No abstract

Kinesins and microtubules are biological motor proteins and rail fibers, respectively. Their combination could be an attractive power source for nanotools. Their movements can be easily reproduced in vitro by the motility assay method. However, the durability of the protein material remains relatively short for use in devices. Herein, regeneration of the active substrate by removing surface-adsorbed kinesins by multi-photon laser ablation and reloading additional kinesin molecules was attempted. The effect of femtosecond laser pulse irradiation to the surface-adsorbed kinesins was investigated by atomic force microscopy and by the microtubule driving performance. The findings of these investigations suggest the successful exchange of kinesins, which leads to improvement of the durability of microtubule driving performance.

In living organisms, many dynamic mechanisms are driven by motor proteins on a wide scale for tasks including the assembly of hierarchical structures at the nano to micrometre scales and macroscopic movements with hierarchical structures. Such complicated assemblies and sophisticated functions are intriguing for applications in nano and microengineering. Using motor proteins may enable multimolecular assembly in artificial systems by reproducing simple molecular movements using established methods such as motility assays of kinesin and microtubules. However, building a multimolecular system and selecting the target functions are key points to consider for potential applications. We use an active matrix consisting of crosslinked microtubules driven by kinesin to agitate microscopic objects that are not moved by thermal fluctuation, that is, non-Brownian particles. This method may contribute to enhance various self-assembly processes for larger objects. The resulting isotropic agitating properties are compared with those of other agitation methods based on external forces exerted by electric motors. The active matrix may provide a new type of mesoscopic scale actuator to perform stochastic mechanical agitation.

Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared (λ = 1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi networks were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.