Cytoplasmic dynein is not a conventional processive motor

Walter, Wilhelm J.; Brenner, Bernhard; Walter, Steffen

doi:10.1016/j.jsb.2009.11.011

Cited by 26 publications

(29 citation statements)

References 23 publications

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“…In a previous study, we showed that cytoplasmic dynein is capable of switching from nonprocessive to processive stepping by increasing the concentration of ATP (28). In the present study we further demonstrate that processivity at a fixed concentration of ATP can be varied by changing the level of Mg 2þ .…”

Section: Discussionsupporting

confidence: 78%

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Two independent switches regulate cytoplasmic dynein’s processivity and directionality

Walter

Koonce²,

Brenner³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionsupporting

confidence: 78%

“…Optical trap experiments were carried out as described previously (26,28). Details are provided in SI Text.…”

Section: Methodsmentioning

confidence: 99%

Two independent switches regulate cytoplasmic dynein’s processivity and directionality

Walter

Koonce²,

Brenner³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Hendricks

Holzbaur

Goldman

2012

Proc. Natl. Acad. Sci. U.S.A.

171

219

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Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus-and minus-end-directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.

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“…The motile behaviour of mammalian dynein has been studied using complexes purified from brain (Mallik et al , 2005; Ross et al , 2006; Miura et al , 2010; Ori‐McKenney et al , 2010; Walter et al , 2010) and tissue culture cells (Ichikawa et al , 2011), as well as complexes reconstituted from individual, recombinant components (Trokter et al , 2012). Movement of individual mammalian dynein complexes has been assayed by adhering the motor to beads (King & Schroer, 2000; Mallik et al , 2005; Walter et al , 2010), labelling accessory proteins (Ross et al , 2006; Miura et al , 2010) or by GFP tagging of the motor (Trokter et al , 2012). The extent to which individual dynein complexes can take multiple successive steps without detaching from the microtubule, a behaviour termed processivity, varied in these studies.…”

Section: Introductionmentioning

confidence: 99%

In vitro reconstitution of a highly processive recombinant human dynein complex

et al. 2014

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Cytoplasmic dynein is an approximately 1.4 MDa multi‐protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein‐associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N‐terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single‐molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long‐distance motor movement.

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Cytoplasmic dynein is not a conventional processive motor

Cited by 26 publications

References 23 publications

Two independent switches regulate cytoplasmic dynein’s processivity and directionality

Two independent switches regulate cytoplasmic dynein’s processivity and directionality

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

In vitro reconstitution of a highly processive recombinant human dynein complex

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