Kinesin-8 motors, which move in a highly processive manner toward microtubule plus ends where they act as depolymerases, are essential regulators of microtubule dynamics in cells. To understand their ...navigation strategy on the microtubule lattice, we studied the 3D motion of single yeast kinesin-8 motors, Kip3, on freely suspended microtubules in vitro. We observed short-pitch, left-handed helical trajectories indicating that kinesin-8 motors frequently switch protofilaments in a directionally biased manner. Intriguingly, sidestepping was not directly coupled to forward stepping but rather depended on the average dwell time per forward step under limiting ATP concentrations. Based on our experimental findings and numerical simulations we propose that effective sidestepping toward the left is regulated by a bifurcation in the Kip3 step cycle, involving a transition from a two-head–bound to a one-head–bound conformation in the ATP-waiting state. Results from a kinesin-1 mutant with extended neck linker hint toward a generic sidestepping mechanism for processive kinesins, facilitating the circumvention of intracellular obstacles on the microtubule surface.
Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which ...cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that the concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that, under conditions of macro-molecular crowding, tau forms liquid-like drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles, which deform the drops while remaining enclosed by diffusible tau molecules exhibiting a liquid-like behavior. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.
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•Tau forms liquid-like drops in vitro under conditions of molecular crowding•Tau drops concentrate tubulin•Microtubule bundles polymerize within drops, deforming them into a rod-like structures•Microtubules in tau drop-derived structures are bundled and stable
Hernández-Vega et al. show that tau forms liquid-like drops in vitro. Tubulin gets enriched in these drops, enabling microtubule bundles polymerization within drops. Microtubule bundles deform tau drops, reshaping them into rod-like structures. Microtubules in these structures remain as stable bundles. The findings have potential implications for tau function in axonal projections.
Plasmonic structures allow the manipulation of light with materials that are smaller than the optical wavelength. Such structures can consist of plasmonically active metal nanoparticles and can be ...fabricated through scalable bottom-up self-assembly on DNA origami templates. To produce functional devices, the precise and high-yield arrangement of each of the nanoparticles on a structure is of vital importance as the absence of a single particle can destroy the functionality of the entire device. Nevertheless, the parameters influencing the yield of the multistep assembly process are still poorly understood. To overcome this deficiency, we employed a test system consisting of a tubular six-helix bundle DNA origami with binding sites for eight oligonucleotide-functionalized gold nanoparticles. We systematically studied the assembly yield as a function of a wide range of parameters such as ionic strength, stoichiometric ratio, oligonucleotide linker chemistry, and assembly kinetics by an automated high-throughput analysis of electron micrographs of the formed heterocomplexes. Our optimized protocols enable particle placement yields up to 98.7% and promise the reliable production of sophisticated DNA-based multiparticle plasmonic devices for applications in photonics, optoelectronics, and nanomedicine.
Over the past ten years, great advancements have been made towards using biomolecular motors for nanotechnological applications. In particular, devices using cytoskeletal motor proteins for molecular ...transport are maturing. First efforts towards designing such devices used motor proteins attached to micro-structured substrates for the directed transport of microtubules and actin filaments. Soon thereafter, the specific capture, transport and detection of target analytes like viruses were demonstrated. Recently, spatial guiding of the gliding filaments was added to increase the sensitivity of detection and allow parallelization. Whereas molecular motor powered devices have not yet demonstrated performance beyond the level of existing detection techniques, the potential is great: Replacing microfluidics with transport powered by molecular motors allows integration of the energy source (ATP) into the assay solution. This opens up the opportunity to design highly integrated, miniaturized, autonomous detection devices. Such devices, in turn, may allow fast and cheap on-site diagnosis of diseases and detection of environmental pathogens and toxins.
Motor proteins in the kinesin-8 family depolymerize microtubules in a length-dependent manner that may be crucial for controlling the length of organelles such as the mitotic spindle. We used ...single-molecule microscopy to understand the mechanism of length-dependent depolymerization by the budding yeast kinesin-8, Kip3p. We found that after binding at a random position on a microtubule and walking to the plus end, an individual Kip3p molecule pauses there until an incoming Kip3p molecule bumps it off. Kip3p dissociation is accompanied by removal of just one or two tubulin dimers (on average). Such a cooperative mechanism leads to a depolymerization rate that is proportional to the flux of motors to the microtubule end and accounts for the length dependence of depolymerization. This type of feedback between length and disassembly may serve as a model for understanding how an ensemble of molecules can measure and control polymer length.
Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 ...performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.
Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be ...adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions densityand bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionary adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.
Microtubule-associated proteins (MAPs) are a functionally highly diverse class of proteins that help to adjust the shape and function of the microtubule cytoskeleton in space and time. For this ...purpose, MAPs structurally support microtubules, modulate their dynamic instability, or regulate the activity of associated molecular motors. The microtubule-binding domains of MAPs are structurally divergent, but often depend on electrostatic interactions with the negatively charged surface of the microtubule. This suggests that the surface exposure of positive charges rather than a certain structural fold is sufficient for a protein to associate with microtubules. Consistently, positively charged artificial objects have been shown to associate with microtubules and to diffuse along their lattice. Natural MAPs, however, show a more sophisticated functionality beyond lattice-diffusion. Here, we asked whether basic electrostatic interactions are sufficient to also support advanced MAP functionality. To test this hypothesis, we studied simple positively charged peptide sequences for the occurrence of typical MAP-like behavior. We found that a multivalent peptide construct featuring four lysine-alanine heptarepeats (starPEG-(KA7)
)-but not its monovalent KA7-subunits-show advanced, biologically relevant MAP-like behavior: starPEG-(KA7)
binds microtubules in the low nanomolar range, diffuses along their lattice with the ability to switch between intersecting microtubules, and tracks depolymerizing microtubule ends. Further, starPEG-(KA7)
promotes microtubule nucleation and growth, mediates depolymerization coupled pulling at plus ends, and bundles microtubules without significantly interfering with other proteins on the microtubule lattice (as exemplified by the motor kinesin-1). Our results show that positive charges and multivalency are sufficient to mimic advanced MAP-like behavior.
The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved ...with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.
Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer’s. However, little is known about how motor ...proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor’s specific intracellular tasks and environments.
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