Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity.

Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of z -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.

The Kinesin-8 Kip3 Depolymerizes Microtubules with a Collective Force-Dependent Mechanism.

Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes, in particular during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors' residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times, suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called "bump-off" and "switching" models. Understanding the mechanics of kinesin-8's microtubule end activity will provide important insights into cell division with implications for cancer research.

Self-Sensing Enzyme-Powered Micromotors Equipped with pH-Responsive DNA Nanoswitches.

Biocatalytic micro- and nanomotors have emerged as a new class of active matter self-propelled through enzymatic reactions. The incorporation of functional nanotools could enable the rational design of multifunctional micromotors for simultaneous real-time monitoring of their environment and activity. Herein, we report the combination of DNA nanotechnology and urease-powered micromotors as multifunctional tools able to swim, simultaneously sense the pH of their surrounding environment, and monitor their intrinsic activity. With this purpose, a FRET-labeled triplex DNA nanoswitch for pH sensing was immobilized onto the surface of mesoporous silica-based micromotors. During self-propulsion, urea decomposition and the subsequent release of ammonia led to a fast pH increase, which was detected by real-time monitoring of the FRET efficiency through confocal laser scanning microscopy at different time points (i.e., 30 s, 2 and 10 min). Furthermore, the analysis of speed, enzymatic activity, and propulsive force displayed a similar exponential decay, matching the trend observed for the FRET efficiency. These results illustrate the potential of using specific DNA nanoswitches not only for sensing the micromotors' surrounding microenvironment but also as an indicator of the micromotor activity status, which may aid to the understanding of their performance in different media and in different applications.

Influence of Enzyme Quantity and Distribution on the Self-Propulsion of Non-Janus Urease-Powered Micromotors.

The use of enzyme catalysis to power micro- and nanomachines offers unique features such as biocompatibility, versatility, and fuel bioavailability. Yet, the key parameters underlying the motion behavior of enzyme-powered motors are not completely understood. Here, we investigate the role of enzyme distribution and quantity on the generation of active motion. Two different micromotor architectures based on either polystyrene (PS) or polystyrene coated with a rough silicon dioxide shell (PS@SiO2) were explored. A directional propulsion with higher speed was observed for PS@SiO2 motors when compared to their PS counterparts. We made use of stochastically optical reconstruction microscopy (STORM) to precisely detect single urease molecules conjugated to the micromotors surface with a high spatial resolution. An asymmetric distribution of enzymes around the micromotor surface was observed for both PS and PS@SiO2 architectures, indicating that the enzyme distribution was not the only parameter affecting the motion behavior. We quantified the number of enzymes present on the micromotor surface and observed a 10-fold increase in the number of urease molecules for PS@SiO2 motors compared to PS-based micromotors. To further investigate the number of enzymes required to generate a self-propulsion, PS@SiO2 particles were functionalized with varying amounts of urease molecules and the resulting speed and propulsive force were measured by optical tracking and optical tweezers, respectively. Surprisingly, both speed and force depended in a nonlinear fashion on the enzyme coverage. To break symmetry for active propulsion, we found that a certain threshold number of enzymes molecules per micromotor was necessary, indicating that activity may be due to a critical phenomenon. Taken together, these results provide new insights into the design features of micro/nanomotors to ensure an efficient development.

Enzyme-Powered Hollow Mesoporous Janus Nanomotors.

The development of synthetic nanomotors for technological applications in particular for life science and nanomedicine is a key focus of current basic research. However, it has been challenging to make active nanosystems based on biocompatible materials consuming nontoxic fuels for providing self-propulsion. Here, we fabricate self-propelled Janus nanomotors based on hollow mesoporous silica nanoparticles (HMSNPs), which are powered by biocatalytic reactions of three different enzymes: catalase, urease, and glucose oxidase (GOx). The active motion is characterized by a mean-square displacement (MSD) analysis of optical video recordings and confirmed by dynamic light scattering (DLS) measurements. We found that the apparent diffusion coefficient was enhanced by up to 83%. In addition, using optical tweezers, we directly measured a holding force of 64 ± 16 fN, which was necessary to counteract the effective self-propulsion force generated by a single nanomotor. The successful demonstration of biocompatible enzyme-powered active nanomotors using biologically benign fuels has a great potential for future biomedical applications.

Kinesin-8 is a low-force motor protein with a weakly bound slip state.

During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.

Measuring the complete force field of an optical trap.

The use of optical traps to measure or apply forces on the molecular level requires a precise knowledge of the trapping force field. Close to the trap center, this field is typically approximated as linear in the displacement of the trapped microsphere. However, applications demanding high forces at low laser intensities can probe the light-microsphere interaction beyond the linear regime. Here, we measured the full nonlinear force and displacement response of an optical trap in two dimensions using a dual-beam optical trap setup with back-focal-plane photodetection. We observed a substantial stiffening of the trap beyond the linear regime that depends on microsphere size, in agreement with Mie theory calculations. Surprisingly, we found that the linear detection range for forces exceeds the one for displacement by far. Our approach allows for a complete calibration of an optical trap.

Inertial effects of a small Brownian particle cause a colored power spectral density of thermal noise.

The random thermal force acting on Brownian particles is often approximated in Langevin models by a "white-noise" process. However, fluid entrainment results in a frequency dependence of this thermal force giving it a "color." While theoretically well understood, direct experimental evidence for this colored nature of the noise term and how it is influenced by a nearby wall is lacking. Here, we directly measured the color of the thermal noise intensity by tracking a particle strongly confined in an ultrastable optical trap. All our measurements are in quantitative agreement with the theoretical predictions. Since Brownian motion is important for microscopic, in particular, biological systems, the colored nature of the noise and its distance dependence to nearby objects need to be accounted for and may even be utilized for advanced sensor applications.

Seeded growth of titania colloids with refractive index tunability and fluorophore-free luminescence.

Titania is an important material in modern materials science, chemistry, and physics because of its special catalytic, electric, and optical properties. Here, we describe a novel method to synthesize colloidal particles with a crystalline titania, anatase core and an amorphous titania-shell structure. We demonstrate seeded growth of titania onto titania particles with accurate particle size tunability. The monodispersity is improved to such an extent so that colloidal crystallization of the grown microspheres becomes feasible. Furthermore, seeded growth provides separate manipulation of the core and shell. We tuned the refractive index of the amorphous shell between 1.55 and 2.3. In addition, the particles show luminescence when trace amounts of aminopropyl-triethoxysilane are incorporated into the titania matrix and are calcined at 450 °C. Our novel colloids may be useful for optical materials and technologies such as photonic crystals and optical trapping.

Single depolymerizing and transport kinesins stabilize microtubule ends.

Microtubules are highly dynamic cellular filaments and an accurate control of their length is important for many intracellular processes like cell division. Among other factors, microtubule length is actively modulated by motors from the kinesin superfamily. For example, yeast kinesin-8, Kip3, motors depolymerize microtubules by a cooperative, force- and length-dependent mechanism. However, whether single motors can also depolymerize microtubules is unclear. Here, we measured how single kinesin motors influenced the stability of microtubules in an in vitro assay. Using label-free interference reflection microscopy, we determined the spontaneous microtubule depolymerization rate of stabilized microtubules in the presence of kinesins. Surprisingly, we found that both single Kip3 and nondepolymerizing kinesin-1 transport motors, used as a control, stabilized microtubules further. For Kip3, this behavior is contrary to the collective force-dependent depolymerization activity of multiple motors. Because of the control measurement, the finding may hint at a more general stabilization mechanism. The complex, concentration-dependent interaction with microtubule ends provides new insights into the molecular mechanism of kinesin-8 and its regulatory function of microtubule length.

Germanium nanospheres for ultraresolution picotensiometry of kinesin motors.

Kinesin motors are essential for the transport of cellular cargo along microtubules. How the motors step, detach, and cooperate with each other is still unclear. To dissect the molecular motion of kinesin-1, we developed germanium nanospheres as ultraresolution optical trapping probes. We found that single motors took 4-nanometer center-of-mass steps. Furthermore, kinesin-1 never detached from microtubules under hindering load conditions. Instead, it slipped on microtubules in microsecond-long, 8-nanometer steps and remained in this slip state before detaching or reengaging in directed motion. Unexpectedly, reengagement and thus rescue of directed motion was more frequent. Our observations broaden our knowledge on the mechanochemical cycle and slip state of kinesin. This state and rescue need to be accounted for to understand long-range transport by teams of motors.

Optical trapping of coated microspheres.

In an optical trap, micron-sized dielectric particles are held by a tightly focused laser beam. The optical force on the particle is composed of an attractive gradient force and a destabilizing scattering force. We hypothesized that using anti-reflection-coated microspheres would reduce scattering and lead to stronger trapping. We found that homogeneous silica and polystyrene microspheres had a sharp maximum trap stiffness at a diameter of around 800 nm--the trapping laser wavelength in water--and that a silica coating on a polystyrene microsphere was a substantial improvement for larger diameters. In addition, we noticed that homogeneous spheres of a correct size demonstrated anti-reflective properties. Our results quantitatively agreed with Mie scattering calculations and serve as a proof of principle. We used a DNA stretching experiment to confirm the large linear range in detection and force of the coated microspheres and performed a high-force motor protein assay. These measurements show that the surfaces of the coated microspheres are compatible with biophysical assays.

Implementation and Tuning of an Optical Tweezers Force-Clamp Feedback System.

Feedback systems can be used to control the value of a system variable. In optical tweezers, active feedback is often implemented to either keep the position or tension applied to a single biomolecule constant. Here, we describe the implementation of the latter: an optical force-clamp setup that can be used to study the motion of processive molecular motors under a constant load. We describe the basics of a software-implemented proportional-integral-derivative (PID) controller, how to tune it, and how to determine its optimal feedback rate. Limitations, possible feed-forward applications, and extensions into two- and three-dimensional optical force clamps are discussed. The feedback is ultimately limited by thermal fluctuations and the compliance of the involved molecules. To investigate a particular mechanical process, understanding the basics and limitations of the feedback system will be helpful for choosing the proper feedback hardware, for optimizing the system parameters, and for the design of the experiment.

Custom-Made Microspheres for Optical Tweezers.

Due to their high position and force sensitivity and the ability to remotely apply forces and torques, optical tweezers are widely used in diverse fields, such as biology, material science, and physics. Often, small dielectric particles are trapped and used as probes, which for experimental convenience are mostly spherical and composed of silica or polystyrene. The optical properties of these materials together with the microsphere size determine the trapping efficiency, and the position and force resolution. However, using only a single, homogeneous, isotropic, and unstructured material limits the range of trapping properties and thereby the applications of optical tweezers. Here, we show how custom-made microspheres composed of coated high-refractive-index materials-titania and nanodiamonds-and birefringent, liquid crystals extend the range and combination of desired trapping properties. These custom-made microspheres either enable the generation of high forces, a high force or time resolution, or the applications of torques. Custom-made probes expand the range of possible experiments and approaches broadening the scope and applicability of optical tweezers.

Kinesin Kip2 enhances microtubule growth in vitro through length-dependent feedback on polymerization and catastrophe.

The size and position of mitotic spindles is determined by the lengths of their constituent microtubules. Regulation of microtubule length requires feedback to set the balance between growth and shrinkage. Whereas negative feedback mechanisms for microtubule length control, based on depolymerizing kinesins and severing proteins, have been studied extensively, positive feedback mechanisms are not known. Here, we report that the budding yeast kinesin Kip2 is a microtubule polymerase and catastrophe inhibitor in vitro that uses its processive motor activity as part of a feedback loop to further promote microtubule growth. Positive feedback arises because longer microtubules bind more motors, which walk to the ends where they reinforce growth and inhibit catastrophe. We propose that positive feedback, common in biochemical pathways to switch between signaling states, can also be used in a mechanical signaling pathway to switch between structural states, in this case between short and long polymers.