Summary Kinesin-2 motors, which are involved in intraflagellar transport and cargo transport along cytoplasmic microtubules, differ from motors in the canonical Kinesin-1 family in having a heterodimeric rather than homodimeric structure and in possessing a three amino acid insertion in their neck linker domain. To determine how these structural features alter the chemomechanical coupling in Kinesin-2, we used single-molecule bead experiments to measure the processivity and velocity of mouse Kinesin-2 heterodimer (KIF3A/B) and the engineered homodimers KIF3A/A and KIF3B/B, and compared their behavior to Drosophila Kinesin-1 heavy chain (KHC). Single motor run lengths of Kinesin-2 were four-fold shorter than Kinesin-1. Extending the Kinesin-1 neck linker by three amino acids led to a similar reduction in processivity. Furthermore, Kinesin-2 processivity varied inversely with ATP concentration. Stochastic simulations of the Kinesin-1 and Kinesin-2 hydrolysis cycles suggest that “front head gating”, in which rearward tension prevents ATP binding to the front head when both heads are bound to the microtubule, is diminished in Kinesin-2. Because the mechanical tension that underlies front head gating must be transmitted through the neck linker domains, we propose that the diminished coordination in Kinesin-2 is a result of its longer and hence more compliant neck linker element.

The conjugation of 14 nm diameter CoFe2O4 nanoparticles to the surface of biotinylated microtubules enables their manipulation with externally applied magnetic fields of small, permanent NdFeB magnets. Microtubules are selectively patterned on kinesin motor-modified glass surfaces in coparallel arrays that mimic the orientation of the magnetic field lines over millimeter distances. The magnetic field is simultaneously used to increase surface loading of microtubules. We demonstrate that motility across the kinesin motor surface is retained following magnetic functionalization of the microtubules, while gliding speed is dependent on loading level of the neutravidin linker as well as magnetic nanoparticles.

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.

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. [1][2][3][4] 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, [5] optical traps, [6] and ATPase assays. [7] Single-molecule visualization is crucial for investigating the motor mechanism and their ability to move and assemble nanoparticles. [8][9][10] 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, [8] and their position resolved to within nearly one nanometer. [11] 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. [11] However, because the spatial resolution is based on the number of photons collected, the temporal resolution using these fluorophores is limit-ed 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, [12] 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. [13] 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 c...

Biomolecular motors have great potential as transporters and actuators in microscale devices. Existing efforts toward harnessing kinesin motors have involved microtubule movements over immobilized motors. The reverse geometry has distinct advantages, but progress has been hindered by the difficulty of immobilizing patterned and aligned microtubules on surfaces. Here we show that microtubules can be reversibly patterned with microscale resolution through DNA hybridization, and that these DNA-functionalized microtubules support the movement of kinesin-coated beads.