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Biomolecular Motor Systems: From Cellular Function to Nanotechnological Applications

Final Report Summary - NANOTRANS (Biomolecular Motor Systems: From Cellular Function to Nanotechnological Applications)

Achievements within the NanoTrans project include (i) new knowledge about intracellular transport processes on a molecular level using novel nano-optical imaging tools and (ii) novel methodologies to use biomolecular motor systems in engineered environments for the fulfillment of nanotechnological tasks.

Building on experience in optical microscopy and single molecule biophysics the research group developed and applied novel tools to study the dynamical functioning of microtubule-based motor proteins in vitro with nanometer precision in three dimensions: (a) a novel image-processing algorithm for the tracking of single particles and elongated filaments with nanometer precision has been developed, (b) the detailed paths of processive kinesin-8 motor proteins on the microtubule surface have been investigated using quantum dots in combination with fluorescence-interference contrast microscopy, (c) molecular crowding, examined by single-molecule microscopy using kinesin-8 motor proteins labeled by green-fluorescent proteins in combination with total-internal reflection fluorescence microscopy, has been shown to cause traffic jams on microtubules, (d) long range transport of microtubules and giant unilamellar vesicles coupled to multiple kinesin-14 and kinesin-1 motor proteins along microtubule networks has been demonstrated, (e) horizontal magnetic tweezers to measure the force-velocity relationship for multiple kinesin-1 motors have been constructed and tested, (f) multi-motor transport in a system of active and inactive kinesin-1 motors has been studied, (g) diffusible crosslinkers have been shown to generate directed forces in microtubule networks, (h) kinesin-1 motors have been directly observed to circumvent permanent roadblocks on microtubules by side-shifting to neighboring protofilaments, (i) the non-processive rice kinesin-14 OsKCH1 has been shown to transport actin filaments along microtubules with two distinct velocities, (j) an impact-free measurement technique of microtubule rotations on kinesin and cytoplasmic-dynein coated surfaces has been devised, and (k) small crowders have been shown to slow down kinesin-1 stepping by hindering motor domain diffusion. The results from these studies broaden our knowledge about the design principles of molecular machines as well as the principles by which they interact with each other. In particular, it has been learnt (1) that the length of the motor neck-linker (i.e. the structural elements which connect the two motor heads) determines if a processive microtubule motor can switch protofilaments on the microtubule surface, (2) that motor processivity and end-residency determine if traffic jams form on microtubules, (3) that flexibility in the motor structure as well as flexibility in the coupling of the motors to each other allows multiple motors of the same directionality to transport large cargo over long distances, and (4) that the contribution of a single kinesin motor engaged in the cooperative transport by multiple motors strongly depends on its actual geometrical arrangement with respect to the microtubule.

This knowledge is expected to pave the road for applications of molecular motors for a wide range of self-organizing nanofunctions in engineered environments. In contrast to conventional "macroscopic" top-down or "atomic" bottom-up approaches, a driving factor for this prospect is the capability of cellular machines to work in parallel, thus enabling the efficient fabrication and detection of nanostructures. Towards this end the research group (l) demonstrated the programmable, spatio-temporal control over protein bioactivity using light and electrical currents in combination with surface-grafted thermoresponsive polymer chains, (m) applied microtubules as molecular rulers to measure axial nanometer distances by fluorescence-lifetime imaging microscopy, (n) demonstrated the programmable patterning of protein bioactivity by visible light, (o) performed the parallel computation with molecular motor-propelled agents in nanofabricated networks, and (p) optimized the expression and purification of kinesin-1 motors for nanotechnological applications. These studies generated novel methodologies (5) towards the dynamic routing of kinesin-driven microtubule transporters at junctions, potentially to be applied in future lab-on-chip systems for molecular sorting and (6) towards applying fluorescently labeled microtubules as optical probes to determine the geometrical size of attached proteins with nanometer precision.

In summary, the NanoTrans project focused on the interface between molecular cell biology and nanotechnology. The obtained results provide a significant step towards letting smart nanomaterials fulfill biological functions in cellular systems and to efficiently operating biomolecular machines in inorganic environments.