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.
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.
We present a stochastic and spatial Monte Carlo model for the growth of a fungal colony in microstructures. This model is based on an "L-system-like" representation of filaments as individual objects. Each of these can both grow in space (and be diverted by obstacles) and can send new branches. All parameters in the model such as filament dimensions, the growth speed, behavior at and around obstacles, branching angle and frequency and others are obtained from experimental studies of growth in artificial microstructures. We investigate four different possible "strategies" the colony might use to achieve the tasks of (a) filling the available space and (2) finding its way out of the structures. The simulation results indicate that a combination of directional memory and a stop-and-branch behavior at corners gives the best results and observe that in fact this is similar to the experimentally observed behavior of the fungi. The model is expected to be of use in studying the colonization of microstructures by fungi and in the design of devices either using fungal growth or aiming to inhibit it.
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 Spitzenkoerper's 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.
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.
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-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 ~ 4500 V m-1 (Δv/ΔE = 0.037 μm2 V-1sec-1), and then increased at a more rapid rate (Δv/ΔE = 0.100 μm2 V-1sec-1) at higher field strengths up to 9000 V m-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-1. Higher electric field strengths caused filament breakage.
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.
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 PDMS microenvironments of a scale similar to that of individual hyphae. Confinement was found to affect filament branching rate and angle. Overall, fungal hyphae demonstrate much more coordinated behavior during confinement than observed during growth on simple planar unconfined substrates. The remarkable difference of fungal growth behaviour observed in the PDMS microenvironments compared to open, unrestricted environments suggests that three-dimensional microstructures could be used to control and alter fungal motility.