Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Modulation of kinesin’s load-bearing capacity by force geometry and the microtubule track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule associated proteins and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long-axis of the microtubule due to contact of the single bead with the underlying surface. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule the median attachment duration between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median attachment duration (median-Δt) of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median-Δt < 0.2 s) to a persistent motor that sustains attachment (median-Δt > 3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice which has implications for a range of cellular activities including cell division and organelle transport.Significance StatementKinesins are cytoskeletal motors responsible for the transport of cargoes along microtubules. It is well known that opposing forces decrease kinesin’s speed and run length. In this study, we found that when the pair of opposing forces applied on the kinesin-microtubule complex are parallel to the microtubule, the ability of kinesin to remain attached to the microtubule can vary by more than an order of magnitude depending on the relative azimuthal position of the pair of forces along the periphery of the microtubule. These results reveal a previously unknown versatility of kinesin’s load bearing capacity and as such have implications for the potential physiological roles of kinesin in a wide range of cell activities, including organelle transport and cell division.

Radio-transmitter attachment methods for monitoring the endangered eastern barred bandicoot (Perameles gunnii)

Radio-tracking is a key technique for monitoring threatened species during ecological research and reintroduction programs. In the case of the endangered eastern barred bandicoot (Perameles gunnii), it has not been possible to radio-track for extended periods (≥3 months) due to difficulties in reliably and safely attaching radio-transmitters. In this study we compared eight attachment methods. Transmitters weighing 1.2–28 g were either mounted with adhesive, attached to a collar or implanted into the peritoneum. Intraperitoneal transmitters were superior in terms of attachment duration, but were considered inferior overall as they could not be fitted in the field and had a very short detection range once implanted (≤50 m). Retention times for external transmitters differed greatly between methods, ranging from 1 to 102 days. One tail-mount attachment technique caused minimal adverse effects but another caused tail amputation in one animal, and both had short retention times (3–33 days). Neither of the glue-on transmitter methods resulted in substantial periods of attachment (2–30 days) and flank-mounted transmitters also caused severe skin trauma. Radio-collars were generally retained for longer periods (42–102 days) but resulted in limb entanglement when they were fitted too loosely or subconjunctival haemorrhages when they were too tight. Cable tie collars are recommended as the most suitable attachment technique for bandicoots, as when fitted correctly they cause minimal impact to individuals and permit long retention times, but precise fitting is essential.