Multiplication of Motor-Driven Microtubules for Nanotechnological Applications.

Microtubules gliding on motor-functionalized surfaces have been explored for various nanotechnological applications. However, when moving over large distances (several millimeters) and long times (tens of minutes), microtubules are lost due to surface detachment. Here, we demonstrate the multiplication of kinesin-1-driven microtubules that comprises two concurrent processes: (i) severing of microtubules by the enzyme spastin and (ii) elongation of microtubules by self-assembly of tubulin dimers at the microtubule ends. We managed to balance the individual processes such that the average length of the microtubules stayed roughly constant over time while their number increased. Moreover, we show microtubule multiplication in physical networks with topographical channel structures. Our method is expected to broaden the toolbox for microtubule-based in vitro applications by counteracting the microtubule loss from substrate surfaces. Among others, this will enable upscaling of network-based biocomputation, where it is vital to increase the number of microtubules during operation.

Regeneration of Assembled, Molecular-Motor-Based Bionanodevices.

The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.

Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.

Molecular motors, highly efficient biological nanomachines, hold the potential to be employed for a wide range of nanotechnological applications. Toward this end, kinesin, dynein, or myosin motor proteins are commonly surface-immobilized within engineered environments in order to transport cargo attached to cytoskeletal filaments. Being able to flexibly control the direction of filament motion, and in particular on planar, non-topographical surfaces, has, however, remained challenging. Here, we demonstrate the applicability of a UV-laser-based ablation technique to programmably generate highly localized patterns of functional kinesin-1 motors with different shapes and sizes on PLL-g-PEG-coated polystyrene surfaces. Straight and curved motor tracks with widths of less than 500 nm could be generated in a highly reproducible manner and proved to reliably guide gliding microtubules. Though dependent on track curvature, the characteristic travel lengths of the microtubules on the tracks significantly exceeded earlier predictions. Moreover, we experimentally verified the performance of complex kinesin-1 patterns, recently designed by evolutionary algorithms for controlling the global directionality of microtubule motion on large-area substrates.

Controlled Retention and Release of Biomolecular Transport Systems Using Shape-Changing Polymer Bilayers.

Biomolecular transport systems based on cytoskeletal filaments and motor proteins have become promising tools for a wide range of nanotechnological applications. In this paper, we report control of such transport systems using substrates with switchable shape. We demonstrate this approach on the example of microtubules gliding on surfaces of self-folding polymer bilayers with adsorbed kinesin motors. The polymer bilayers are able to undergo reversible transitions between flat and tube-like shapes that allow the externally controlled retention and release of gliding microtubules. The demonstrated approach, based on surfaces with reconfigurable topography, opens broad perspectives to control biomolecular transport systems for bioanalytical and sensing applications, as well as for the construction of subcellular compartments in the field of synthetic biology.

Programmable patterning of protein bioactivity by visible light.

The simple and quick patterning of functional proteins on engineered surfaces affords an opportunity to fabricate protein microarrays in lab-on-chip systems. We report on the programmable patterning of proteins as well as the local activation of enzymes by visible light. We successfully generated functional protein patterns with different geometries in situ and demonstrated the specific patterning of multiple kinds of proteins side-by-side without the need for specific linker molecules or elaborate surface preparation.

The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding.

During mitosis and meiosis, the bipolar spindle facilitates chromosome segregation through microtubule sliding as well as microtubule growth and shrinkage. Kinesin-14, one of the motors involved, causes spindle collapse in the absence of kinesin-5 (Refs 2, 3), participates in spindle assembly and modulates spindle length. However, the molecular mechanisms underlying these activities are not known. Here, we report that Drosophila melanogaster kinesin-14 (Ncd) alone causes sliding of anti-parallel microtubules but locks together (that is, statically crosslinks) those that are parallel. Using single molecule imaging we show that Ncd diffuses along microtubules in a tail-dependent manner and switches its orientation between sliding microtubules. Our results show that kinesin-14 causes sliding and expansion of an anti-parallel microtubule array by dynamic interactions through the motor domain on the one side and the tail domain on the other. This mechanism accounts for the roles of kinesin-14 in spindle organization.

Biotemplated nanopatterning of planar surfaces with molecular motors.

We report on the generation of nanometer-wide, non-topographical patterns of proteins on planar surfaces. In particular, we used the regular lattice of reconstituted microtubules as template structures to specifically bind and transfer kinesin-1 and nonclaret disjunctional motor proteins. The generated tracks, which comprise dense and structurally oriented arrays of functional motor proteins, proved to be highly efficient for the guiding of microtubule transporters.