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Posted: Aug 29, 2008
Nanotechnology transport systems get a closer look
(Nanowerk Spotlight) In much the same way that each of our bodies depends on bones for mechanical integrity and strength, each cell within our bodies is governed mechanically by a skeleton – called the cytoskeleton – of composite materials including protein polymers and motor proteins. The cytoskeleton is an active material that maintains cell shape, enables some cell motion, and plays important roles in both intra-cellular transport and cellular division. The cytoskeletal system is not at thermodynamic equilibrium and this non-equilibrium drives motor proteins that are the force generators in cells.
These motor proteins of the cytoskeleton accomplish nanotransport tasks by moving 'cargo' along microtubules that are about 25 nm wide but can grow up to 1,000 times as long. Nanotechnology engineers are fascinated by this transport mechanism and several efforts are underway in various labs to unravel and, researchers hope, eventually copy nature's engineering feat. A particularly promising setup consists of surface-attached linear motor proteins that drive the motion of cytoskeletal filaments. Some researchers expect that artificial molecular transport systems which utilize microtubules motility will be an alternative to pressure-driven or electrokinetic flow-based microfluidic devices.
"The principle is very reminiscent of crowd surfing" Dr. Stefan Diez explains to Nanowerk. "The moving filaments serve as nanoshuttles, which transport and deliver cargo in a controlled manner. However, cytoskeletal filaments rotate around their longitudinal axis due to intrinsic properties of filaments and motors. We have found that this rotation is not disturbed in the case of small cargo (quantum dots)."
Schematic representation of moving filaments that serve as nanoshuttles, transporting and delivering cargo in a controlled manner. (Image: Diez group, Max Planck Institute of Molecular Cell Biology and Genetics)
While the linear motion of motor proteins along microtubules has been studied in detail using stepping assays with motor-coated microbeads, motor-attached quantum dots or single fluorescently labeled motors, a complication arises from the fact that these microtubules are three-dimensional nanostructures. So a motor attached to the surface doesn't necessarily have to propel the microtubule straight ahead, as you would want it to, but could end up rotating the filament and go off-axis. This could result in the cargo being stripped off or the forward motion of the gliding microtubules to be impaired.
Investigating this rotation behavior with high accuracy is therefore an important issue.
Diez, who leads the Molecular Transport in Cell Biology and Nanotechnology group at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, and his group describe a novel method to characterize the rotational movement of cytoskeletal filaments gliding over motor-coated substrate surfaces. This technique allows exploring the detailed paths that motors take on cytoskeletal filaments. This is also important in understanding situations of heavy intracellular traffic, where motors might have to switch lanes.
Diez says that, although the labeling of the microtubules with quantum dots did not impede the rotational movement, the pick-up of larger cargo did. "However, the fact that the velocity of microtubule gliding was not affected shows that kinesin-driven microtubules make flexible, responsive and effective molecular shuttles for nanotransport applications."
The Max Planck scientists concluded that this shows that kinesin-1 driven microtubules make responsive and effective nanoshuttles.
"When filament rotation had been studied previously, optical detection relied on filament supercoiling or periodic sideways deflections of distinctive microtubule structures," Bert Nitzsche, first author of the paper and a PhD student in Diez's group, explains. "As a consequence, these experiments had a limited accuracy with respect to quantification of the rotational periodicities and were based on the deliberate construction of impaired gliding assays or defective filaments. In contrast, in our experiments, given the contour length of ∼60 nm for the kinesin-1 constructs, we expected that the quantum dots used as optical reporters would have no impact on the rotations."
In a novel approach, the scientists combined 2-D nanometer tracking and interference-based fluorescence imaging for the third dimension, which allows the 3-D tracking of fluorescent particles. The technique is easy to set up and easy to use. It only requires a standard fluorescence microscope and the imaging to be performed on a reflective surface.
"Specifically, we have used the technique to characterize the movement of filamentous nanoshuttles that glide over surfaces coated with linear molecular motor proteins" says Nitzsche. "This was being done by tracking the 3-D paths of quantum dots being attached to the filaments."
The kinesin-1 motor proteins used by Diez and his team are processive i.e. they have the tendency to remain bound to the microtubules which they propel. If large cargo is attached to the nanoshuttle and prohibits rotation, the motors would be expected to build up strain thereby dramatically affecting microtubule motility.
"To our knowledge we are the first ones to report that although rotation is stopped, transport of large cargo is still perfectly possible using a processive motor" says Diez.
This method and the resulting observations are interesting for the design and understanding of transport of cargo, but the 3-D nanometry technique could generally be applied to observe single molecule interactions in 3-D with nanometer precision.
As far as the nanotransport aspect is concerned, enhanced spatio-temporal control and the loading and delivery of cargo are future challenges that the team is planning to tackle. Diez says that they are going to explore ways to take advantage of the rotation of nanoshuttles, for example for loading and unloading of cargo.
With regard to the 3-D nanometry technique, a future challenge is to achieve better resolution in space and time. In particular, better optical labels are a key to further improvement. One example is the optimization of the optical properties of quantum dots, which so far exhibit blinking and thereby add considerable noise to the positions of the tracked objects.
By the way, in case you still haven't seen the absolutely amazing animation Cellular Visions: The Inner Life of a Cell yet, go watch it now. In it, there is a sequence where a motor protein is sort of 'walking' along a filament, dragging this round sphere of lipids behind it. This kind of nanoscale biological motor is able to load/unload particular types of cargo without external stimuli, and transport them along cytoskeletal filaments by using the energy of adenosine triphosphate (ATP) hydrolysis within cells. Nanotechnology researchers are fascinated by the various molecular delivery systems that have evolved in nature and they are receiving increasing attention as blueprints for nanoscale actuators and building blocks to construct artificially-engineered bio-hybrid systems.