A platform to parallelize planar surfaces and control their spatial separation with nanometer resolution.

Parallelizing planar surfaces and manipulating them into close proximity with spatial separation of nanoscale dimensions is critical for probing phenomena such as near-field radiative heat transport and Casimir forces. Here, we report on a novel platform, with an integrated reflected light microscope, that is capable of parallelizing two planar surfaces such that the angular deviation is <6 µrad, while simultaneously allowing control of the gap from 15 µm down to contact with ∼0.15 nm resolution. The capabilities of this platform were verified by using two custom-fabricated micro-devices with planar surfaces, 60 × 60 µm(2) each, whose flatness and surface roughness were experimentally quantified. We first parallelized the two micro-devices by using the developed platform in conjunction with a simple optical approach that relies on the shallow depth of field (∼2 µm) of a long working distance microscope objective. Subsequently, we experimentally tested the parallelism achieved via the optical alignment procedure by taking advantage of electrodes integrated into the micro-devices. Our measurements unambiguously show that the simple depth-of-field based optical approach enables parallelization such that the angular deviation between the two surfaces is within ∼500 µrad. This ensures that the separation between any two corresponding points on the parallel surfaces deviate by ∼30 nm or less from the expected value. Further, we show that improved parallelization can be achieved using the integrated micro-electrodes which enable surface roughness limited parallelization with deviations of ∼5 nm from parallelism.

High resolution resistive thermometry for micro/nanoscale measurements.

High resolution thermometry plays an important role in several micro/nanoscale studies. Here, we present a detailed analysis of the resolution of resistance thermometry schemes that employ an electrical sensing current to monitor the temperature-dependent resistance. Specifically, we theoretically and experimentally analyze four different schemes where modulated or unmodulated temperatures in microdevices are measured using modulated or unmodulated sensing currents. Our analysis and experiments suggest that measurement of unmodulated temperatures using a modulated sensing current improves the resolution in comparison to a scenario where an unmodulated sensing current is used. However, depending on the exact measurement conditions, such improvements might be modest as the overall resolution may be limited by random low frequency environmental temperature fluctuations. More importantly, we find that high-resolution thermometry can be achieved in the measurement of modulated temperatures. Specifically, we show that by using appropriate instrumentation and a 10 kΩ platinum resistance thermometer it is possible to measure modulated temperatures (0.5-20 Hz) with a resolution of about 20-100 µK. The advances described here will enable a dramatic improvement in the heat-current resolution of resistive thermometry based microdevices that are used for probing nanoscale phonon and photon transport.

Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa.

Fungal conventional kinesins are unusually fast microtubule motor proteins. To compare the functional organization of fungal and animal conventional kinesins, a set of C-terminal deletion mutants of the Neurospora crassa conventional kinesin, NcKin, was investigated for its biochemical and biophysical properties. While the shortest, monomeric construct comprising the catalytic core and the neck-linker (NcKin343) displays very high steady-state ATPase (k(cat) = 260/s), constructs including both the full neck and adjacent hinge domains (NcKin400, NcKin433 and NcKin480) show wild-type behaviour: they are dimeric, show fast gliding and slower ATP turnover rates (k(cat) = 60-84/s), and are chemically processive. Unexpectedly, a construct (NcKin378, corresponding to Drosophila KHC381) that includes just the entire coiled-coil neck is a monomer. Its ATPase activity is slow (k(cat) = 27/s), and chemical processivity is abolished. Together with a structural analysis of synthetic neck peptides, our data demonstrate that the NcKin neck domain behaves differently from that of animal conventional kinesins and may be tuned to drive fast, processive motility.

Single-molecule tracking of myosins with genetically engineered amplifier domains.

We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was approximately 30 degrees. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.

A force transducer for measuring mechanical properties of single cardiac myocytes.

We have described a transducer design capable of recording forces generated by single cardiac myocytes with sufficient temporal resolution to detect force responses to rapid length changes. Our force sensors were made from thin steel foils that act as cantilevers whose bending is monitored by reflection off a laser beam. Deflection of the laser beam is measured by a differential photodiode detector. A small, 50-micron-thick tungsten needle attached to the free end of the steel foil allowed us to glue single cardiac cells to the force transducer. The transducers have compliances of approximately 0.02 m/N and resonance frequencies between 2 and 3 kHz. The resolution is approximately 18 nN rms at a detector bandwidth of 16 kHz, so we were able to resolve 0.2% of the maximum isometric force ( approximately 12 microN) developed by a single cardiac myocyte. We have demonstrated that the transducer is well suited to analysis of mechanical properties of single ventricular myocytes, for example, the recording of isometric forces and rate constants of force redevelopment after rapid release-restretch maneuvers.

Directional loading of the kinesin motor molecule as it buckles a microtubule.

Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.

The force generated by a single kinesin molecule against an elastic load.

To probe the mechanism by which the motor protein kinesin moves along microtubules, we have developed a highly sensitive technique for measuring the force exerted by a single motor molecule. In this technique, one end of a microtubule is attached to the tip of a flexible glass fiber of calibrated stiffness. The other end of the microtubule makes contact with a surface sparsely coated with kinesin. By imaging the tip of the glass fiber on a photodiode detector, displacement of the microtubule by kinesin through as little as 1 nm can be detected and forces as small as 1 pN resolved. Using this force-fiber apparatus we have characterized the mechanical output of this molecular motor. The speed at which a molecule of kinesin moved along the surface of a microtubule decreased linearly as the elastic force was increased. The force required to stop a single kinesin molecule was 5.4 +/- 1.0 pN (mean +/- SD; n = 16), independent of the stiffness of the fiber, the damping from the fluid, and whether the ATP concentration was high or low.

Kinesin follows the microtubule's protofilament axis.

We tested the hypothesis that kinesin moves parallel to the microtubule's protofilament axis. We polymerized microtubules with protofilaments that ran either parallel to the microtubule's long axis or that ran along shallow helical paths around the cylindrical surface of the microtubule. When gliding across a kinesin-coated surface, the former microtubules did not rotate. The latter microtubules, those with supertwisted protofilaments, did rotate; the pitch and handedness of the rotation accorded with the supertwist measured by electron cryo-microscopy. The results show that kinesin follows a path parallel to the protofilaments with high fidelity. This implies that the distance between consecutive kinesin-binding sites along the microtubule must be an integral multiple of 4.1 nm, the tubulin monomer spacing along the protofilament, or a multiple of 8.2 nm, the dimer spacing.

Bivalve hemocyanins--a comparison with other molluscan hemocyanins.

1. The hemocyanins of the protobranch bivalves Yoldia thraciaeformis, Yoldia limatula and Acila castrensis have absorption spectra similar to other hemocyanins. 2. Hemocyanins from all three bivalves appear as six-tiered cylinders in the electron microscope (30-32 nm in diameter by 34-38 nm in height). Yoldia thraciaeformis and A. castrensis hemocyanins tend to dissociate to three-tiered half molecules with polar images and also to associate into long tubular polymers. 3. Yoldia thraciaeformis and A. castrensis hemocyanins chromatograph on Sepharose 4B gel close to gastropod hemocyanin (Mr = 9 x 10(6] rather than chiton hemocyanin (Mr = 4 x 10(6]. 4. Hemocyanins from all three vivalves have subunits with electrophoretic mobilities similar to gastropod and polyplacophoran hemocyanin subunits and slower than octopodan hemocyanin subunits. 5. These similarities between bivalve and gastropod hemocyanins are consistent with the hypothesis that bivalves and gastropods have shared a common ancestor.

Autoradiographic measurement of tritiated agmatine as an indicator of physiologic activity in Hermissenda visual and vestibular neurons.

[3H]Agmatine (amino-4-guanidobutane) has been shown to be potentially useful for identifying and assessing the ACh sensitivity of specific neurons. Small cationic amines are able to permeate ACh-activated ion channels in sympathetic neurons and vertebrate endplates. Sensory neurons of the photic pathway in the nudibranch mollusc Hermissenda crassicornis are cholinergic and the synaptic interactions between the photic and vestibular systems have been well characterized electrophysiologically. We have therefore tested the feasibility of using autoradiography with [3H]agmatine, (a) to identify known ACh-responsive postsynaptic cells and (b) to examine its ability to serve as an indicator of physiologic activity within the photic and vestibular pathways under conditions of darkness and light stimulation. Scintillation counting revealed that approximately 70% of the radioactivity was associated with the CNS while approximately 30% was found in the processing fluids, indicating that routine glutaraldehyde-osmium fixation and subsequent processing for epoxy embedding allows retention of substantial amounts of the radiolabel. The autoradiographic results consistently demonstrated that the uptake patterns for [3H]agmatine did reflect some of the known neuronal interactions under the experimental conditions of light and dark. The accuracy extended to the second order cells of the optic ganglion and to putative interneurons along the photic tract in the cerebropleural ganglion. Since all the neurons in these pathways are unipolar with their synaptic interactions occurring only at the terminal endings, the radiolabel accumulated in the somata resulted from retrograde axonal transport. In the photic-vestibular pathways, the highest silver grain densities were found over structures (cell bodies or axon tracts) with increased synaptic activity coupled with higher levels of cellular activity (i.e. increased excitatory postsynaptic potentials or increased spontaneous impulse activity). Slightly less label was found in cells which received increased numbers of inhibitory postsynaptic potentials that produced hyperpolarization and a transient cessation of impulse activity under conditions of illumination. Therefore, the uptake levels of [3H]agmatine as revealed by autoradiography appear to reflect not only changes in sensitivity or density of ACh-activated channels but also changes in cellular activity as indicated by increased amounts of retrograde transport. These results represent the first example of the effective use of this radiolabel as an indicator of synaptic activity in invertebrates and in sensory systems.

Hemocyanin respiratory pigment in bivalve mollusks.

Hemocyanins, high molecular weight oxygen-binding proteins, were identified in two species of protobranch bivalve mollusks, Acila castrensis and Yoldia limatula. Although hemocyanins have been reported in chitons, gastropods, and cephalopods, they have not been observed in the Class Bivalvia. In A. castrensis the dissociation products of hemocyanin, characterized by gel electrophoresis, had a subunit molecular weight of approximately 250K. Negatively stained preparations of extracted hemocyanin formed protein aggregates in the shape of cylinders measuring 35 by 38 nanometers. X-ray microanalysis of hemocyanin aggregates in thin sections of Y. limatula demonstrated the presence of copper in the molecules. The discovery of hemocyanin in the protobranchs reinforces the primitive nature of the taxon and is further evidence that the major molluscan classes have a common ancestry.