IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

Intraflagellar transport: Derailing causes turnarounds.

In intraflagellar transport, protein complexes travel along cilia from base to tip without stopping and change direction only at the tip. A new study shows that this directional change does not depend on any tip-associated machinery.

Mechanisms of Regulation in Intraflagellar Transport.

Cilia are eukaryotic organelles essential for movement, signaling or sensing. Primary cilia act as antennae to sense a cell's environment and are involved in a wide range of signaling pathways essential for development. Motile cilia drive cell locomotion or liquid flow around the cell. Proper functioning of both types of cilia requires a highly orchestrated bi-directional transport system, intraflagellar transport (IFT), which is driven by motor proteins, kinesin-2 and IFT dynein. In this review, we explore how IFT is regulated in cilia, focusing from three different perspectives on the issue. First, we reflect on how the motor track, the microtubule-based axoneme, affects IFT. Second, we focus on the motor proteins, considering the role motor action, cooperation and motor-train interaction plays in the regulation of IFT. Third, we discuss the role of kinases in the regulation of the motor proteins. Our goal is to provide mechanistic insights in IFT regulation in cilia and to suggest directions of future research.

Evaluation of a Filtering Facepiece Respirator and a Pleated Particulate Respirator in Filtering Ultrafine Particles and Submicron Particles in Welding and Asphalt Plant Work Environments.

Manufacturing sites, such as welding, casting, and asphalt production (fumes), generate vast numbers of ultrafine particles of <0.1 µm in size and submicron particles close to the ultrafine range (0.1-0.5 µm). Although cumulative masses of these particles are negligible in comparison to the larger particles, the health effects are more severe due to the higher penetration in the human lower respiratory tract, other body parts crossing the respiratory epithelial layers, and the larger surface area. This research investigates the effectiveness of two common commercially available N95 filtering facepieces and N95 pleated particulate respirator models against ultrafine and submicron particles. Two specific types of respirators, the N95 filtering facepiece and the N95 pleated particulate models, in both sealed and unsealed conditions to the manikin face, were tested at various commercial and academic manufacturing sites, a welding and foundry site, and an asphalt production plant. Two TSI Nanoscan SMPS nanoparticle counters were used simultaneously to collect data for particles of 10-420 nm in size from inside and outside of the respirators. While one of them represented the workplace exposure levels, the other one accounted for the exposure upon filtration through the respiratory surfaces. The results showed the particles generated by these manufacturing operations were mostly within the range of from 40 to 200 nm. Results also indicated that while the percentage of filtration levels varied based on the particle size, it remained mostly within the desired protection level of 95% for both of the N95 respirator models in sealed conditions and even for the N95 pleated particulate model in the unsealed condition. However, in the case of the N95 filtering facepiece model, unsealed respirators showed that the percentage of penetration was very high, decreasing the protection levels to 60% in some cases. Although the number of workplace airborne particle levels varied considerably, the filtration percentages were relatively consistent.

Radiostereometric Analysis Allows Assessment of the Stability and Inducible Displacement of Pelvic Ring Disruptions during Healing: A Case Series.

There is currently no accurate data on fracture displacement during the rehabilitation of pelvic ring injuries. This study investigated the use of radiostereometric analysis (RSA) in assessing the stability of C1 pelvic ring injuries stabilised with a posterior plate and an anterior external fixator. Six patients, instructed to weight-bear as tolerated after surgery, were reviewed at 2, 4, 6, 12, 26, 52 and 104 weeks. The external fixators were removed at 6 weeks. Outcomes, including the Iowa Pelvic Score (IPS), and complications were recorded. Fracture stability was assessed using measurements on plain radiographs and RSA. All patients progressed to full weight-bearing without support within 6 weeks. At 104 weeks, the IPS was excellent in four patients, good in one patient and fair in one patient. Plain radiographs showed that all fractures were well reduced, and no loss of reduction occurred over time. By contrast, RSA measurements identified displacement in all cases. The maximum three-dimensional (3D) displacement at any time point in each patient ranged from 2 to 10 mm. Two patients with the largest displacement over time had the lowest IPS. RSA also demonstrated displacements above the currently defined normal threshold through the 'un-injured' sacroiliac joint in the same two patients, suggesting a subtle C2 injury, missed at initial assessment. This study demonstrates the limitations of plain radiographs in assessing pelvic fracture stability and displacement during healing, and the potential of RSA to monitor more accurately the effects of stabilisation and weight-bearing on fracture stability.

Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around each other.

Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

Motor Proteins: It Runs in the Family, but at Different Speeds.

The velocity of intraflagellar transport among evolutionarily distant organisms differs substantially, while the transport machinery is well conserved. A new in vitro study finds that the velocity difference is encoded in the motor proteins driving transport.

A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A.

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5µm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

LPS perception through taste-induced reflex in Drosophila melanogaster.

In flies, grooming serves several purposes, including protection against pathogens and parasites. Previously, we found Escherichia coli or lipopolysaccharides (LPS) can induce grooming behavior via activation of contact chemoreceptors on Drosophila wing. This suggested that specific taste receptors may contribute to this detection. In this study, we examined the perception of commercially available LPS on Drosophila wing chemoreceptors in grooming reflex. Behavioral tests conducted with bitter, sweet and salty gustation such as caffeine, sucrose and salt, using flies carrying a defect in one of their taste receptors related to the detection of bitter molecules (Gr66a, Gr33a), sugars (Gr5a, Gr64f), or salt (IR76b). LPS and tastants of each category were applied to wing sensilla of these taste defectflies and to wild-type Canton Special (CS) flies. Our results indicate that the grooming reflex induced by LPS requires a wide range of gustatory genes, and the inactivation of any of tested genes expressing cells causes a significant reduction of the behavior. This suggests that, while the grooming reflex is strongly regulated by cues perceived as aversive, other sapid cues traditionally related to sweet and salty tastes are also contributing to this behavior.

Field Evaluation of N95 Filtering Facepiece Respirators on Construction Jobsites for Protection against Airborne Ultrafine Particles.

Exposure to high concentrations of airborne ultrafine particles in construction jobsites may play an important role in the adverse health effects among construction workers, therefore adequate respiratory protection is required. The performance of particulate respirators has never been evaluated in field conditions against ultrafine particles on construction jobsites. In this study, respiratory protection levels against ultrafine particles of different size ranges were assessed during three common construction related jobs using a manikin-based set-up at 85 L/min air flow rate. Two NanoScan SMPS nanoparticle counters were utilized for measuring ultrafine particles in two sampling lines of the test filtering facepiece respirator-one from inside the respirator and one from outside the respirator. Particle size distributions were characterized using the NanoScan data collected from outside of the respirator. Two models of N95 respirators were tested-foldable and pleated. Collected data indicate that penetration of all categories of ultrafine particles can exceed 5% and smaller ultrafine particles of <36.5 nm size generally penetrated least. Foldable N95 filtering facepiece respirators were found to be less efficient than pleated N95 respirators in filtering nanoparticles mostly at the soil moving site and the wooden building frameworks construction site. Upon charge neutralization by isopropanol treatment, the ultrafine particles of larger sizes penetrated more compared to particles of smaller sizes. Our findings, therefore, indicate that N95 filtering facepiece respirators may not provide desirable 95% protection for most categories of ultrafine particles and generally, 95% protection is achievable for smaller particles of 11.5 to 20.5 nm sizes. We also conclude that foldable N95 respirators are less efficient than pleated N95 respirators in filtering ultrafine particles, mostly in the soil moving site and the wooden building framework construction site.

Directionally biased sidestepping of Kip3/kinesin-8 is regulated by ATP waiting time and motor-microtubule interaction strength.

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.

Limited Resources Induce Bistability in Microtubule Length Regulation.

The availability of protein is an important factor for the determination of the size of the mitotic spindle. Involved in spindle-size regulation is kinesin-8, a molecular motor and microtubule (MT) depolymerase, which is known to tightly control MT length. Here, we propose and analyze a theoretical model in which kinesin-induced MT depolymerization competes with spontaneous polymerization while supplies of both tubulin and kinesin are limited. In contrast to previous studies where resources were unconstrained, we find that, for a wide range of concentrations, MT length regulation is bistable. We test our predictions by conducting in vitro experiments and find that the bistable behavior manifests in a bimodal MT length distribution.

Measuring Microtubule Supertwist and Defects by Three-Dimensional-Force-Clamp Tracking of Single Kinesin-1 Motors.

Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it is hypothesized that microtubule supertwist and defects can severely influence the function of motors and other microtubule-associated proteins, the presented method allows for the first time to fully map the microtubule lattice in situ. This mapping allows the correlation of motor-filament interactions with the microtubule fine-structure. With the additional ability to apply loads, we expect our 3D-optical-tweezers force clamp to become a valuable tool for obtaining a wide range of information from other biological systems, inaccessible by two-dimensional and/or ensemble measurements.

Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding.

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.

Hornets Have It: A Conserved Olfactory Subsystem for Social Recognition in Hymenoptera?

Eusocial Hymenoptera colonies are characterized by the presence of altruistic individuals, which rear their siblings instead of their own offspring. In the course of evolution, such sterile castes are thought to have emerged through the process of kin selection, altruistic traits being transmitted to following generation if they benefit relatives. By allowing kinship recognition, the detection of cuticular hydrocarbons (CHCs) might be instrumental for kin selection. In carpenter ants, a female-specific olfactory subsystem processes CHC information through antennal detection by basiconic sensilla. It is still unclear if other families of eusocial Hymenoptera use the same subsystem for sensing CHCs. Here, we examined the existence of such a subsystem in Vespidae (using the hornet Vespa velutina), a family in which eusociality emerged independently of ants. The antennae of both males and female hornets contain large basiconic sensilla. Sensory neurons from the large basiconic sensilla exclusively project to a conspicuous cluster of small glomeruli in the antennal lobe, with anatomical and immunoreactive features that are strikingly similar to those of the ant CHC-sensitive subsystem. Extracellular electrophysiological recordings further show that sensory neurons within hornet basiconic sensilla preferentially respond to CHCs. Although this subsystem is not female-specific in hornets, the observed similarities with the olfactory system of ants are striking. They suggest that the basiconic sensilla subsystem could be an ancestral trait, which may have played a key role in the advent of eusociality in these hymenopteran families by allowing kin recognition and the production of altruistic behaviors toward relatives.

Drosophila Bitter Taste(s).

Most animals possess taste receptors neurons detecting potentially noxious compounds. In humans, the ligands which activate these neurons define a sensory space called "bitter". By extension, this term has been used in animals and insects to define molecules which induce aversive responses. In this review, based on our observations carried out in Drosophila, we examine how bitter compounds are detected and if bitter-sensitive neurons respond only to molecules bitter to humans. Like most animals, flies detect bitter chemicals through a specific population of taste neurons, distinct from those responding to sugars or to other modalities. Activating bitter-sensitive taste neurons induces aversive reactions and inhibits feeding. Bitter molecules also contribute to the suppression of sugar-neuron responses and can lead to a complete inhibition of the responses to sugar at the periphery. Since some bitter molecules activate bitter-sensitive neurons and some inhibit sugar detection, bitter molecules are represented by two sensory spaces which are only partially congruent. In addition to molecules which impact feeding, we recently discovered that the activation of bitter-sensitive neurons also induces grooming. Bitter-sensitive neurons of the wings and of the legs can sense chemicals from the gram negative bacteria, Escherichia coli, thus adding another biological function to these receptors. Bitter-sensitive neurons of the proboscis also respond to the inhibitory pheromone, 7-tricosene. Activating these neurons by bitter molecules in the context of sexual encounter inhibits courting and sexual reproduction, while activating these neurons with 7-tricosene in a feeding context will inhibit feeding. The picture that emerges from these observations is that the taste system is composed of detectors which monitor different "categories" of ligands, which facilitate or inhibit behaviors depending on the context (feeding, sexual reproduction, hygienic behavior), thus considerably extending the initial definition of "bitter" tasting.

Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces.

Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.

Dual mechanism for bitter avoidance in Drosophila.

In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, we assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or with L-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detect L-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, we established that bitter molecules differ in their potency to inhibit sucrose detection and that sugar-sensing inhibition affects taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. We postulate that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.

The use of weekly departmental review of all orthopaedic intra-operative radiographs in order to improve quality, due to standardized peer expectations and the "Hawthorne effect".

INTRODUCTION: Clinical governance highlights risk management, clinical effectiveness and use of evidence based practice as key elements in the provision of a quality service. A change in the method of quality control in our orthopaedic trauma unit allowed us the opportunity to study if the quality of operative outcomes had changed as a result. The Hawthorne effect refers to phenomenon whereby employees work quality improves by virtue of their awareness that their labour is being assessed. METHODS: A new outcome appraisal forum was introduced in our department in 2009. This forum involved a weekly whole department review of all the previous week's intraoperative radiographs. We used the tip apex distance (TAD) of the dynamic hip screw (DHS) procedures in hip fracture patients as a surrogate marker, of any objective change in the quality and consistency of intra-operative radiographs, in the year prior to and after the introduction of this review system. RESULTS: We found that the mean TAD and the number of TAD measurements over 25 mm decreased significantly in the year after the new quality control mechanism was introduced. CONCLUSION: We would recommend the use of a weekly quality control meeting scrutinizing every intraoperative radiograph as a simple, cost effective method of incorporating many aspects of clinical governance, as well as fostering a culture of quality.