Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking.

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

Optical measurement of mechanical forces inside short DNA loops.

Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.

Calibration of dynamic molecular rulers based on plasmon coupling between gold nanoparticles.

Pairs of noble metal nanoparticles can be used to measure distances via the distance dependence of their plasmon coupling. These "plasmon rulers" offer exceptional photostability and brightness; however, the advantages and limitations of this approach remain to be explored. Here we report detailed plasmon peak versus separation calibration curves for 42- and 87-nm-diameter particle pairs, determine their measurement errors, and describe experimental procedures to improve their performance in biology, nanotechnology, and materials sciences.

Biocompatible force sensor with optical readout and dimensions of 6 nm3.

We have developed a nanoscopic force sensor with optical readout. The sensor consists of a single-stranded DNA oligomer flanked by two dyes. The DNA acts as a nonlinear spring: when the spring is stretched, the distance between the two dyes increases, resulting in reduced Förster resonance energy transfer. The sensor was calibrated between 0 and 20 pN using a combined magnetic tweezers/single-molecule fluorescence microscope. We show that it is possible to tune the sensor's force response by varying the interdye spacing and that the FRET efficiency of the sensors decreases with increasing force. We demonstrate the usefulness of these sensors by using them to measure the forces internal to a single polymer molecule, a small DNA loop. Partial conversion of the single-stranded DNA loop to a double-stranded form results in the accumulation of strain: a force of approximately 6 pN was measured in the loop upon hybridization. The sensors should allow measurement of forces internal to various materials, including programmable DNA self-assemblies, polymer meshes, and DNA-based machines.

Intrinsic activation of human motoneurons: possible contribution to motor unit excitation.

The main purpose of this study was to estimate the contribution of intrinsic activation of human motoneurons (e.g., by plateau potentials) during voluntary and reflexive muscle contractions. Pairs of motor units were recorded from either the tibialis anterior or soleus muscle during three different conditions: 1) during a brief muscle vibration followed by a slow relaxation of a steady isometric contraction; 2) during a triangular isometric torque contraction; and 3) during passive sinusoidal muscle stretch superimposed on a steady isometric contraction. In each case, the firing rate of a tonically firing control motor unit was used as a measure of the effective synaptic excitation (i.e., synaptic drive) to a slightly higher-threshold test motor unit that was recruited and de-recruited during a contraction trial. The firing rate of the control unit was compared at recruitment and de-recruitment of the test unit. This was done to determine whether the estimated synaptic drive needed to recruit a motor unit was less than the amount needed to sustain firing as a result of an added depolarization produced from intrinsic sources. After test unit recruitment, the firing rate of the control unit could be decreased significantly (on average by 3.6 Hz from an initial recruitment rate of 9.8 Hz) before the test unit was de-recruited during a descending synaptic drive. Similar decreases in control unit rate occurred in all three experimental conditions. This represents a possible 40% reduction in the estimated synaptic drive needed to maintain firing of a motor unit compared with the estimated amount needed to recruit the unit initially. The firing rates of both the control and test units were modulated together in a highly parallel fashion, suggesting that the unit pairs were driven by common synaptic inputs. This tight correlation further validated the use of the control unit firing rate as a monitor of synaptic drive to the test motor unit. The estimates of intrinsically mediated depolarization of human motoneurons ( approximately 40% during moderate contractions) are consistent with values obtained for plateau potentials obtained from intracellular recordings of motoneurons in reduced animal preparations, although various alternative mechanisms are discussed. This suggests that similar intrinsic conductances provide a substantial activation of human motoneurons during moderate physiological activity.

Intrinsic activation of human motoneurons: reduction of motor unit recruitment thresholds by repeated contractions.

The main purpose of this study was to examine whether facilitation of human motor unit recruitment by repeated voluntary contractions is mediated, in part, by time and activity-dependent increases in the intrinsic excitability of the parent motoneuron. To do this, pairs of tibialis anterior or soleus motor units were recorded during slowly increasing and then decreasing voluntary contractions. The firing rate of the lower-threshold motor unit of the pair (control unit) was used as a measure of effective synaptic excitation (i.e., drive) to the motoneurons. This rate was used to estimate the recruitment threshold of the higher-threshold unit of the pair (test unit). The test unit was repeatedly recruited and de-recruited in a series of contractions, and the interval between the de-recruitment and re-recruitment of the test unit (interactivation interval) was systematically varied between 0.6 and 60 s. An increase in intrinsic excitability of a unit was considered to have occurred if the level of estimated synaptic input (as measured by the firing rate of the control motor unit) needed to recruit a unit was reduced. At short interactivation intervals (1-2 s), the control unit firing frequency was significantly lower when the test unit was recruited on the second contraction, compared with the first (by 3.9 Hz or a 64% reduction). This suggested that the intrinsic excitability of the test motor unit had increased during the second contraction because it could be recruited at a much lower level of estimated synaptic drive. Longer interaction intervals (2-6 s) produced less recruitment facilitation. At even longer interactivation intervals (>6 s) there was no significant facilitation (time constant of effect was 4.8 s). In some motor units, the effect of this short-term facilitation appeared to be so pronounced that it resulted in reversing the order of de-recruitment with the other initially lower-threshold motor units. Such reversals were occasionally observed for orderly re-recruitment. The time course and behavior of the observed short-term facilitation of motor unit discharge was qualitatively similar to the warm-up phenomenon of plateau potentials seen in motoneurons of reduced preparations (e.g., 4-6 s). The possibility of warm-up contributing to the time and activity-dependent facilitation of human motor unit recruitment is discussed.