Two independent switches regulate cytoplasmic dynein’s processivity and directionality

Abstract

Cytoplasmic dynein is a microtubule-based molecular motor that participates in a multitude of cell activities, from cell division to organelle transport. Unlike kinesin and myosin, where different tasks are performed by highly specialized members of these superfamilies, a single form of the dynein heavy chain is utilized for different functions. This versatility demands an extensive regulation of motor function. Using an improved application of an optical trap, we were now able to demonstrate that cytoplasmic dynein can generate a discrete power stroke as well as a processive walk in either direction; i.e., towards the plus-or towards the minus-end of a microtubule. Thus, dynein's motor functions can be described by four basic modes of motion: processive and nonprocessive movement, and movement in the forward and reverse directions. Importantly, these four modes of movement can be controlled by two switches. One switch, based on phosphate, determines the directionality of movement. The second switch, depending on magnesium, converts cytoplasmic dynein from a nonprocessive to a processive motor. The two switches can be triggered separately or jointly by changing concentrations of phosphate and magnesium in the local environment. The control of four modes of movement by two switches has major implications for our understanding of the cellular functions and regulation of cytoplasmic dynein. Based on recent studies of dynein's structure we are able to draw new conclusions on cytoplasmic dynein's stepping mechanism. molecular motors | motor mechanics | single molecule I n eukaryotic cells almost all organelle transport is performed by the three families of cytoskeleton-based molecular motors, myosin, kinesin, and dynein (1-3). While myosin and kinesin have evolved into large families with multiple members, each of which specialized for different tasks, a single dynein heavy chain performs all of the dynein related activities in the cytoplasm of eukaryotic cells (1).One key function of cytoplasmic dynein is to power cargo transport over long distances. Here, motor processivity is required if long distance transport is to be achieved by a single motor molecule. To facilitate cargo delivery processivity must be switched off, when the target area is reached. Regulation becomes even more important when multiple motors of the same or even different type act together. For example, a model situation for the cooperation of different motors occurs during axonal transport driven by kinesin-1 and cytoplasmic dynein. During long distance travel, frequent changes in direction are observed. It is generally thought that the reversals of direction result from counteracting motor activity (4, 5). Though, a simple tug-of-war model in which only the winner determines the direction of movement appears rather inefficient to deliver cargo to specific locations. Moreover, it has been demonstrated in vitro and in vivo that dynein's activity dominates over kinesin (6, 7). Therefore, being able to modulate dynein's processivity and directional...

Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

“…Although there is some controversy over the unitary stall force of mammalian dynein (29) likely arising from the sensitivity of dynein's biophysical properties to buffer conditions (30,31), our laboratory and others find that individual dynein motors isolated from mammalian tissue produce ∼1 pN (25,32,33). As clear stall events are rare in the cellular data, we define a force event as an excursion from the trap center greater than ±0.5 pN, including both events in which motors stall and ones in which motors detach before reaching stall.…”

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

“…Polarity-marked microtubules were made with rhodamine-labeled microtubules and HiLyte TM 488 microtubules (Cytoskeleton Inc.) by adapting a protocol as described previously by Walter et al (32). Homemade perfusion chambers were constructed by sandwiching 22 ϫ 60-and 22 ϫ 22-mm glass coverslips using double-sided tape.…”

Ensemble velocity of non-processive molecular motors with multiple chemical states

“…To the best of our knowledge, no such behaviour has been reported in natural motor proteins so far. There are, however, kinesin-5 Cin8 rsfs.royalsocietypublishing.org Interface Focus 4: 20140032 that switches direction depending on the ionic strength [46,47] and dynein that reverses upon addition of phosphate [48]. Notable achievements involving artificial inversion and/or direction switching include an insertion in the myosin lever arm that reversed its direction [49] and a myosin construct that can switch direction depending on the calcium concentration [50].…”