Transport of semiconductor nanocrystals by kinesin molecular motors.

TLDR: Semiconductor nanocrystals are synthesized, attached to kinesin biomolecular motors, demonstrated that single motors can be visualized by simple epifluorescence or evanescent wave microscopy, and shown that motor function is unaffected by particle functionalization.

Abstract: Kinesin molecular motors harness the energy of ATP hydrolysis to transport cargo such as vesicles and organelles along intracellular microtubules. Purified components of this system can be used for nanoscale transport by integrating the motors and filaments into MEMS and NEMS devices. Hence, it is important to understand the function of these proteins for biological, therapeutic, and nanotechnological applications. Existing techniques for studying motors include the microtubule gliding assay, optical traps, and ATPase assays. Single-molecule visualization is crucial for investigating the motor mechanism and their ability to move and assemble nanoparticles. In this report, we synthesize semiconductor nanocrystals, attach them to kinesins, demonstrate that single motors can be visualized by simple epifluorescence or evanescent wave microscopy, and show that motor function is unaffected by particle functionalization. Single kinesin motors functionalized with green fluorescent protein (GFP) or synthetic fluorophores can be imaged by total internal reflection fluorescence (TIRF) microscopy, and their position resolved to within nearly one nanometer. By tracking kinesins in which one of the two motor domains (heads) was labeled, this technique was used to show that at limiting ATP concentrations each head takes 16-nm steps along a microtubule, ruling out the “inchworm” model of kinesin motility. However, because the spatial resolution is based on the number of photons collected, the temporal resolution using these fluorophores is limited to roughly 300 ms. Brighter fluorophores are needed to measure faster events. While fluorescent beads have higher signal intensities, their size alters the diffusion properties of the tagged molecule and complicates intracellular experiments. Semiconductor nanocrystals (quantum dots) have great potential in biological imaging due to their small size ( 5– 10 nm radius with functionalization), high quantum yield, large excitation band, and negligible photobleaching. Quantum dots with different optical properties can be synthesized with ease by growing them to different sizes, and single fluorophores can be visualized by simple epifluorescence microscopy rather than the evanescent wave microscopy that is generally required for GFP and other synthetic fluorophores. In addition, they can be introduced into cells by a variety of methods. By synthesizing our own quantum dots, we have the advantage of being able to separately tune the emission wavelength and control the surface functionality. The goal of this study is to functionalize quantum dots with active kinesin biomolecular motors and transport these dots along immobilized microtubules. This new labeling approach will open up a number of avenues of investigation. First, it will enable more precise tracking of motors in vitro to understand motor stepping and detachment under controlled conditions. Second, these bright particles should enable individual kinesins to be followed in cells, which is very difficult with current labeling procedures. Third, quantum dots can be used as models for biomotor-driven nanoparticle assembly in vitro. More and more materials are being synthesized at nanoscale geometries that confer novel and enhanced functionality. However, despite the success of various self-assembly processes, organization of these nanoparticles into configurations far from their thermodynamic minima is a continuing hurdle. Because kinesins are specialized transport motors that have evolved to organize the intracellular environment, they provide a powerful tool for transport and assembly of synthetic nanomaterials. Harnessing these biological motors for this purpose requires a model system that can be easily visualized and quantified. At present, microtubules that have been coated with quantum dots have been shown to move along immobilized motors so long as the region of functionalization is limited. Furthermore, in a related and impressive recent study, individual myosin V motors were labeled with a different-colored quantum dot on each head to definitively show that the two heads alternately step along an immobilized actin filament. Here, we demonstrate for the first time that individual kinesin motors can be functionalized with quantum dots, and their movement along microtubules easily tracked by either TIRF or epifluorescence microscopy. Using quantum dots for this purpose comes with a number of hurdles. Generally, quantum-dot cores are synthesized in an organic phase and usually with cytotoxic compounds, so for biological applications the cores need to be protected and transferred to an aqueous phase by coating with a shell of a second semiconductor with a larger bandgap and with protective ligands. Additional ligands must be [*] G. Muthukrishnan, Dr. W. O. Hancock Department of Bioengineering, 229 Hallowell Bldg. The Pennsylvania State University University Park, PA 16802 (USA) Fax: (+1)814-863-0490 E-mail: mbw@chem.psu.edu