SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

Motor Dynamics Underlying Cargo Transport by Pairs of Kinesin-1 and Kinesin-3 Motors.

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this "K-loop" is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.

Processive Kinesin-14 HSET Exhibits Directional Flexibility Depending on Motor Traffic.

A common mitotic defect observed in cancer cells that possess supernumerary (more than two) centrosomes is multipolar spindle formation [1, 2]. Such structures are resolved into a bipolar geometry by minus-end-directed motor proteins, such as cytoplasmic dynein and the kinesin-14 HSET [3-8]. HSET is also thought to antagonize plus-end-directed kinesin-5 Eg5 to balance spindle forces [4, 5, 7, 9]. However, the biomechanics of this force opposition are unclear, as HSET has previously been defined as a non-processive motor [10-16]. Here, we use optical trapping to elucidate the mechanism of force generation by HSET. We show that a single HSET motor has a processive nature with the ability to complete multiple steps while trapped along a microtubule and when unloaded can move in both directions for microns. Compared to other kinesins, HSET has a relatively weak stall force of 1.1 pN [17, 18]. Moreover, HSET's tail domain and its interaction with the E-hook of tubulin are necessary for long-range motility. In vitro polarity-marked bundle assays revealed that HSET selectively generates force in anti-parallel bundles on the order of its stall force. When combined with varied ratios of Eg5, HSET adopts Eg5's directionality while acting as an antagonizing force brake, requiring at least a 10-fold higher Eg5 concentration to surpass HSET's sliding force. These results reveal HSET's ability to change roles within the spindle from acting as an adjustable microtubule slider and force regulator to a processive motor that aids in minus end focusing.

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters.

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.

Human CLASP2 specifically regulates microtubule catastrophe and rescue.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

Two mechanisms coordinate the recruitment of the chromosomal passenger complex to the plane of cell division.

During cytokinesis, the chromosomal passenger complex (CPC) promotes midzone organization, specifies the cleavage plane, and regulates furrow contractility. The localizations of the CPC are coupled to its cytokinetic functions. At the metaphase-to-anaphase transition, the CPC dissociates from centromeres and localizes to midzone microtubules and the equatorial cortex. CPC relocalization to the cell middle is thought to depend on MKlp2-driven, plus end-directed transport. In support of this idea, MKlp2 depletion impairs cytokinesis; however, cytokinesis failure stems from furrow regression rather than failed initiation of furrowing. This suggests that an alternative mechanism(s) may concentrate the CPC at the division plane. We show here that direct actin binding, via the inner centromere protein (INCENP), enhances CPC enrichment at the equatorial cortex, thus acting in tandem with MKlp2. INCENP overexpression rescues furrowing in MKlp2-depleted cells in an INCENP-actin binding-dependent manner. Using live-cell imaging, we also find that MKlp2-dependent targeting of the CPC is biphasic. MKlp2 targets the CPC to the anti-parallel microtubule overlap of the midzone, after which the MKlp2-CPC complex moves in a nondirected manner. Collectively, our work suggests that both actin binding and MKlp2-dependent midzone targeting cooperate to precisely position the CPC during mitotic exit, and that these pathways converge to ensure successful cleavage furrow ingression.

Cell Division: Centrosomes Have Separation Anxiety.

Prior to mitosis, duplicated centrosomes are tethered together, which is thought to prevent mitotic defects. A new study establishes the role of tetrameric Kif25, a microtubule minus-end-directed kinesin-14 motor, in preventing premature centrosome separation through a microtubule-dependent pathway.

Kinesin-5 inhibitor resistance is driven by kinesin-12.

The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10(7) cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process.

Influence of fluorescent tag on the motility properties of kinesin-1 in single-molecule assays.

Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SNAP and HALO ligands. We found that although both green and red SNAP ligands provide sufficient fluorescent signal, only the tetramethyl rhodamine (TMR) HALO ligand provides sufficient signal for detection in these assays. This study will serve as a valuable reference for choosing fluorescent labels for single-molecule motility assays.

Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia.

Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet-Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.

A method for multiprotein assembly in cells reveals independent action of kinesins in complex.

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor-cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.

Dimerization of mammalian kinesin-3 motors results in superprocessive motion.

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 µm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.

Quantification of training load in Canadian football: application of session-RPE in collision-based team sports.

The session-rating of perceived exertion (Session-RPE) method for quantifying internal training load (TL) has proven to be a highly valuable and accurate monitoring tool in numerous team sports. However, the influence of frequent impact during Canadian football on the validity of this subjective rating tool remains unclear. The aim of this study was to validate Session-RPE application to a prolonged, intermittent, high-intensity collision-based team sport through correlation of internal TL data collected using 2 criterion heart rate-based measures known as Polar Training-Impulse (TRIMP) and Edwards' TL. Twenty male participants (age = 22.0 ± 1.4 years) from the competitive roster of the University of Saskatchewan Canadian football team were recruited. Session-RPE, Polar TRIMP, and Edwards' TL data were collected daily over the 2011 Canadian Interuniversity Sport pre-competitive and competitive season (11 weeks; 713 total practice sessions). On average, each player contributed 36 sessions of data to the analysis. Statistically significant correlations (p < 0.01) between Session-RPE with Polar TRIMP (r = 0.65-0.91) and with Edwards' TL (r = 0.69-0.91) were found for all individual players. This study provides confirmation that Session-RPE is an inexpensive and simple tool, which is highly practical and accurately measures an individual's response (internal TL) to the Canadian football practice. Furthermore, when considering the number of individuals involved worldwide in collision-based team sports, this tool has the potential to impact a large proportion of the global sporting community.

Long-term athlete development Canada: attempting system change and multi-agency cooperation.

This text provides a synopsis, as well as some greater detail, concerning the "Canadian Sport for Life" project Long-Term Athlete Development Canada (LTAD) initiated in 2004. The genesis of the project may be found in the Canadian Sport Policy released in 2002 by Sport Canada, the sport participation and performance agency within the Canadian Heritage Ministry of the Canadian Government. The project has grown from relatively humble beginnings to become a system-wide movement and catalyst for change that encompasses not only sport participation and excellence, but also aspects to do with education, health, and general recreation. Additionally, it involves all age groups (cradle to grave). Although the project was initiated on behalf of performance sport, it is a clear example of how sport can influence and interact with many facets of a society. In Canada, LTAD clearly is tied to a philosophy that spans a broad narrative from healthy active lives to elite sport performance.

Assessment of physiological capacities of elite athletes & respiratory limitations to exercise performance.

Physiological assessment of athletes is an important process for the characterization of the athlete, monitoring progress and the trained state or 'level of preparedness' of an athlete, as well as aiding the process of training program design. Interestingly, the majority of physiological assessments performed on athletes can also be performed on children with disease, and therefore clinicians can learn a great deal about physiology and assessment of patient populations through the examination of the physiological responses of elite athletes. This review describes typical physiological responses of elite athletes to tests of aerobic and anaerobic metabolism and provides a specific focus upon respiratory limitations to exercise performance. Typical responses of elite athletes are described to provide the scientist and clinician with a perspective of the upper range of physiological capacities of elite athletes.

Diurnal normobaric moderate hypoxia raises serum erythropoietin concentration but does not stimulate accelerated erythrocyte production.

This study was performed to examine the effect of diurnal normobaric hypoxia on hematological parameters. Eleven healthy male volunteers were randomly selected to be in either the hypoxic group (n=6) or the control group (n=5). The hypoxic group was exposed to 8 h of normobaric hypoxia in hypoxic tent systems that elicited a target peripheral O(2) saturation of 81+/-2% on three consecutive days. The control group spent three consecutive 8-h days in modified tent systems that delivered normoxic air into the tent. Venous blood samples were collected before the exposure (days -5, 0), after each day of the exposure (days 1, 2, 3), and for 3 weeks after the exposure (days 7, 10, 13, 17, 24). Serum erythropoietin concentration significantly increased from 9.1+/-3.3 U.L(-1) to 30.7+/-8.6 U.L(-1) in the hypoxic group. Although there were significant increases in hematocrit (4%), hemoglobin concentration (5%), red blood cell count (4%) on day 7 in the hypoxic group, these observations were likely due to dehydration or biological variation over time. There was no significant change in early erythropoietic markers (reticulocyte counts or serum ferritin concentration), which provided inconclusive evidence of accelerated erythroid differentiation and proliferation. The results suggest that the degree of hypoxia was sufficient to stimulate increased erythropoietin production and release. However, the duration of hypoxic exposure was insufficient to propagate the erythropoietic cascade.

Vibration training: an overview of the area, training consequences, and future considerations.

The effects of vibration on the human body have been documented for many years. Recently, the use of vibration for improving the training regimes of athletes has been investigated. Vibration has been used during strength-training movements such as elbow flexion, and vibration has also been applied to the entire body by having subjects stand on vibration platforms. Exposure to whole-body vibration has also resulted in a significant improvement in power output in the postvibratory period and has been demonstrated to induce significant changes in the resting hormonal profiles of men. In addition to the potential training effects of vibration, the improvement in power output that is observed in the postvibratory period may also lead to better warm-up protocols for athletes competing in sporting events that require high amounts of power output. These observations provide the possibility of new and improved methods of augmenting the training and performance of athletes through the use vibration training. Despite the potential benefits of vibration training, there is substantial evidence regarding the negative effects of vibration on the human body. In conclusion, the potential of vibration treatment to enhance the training regimes of athletes appears quite promising. It is essential though that a thorough understanding of the implications of this type of treatment be acquired prior to its use in athletic situations. Future research should be done with the aim of understanding the biological effects of vibration on muscle performance and also the effects of different vibration protocols on muscle performance.

Validation of pulse oximetry during progressive normobaric hypoxia utilizing a portable chamber.

Validation of pulse oximetry in commercially available normobaric hypoxic chambers (NHC) has not been previously reported. The present study examined the validity of pulse oximetry (SpO2) against direct measurements of arterial oxygen saturation (SaO2) via co-oximetry (AVOXimeter 4000) in 13 young adults age 21.3 +/- 0.6 years. Over a period of 2.5 hrs, the inspired fraction of oxygen inside a NHC (Hypoxico, Inc.) was progressively reduced from 20.9% to 11.5%. Measurements of SaO2 at baseline and at 15, 30, 60, 90, 120, and 150 min during the hypoxic exposures were compared with SpO2 estimates of oxygen saturation (Nellcor 295) using reflectance (RS-10, temporal) and transmission (D-25, finger) sensors. Regression analysis and methods for assessing agreement (bias, b; precision, p) of SaO2 with SpO2 were similar (R2 = 0.92, 0.89; b = 0.016, -0.47; p = 2.47, 3.03; RS-10 and D-25, respectively). When SaO2 < 85%, RS-10 had greater validity than D-25 (R2 = 0.73, 0.56; b = 1.38, 1.13; p = 2.72, 4.34; RS-10 and D-25, respectively). In light of these findings, caution should be exercised when monitoring individuals with pulse oximetry during desaturation episodes below 85%. When employing frequent NHC exposures, a priori validation of SpO2 utilized to assess blood oxygen status appears warranted.

Performance evaluation of swimmers: scientific tools.

The purpose of this article is to provide a critical commentary of the physiological and psychological tools used in the evaluation of swimmers. The first-level evaluation should be the competitive performance itself, since it is at this juncture that all elements interplay and provide the 'highest form' of assessment. Competition video analysis of major swimming events has progressed to the point where it has become an indispensable tool for coaches, athletes, sport scientists, equipment manufacturers, and even the media. The breakdown of each swimming performance at the individual level to its constituent parts allows for comparison with the predicted or sought after execution, as well as allowing for comparison with identified world competition levels. The use of other 'on-going' monitoring protocols to evaluate training efficacy typically involves criterion 'effort' swims and specific training sets where certain aspects are scrutinised in depth. Physiological parameters that are often examined alongside swimming speed and technical aspects include oxygen uptake, heart rate, blood lactate concentration, blood lactate accumulation and clearance rates. Simple and more complex procedures are available for in-training examination of technical issues. Strength and power may be quantified via several modalities although, typically, tethered swimming and dry-land isokinetic devices are used. The availability of a 'swimming flume' does afford coaches and sport scientists a higher degree of flexibility in the type of monitoring and evaluation that can be undertaken. There is convincing evidence that athletes can be distinguished on the basis of their psychological skills and emotional competencies and that these differences become further accentuated as the athlete improves. No matter what test format is used (physiological, biomechanical or psychological), similar criteria of validity must be ensured so that the test provides useful and associative information concerning current or future performance. The practical worth of any proposed testing or monitoring protocol should be carefully evaluated. In addition, the developmental stage of the athlete(s) in question should be reflected in the testing/monitoring programme. Finally, increasing technological innovations will bring to the pool deck or dry-land training area simple, fast and advanced diagnostic tools, particularly in the areas of blood-borne markers of training response and neuromuscular excitability.