Tail Swing Research Articles

Two modes of microtubule sliding driven by cytoplasmic dynein

Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin-streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.

Thermodynamics of Nucleotide Binding to Actomyosin V and VI: A Positive Heat Capacity Change Accompanies Strong ADP Binding

We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.

Indicators of Acute Pain and Fly Avoidance Behaviors in Holstein Calves Following Tail-docking

Previous work showed that the banding process of docking minimally affected mature cows’ behavior and physiology, but cutting off the necrotic tail increased haptoglobin. Additionally the docked cows had more flies on the rear legs and exhibited more fly avoidance behaviors. Because many producers dock young calves while they are in hutches where fly problems are more pronounced, we investigated changes in behavior and physiology of young calves following docking by banding. Twenty calves (3 to 5wk of age) were assigned to a docked or control group, at each of two locations (Indiana and Wisconsin). After applying a band to dock the tail, calves were tested every 15min for sensitivity to heat below the band at the Indiana location. Calf behavior was recorded for 2h postbanding and analyzed continuously for that period. After 3wk, tails were removed and then 1wk later, fly counts and fly avoidance behaviors were observed at both locations. Tails were sensitive to heat below the banding site, for 60 to 120min postbanding (mean 87min). Banded calves were more active than control calves during the 2h following banding. Percentage of time spent lying was greater for control calves, and the percentage of time spent walking was greater for docked than control calves. More importantly, movements of the head to touch the tail were increased for banded calves (eight-fold more movements). Fly avoidance behaviors directed toward the rear of the calf were evident at noon or in the afternoon. Ear twitches were more frequent for the docked calves and less frequent in the morning for all calves. Licking was more frequent for the docked calves at 1200 and 1600h. Tail swings were most frequent at 1200 and more frequent for control calves. Two acute phase proteins, haptoglobin and α1 acid-glycoprotein, were not different at any time. In this study, calves that were banded at 3-wk-of-age showed behaviors indicative of discomfort for 2h, were attacked by more flies, and showed increased fly avoidance behaviors when docked.

Tail-Docking Alters Fly Numbers, Fly-avoidance behaviors, and Cleanliness, but not Physiological Measures

Tail docking is an animal well-being issue not only regarding the docking procedures but also because of concerns during fly season. To address the latter question, we selected eight cows that had been tail-docked in a previous experiment and eight nondocked cows matched by stage of lactation. Physiological, immunological, and behavioral measures were used to evaluate the well being of those cows housed in a tie-stall barn during fly season for 5 consecutive days. Behavior was observed for 5-min interval instantaneous scan samples for 1h each at 0800, 1200, and 1600h. Flies were counted before behavior observations. Blood samples were taken daily for plasma and leukocyte separation. Cows were scored on d 5 for cleanliness on a five-point scale. Docked cows were cleaner, but fly counts of docked cows were greater for total fly counts and rear leg counts. However, counts were not different on front legs. Time of day was significant, so each time of day was analyzed separately. Docked cows were observed to exhibit fewer tail swings at 0800h, but docked cows tended to ruminate more at that time. Docked cows tended to stand less at the 1200h observation. Total fly-avoidance behaviors were greater for all cows at the 1600-h observation. Only tail swings tended to be more frequent with docked cows, but foot stomps occurred only in the docked cows. Lymphocyte phenotypes, acute-phase proteins, and immunoglobulin concentrations did not differ. In conclusion, although docked cows were cleaner, as the fly numbers increase throughout the day, fly-avoidance behaviors also increased and foot stomping appeared as an alternative method for fly avoidance by docked cows.

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

A 32 degree tail swing in brush border myosin I on ADP release.

Brush border myosin I (BBMI) is a single-headed, unconventional myosin from intestinal microvilli, composed of a heavy chain of relative molecular mass 119,000 (M(r) 119K) and three calmodulin light chains. Although believed to have a largely structural role, it exhibits the normal actin-activated ATPase and motility properties of a member of the myosin superfamily. Here we present three-dimensional maps of BBMI-decorated actin filaments with and without bound MgADP. While the motor domain remains in a state similar to rigor, the light-chain-binding domain swings through approximately 32 degrees, resulting in a approximately 50-A movement at the end of the region visualized (the second calmodulin light chain). This could correspond to approximately 72-A movement of the entire domain. Although qualitatively similar to the movement observed in myosin II, the magnitude of the change is sufficiently different to suggest that structural changes during the actomyosin ATPase cycle differ among myosins, possibly reflecting adaptation for specialized functional demands.