscle contraction, is also the force-generating element in dynamic nanodevices operating with surface-immobilized motor proteins. These devices require materials that are amenable to micro- and nano-fabrication, but also preserve the bioactivity of molecular motors. The complexity of the protein-surface systems is greatly amplified by those of the polymer-fluid interface; and of the structure and function of molecular motors, making the study of these interactions critical to the success of molecular motor-based nanodevices. We measured the density of the adsorbed motor protein (heavy meromyosin, HMM) using quartz crystal microbalance; and motor bioactivity with ATPase assay, on a set of model surfaces, i.e., nitrocellulose, polystyrene, poly(methyl methacrylate), and poly(butyl methacrylate), poly(tert-butyl methacrylate). A higher hydrophobicity of the adsorbing material translates in a higher total number of HMM molecules per unit area, but also in a lower uptake of water, and a lower ratio of active per total HMM molecules per unit area. We also measured the motility characteristics of actin filaments on the model surfaces, i.e., velocity, smoothness and deflection of movement, determined via in vitro motility assays. The filament velocities were found to be controlled by the relative number of active HMM per total motors, rather than their absolute surface density. The study allowed the formulation of the general engineering principles for the selection of polymeric materials for the manufacturing of dynamic nanodevices using protein molecular motors.

Electrophoretic control of actomyosin motility

The effect of DC electric field strength on in vitro actomyosin motility was examined. Rabbit skeletal muscle heavy meromyosin (HMM) was adsorbed to nitrocellulose-coated glass, and the myosin driven movement of fluorescently labeled actin filaments was recorded in the presence of 0 to 9000 V m/sup -1/ applied DC voltage. The applied electric field resulted in increased filament velocity and oriented actin movement, with leading heads of filaments directed towards the positive electrode. Velocity (v) was found to increase moderately with electric field strength at applied fields up to /spl sim/4500 V m/sup -1/, and then increased more rapidly and irregularly at higher field strengths up to 9000 V m/sup -1/. The electrophoretic effect caused up to 70% of actin motion to be oriented within 30 degrees of the positive electrode, with the largest effect observed using an applied field of 6000 V m/sup -1/. Higher electric field strengths were found to cause extensive filament breakage.

Motility of Actin Filaments on Micro-Contact Printed Myosin Patterns

Protein molecular motors, which convert, directly and efficiently, chemical energy into motion, are excellent candidates for integration in hybrid dynamic nanodevices. To integrate and use the full potential of molecular motors in these devices, their design requires a quantitative and precise prediction of the fundamental mechanical and physicochemical features of cytoskeletal proteins operating in artificial environments. In that regard, the behavior of protein molecular motors constructs in/on nano-confined spaces or nanostructured surfaces that aim to control their motility is of critical interest. Here, we used a standard gliding motility assay to study the actin filaments sliding on a surface comprising heavy mero myosin (HMM) micro- and nano-patterns. To print HMM, we used negative tone, micro contact printing of a blocking protein (bovine serum albumin, BSA) on a nitrocellulose surface, followed by specific adsorption of HMM on BSA-free surfaces. While the large BSA-free patterns allowed for selective confinement of actin filaments motility, the BSA-stamped areas displayed intricate nano-sized HMM patterns, which enabled a deeper analysis of the nano-mechanics of actomyosin motility in confined spaces.

Negotiation of obstacles by fungi in micro-fabricated structures: to turn or not to turn?

Polymer microstructures were used to examine the manner in which fungal filaments negotiate obstacles in confined environments. When faced with an obstacle requiring a right-angle turn, two different responses were observed. In 21% of cases, hyphae turned around the corner and continued growth, while in the remaining 79% of cases, filaments continued apical growth into the corner, resulting in bending of the distal portions of the filament. The different reactions could not be linked to physical constraints (e.g., filament flexibility) since the filament deflection required to negotiate the obstacle was the same in all cases. Instead, the response appeared to be related to the original direction of growth at the time of filament formation (branching), with filaments turning only if the resultant growth vector was no more than 90/spl deg/ from their original branching vector. The results suggest that filaments are somehow able to retain a memory of their original branching direction, consistent with an overall survival strategy based on continued growth away from the colony center and into the surrounding environment.

Actomyosin motility detection using quartz crystal microbalance

A 5 MHz Quartz Crystal Microbalance was used to investigate changes in resonant frequency and motional resistance of the protein film during in vitro actomyosin motility on a poly (tert-butyl methacrylate) surface. QCM crystal frequency was found to decrease with adsorption of heavy meromyosin (HMM) to the crystal surface, and with binding of additional protein during the standard BSA blocking step. The frequency and resistance signals after binding of dead HMM heads in actin rigor complexes were consistent with those expected for a film becoming more rigid, but suggested that little mass was added during this step. Addiion of the low concentration of actin used for motility did not cause a significant signal response, but addition of ATP to initiate actin filament movement caused both frequency and resistance signals to increase slightly, consistent with a less rigid protein film of lower apparent mass, suggesting that moving filaments are felt with a lower effective mass than strongly bound static filaments. The frequency signal also fluctuated significantly during motility, consistent with a dynamic process occurring on the crystal surface.

Effect of surface chemistry on in vitro actomyosin motility

A variety of surface coatings were evaluated for their ability to promote in vitro actomyosin motility. Rabbit skeletal muscle heavy meromyosin (HMM) was adsorbed to uncoated glass and to surfaces coated with nitrocellulose, poly(methyl methacrylate) (PMMA), poly(butyl methacrylate) (PBMA), poly(tert-butyl methacrylate (PtBMA), polystyrene (PS) and hexamethyldisilazane (HMDS), and the myosin driven movement of fluorescently labeled actin filaments was recorded using epifluorescence microscopy. HMDS and uncoated glass did not support actomyosin motility, while mean velocities on other surfaces ranged from 1.7 μm sec-1 (PtBMA) to 3.5 μm sec-1 (NC). Nitrocellulose supported the highest proportion of motile filaments (75%), while 47 - 61% of filaments were motile on other surfaces. Within the methacrylate polymers, average filament velocities increased with decreasing hydrophobicity of the surface. Distributions of instantaneous acceleration values and angle deviations suggested more erratic and stuttered movement on the methacrylates and polystyrene than on NC, in line with qualitative visual observations. Despite the higher velocities and high proportion of motile filaments on NC, this surface resulted in a high proportion of small filaments and high rates of filament breakage during motility. Similar effects were observed on PS and PtBMA, while PBMA and PMMA supported longer filaments with less observed breakage.

Laser-based manipulation of growth direction and intracellular processes in fungal hyphae

Fungal growth is concentrated in elongated tips, called hyphae, which have the tendency to maintain their direction of growth. Hyphal tips exhibit a number of tropisms in response to various factors e.g. nutrients, light, physical contact.Irradiation in the area of hyphal tips with a 1064 nm laser affected shown a sensing mechanism within the fungal tip. The result of this was a change of growth direction caused by Spitzenkoerpers tendency to move away from the trap. The manipulation of the growth orientation of fungi in microstructures using focused laser beam has the potential to help the understanding of space search algorithms used by microorganisms.

Fungal growth in confined microfabricated networks

The understanding and control of cell growth in confined microenvironments has application to a variety of fields including cell biosensor development, medical device fabrication, and pathogen control. While the majority of work in these areas has focused on mammalian and bacterial cell growth, this study reports on the growth behavior of fungal cells in three-dimensionally confined PDMS microenvironments of a scale similar to that of individual hyphae. The general responses of hyphae to physical confinement included continued apical extension against barriers, resultant filament bending and increased rates of subapical branching with apparent directionality towards structure openings. Overall, these responses promoted continued extension of hyphae through the confined areas and away from the distal regions of the fungal colony. The induction of branching by apical obstruction provides a means of controlling the growth and branching of fungal hyphae through purposefully designed microstructures.

Determination of the persistence length of actin filaments on microcontact printed myosin patterns

Protein molecular motors, which convert chemical energy into kinetic energy, are prime candidates for use in nanodevice in which active transport is required. To be able to design these devices it is essential that the properties of the cytoskeletal filaments propelled by the molecular motors are well established. Here we used micro-contact printed BSA to limit the amount of HMM that can adsorb creating a tightly confined pathway for the filaments to travel. Both the image and statistical analysis of the movement of the filaments through these structures have been used to new insights into the motility behaviour of actomyosin on topographically homogenous, but motor-heterogeneous planar systems. It will be shown that it is possible to determine the persistence length of the filaments and that it is related to the amount of locally adsorbed HMM. This provides a basis that can be used to optimize the design of future nanodevices incorporating the actomyosin system for the active transport.

Adsorption-induced inactivation of heavy meromyosin on polymer surfaces imposes effective drag force on sliding actin filaments in vitro

Actin and myosin are of interest as potential force-generating elements in engineered nanodevices. Such applications require surface coatings which are both biocompatible and amenable to nanolithographic processing, but the manner in which surfaces modulate motor protein function has not been rigorously studied. Here we examine motor protein surface density and bioactivity on a variety of polymer surfaces, and compare the results to in vitro actomyosin motility characteristics. Filament velocities were found to be controlled by the proportion, rather than density, of active heavy meromyosin (HMM), consistent with the imposition of an effective drag force by inactivated HMM due to weak actin-binding interactions. Interpretation of the results with respect to previous models suggests that the inactive HMM fraction has no force-generating ability, and that the effective drag imposed on polystyrene is lower than that on methacrylate polymers and nitrocellulose, consistent with a higher degree of protein denaturation on aromatic surface structures