Vladimir I. Gelfand

Dynactin is required for bidirectional organelle transport

Kinesin II is a heterotrimeric plus end–directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II–mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II–associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530–793 of XKAP and aa 600–811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

Interactions and regulation of molecular motors in Xenopus melanophores

Many cellular components are transported using a combination of the actin- and microtubule-based transport systems. However, how these two systems work together to allow well-regulated transport is not clearly understood. We investigate this question in the Xenopus melanophore model system, where three motors, kinesin II, cytoplasmic dynein, and myosin V, drive aggregation or dispersion of pigment organelles called melanosomes. During dispersion, myosin V functions as a “molecular ratchet” to increase outward transport by selectively terminating dynein-driven minus end runs. We show that there is a continual tug-of-war between the actin and microtubule transport systems, but the microtubule motors kinesin II and dynein are likely coordinated. Finally, we find that the transition from dispersion to aggregation increases dynein-mediated motion, decreases myosin V–mediated motion, and does not change kinesin II–dependent motion. Down-regulation of myosin V contributes to aggregation by impairing its ability to effectively compete with movement along microtubules.

Myosin cooperates with microtubule motors during organelle transport in melanophores

Melanophores offer an outstanding system for the study of intracellular motility. These cells aggregate their pigment-filled melanosomes to the cell center or disperse them throughout the cytoplasm in response to hormonal modulation of intracellular cyclic AMP levels in order to effect color changes in lower vertebrates [1]. Previous work from our laboratory demonstrated a role for microtubule-based motors in melanosome transport and we succeeded in reconstituting their regulated motility along microtubules in vitro [2,3]. Here we demonstrate that, in addition to microtubule-mediated motility, melanosomes purified from Xenopus melanophores exhibit unidirectional movement along actin filaments in vitro as well. Immunoblotting analysis shows that these organelles possess the actin-based organelle motor, myosin-V. In vivo, melanosomes are able to slowly disperse in the absence of microtubules, and this slow dispersion requires the integrity of the actin cytoskeleton. Furthermore, in cells with dispersed pigment, disruption of filamentous actin induces a rapid, microtubule-dependent aggregation of melanosomes to the cell center. Our results, together with the accompanying paper by Rodionov et al. [4], demonstrate that the concerted efforts of both microtubule-based and actin-based motors are required for proper melanosome distribution in melanophores. This is the first example of a biochemically defined organelle in possession of both plus-end and minus-end directed microtubule motors and a myosin; coordinated activity of all three motors is essential for organelle motility in vivo.

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore–microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

Motor–cargo interactions: the key to transport specificity

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.

Opposite-Polarity Motors Activate One Another to Trigger Cargo Transport in Live Cells

Mechanical interactions between any two opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.

Membrane trafficking, organelle transport, and the cytoskeleton

Cytoskeleton-associated motor proteins typically drive organelle movements in eukaryotic cells in a manner that is tightly regulated, both spatially and temporally. In the past year, a novel organelle transport mechanism utilizing actin polymerization was described. Important advances were also made in the assignment of functions to several new motors and in our understanding of how motor proteins are regulated during organelle transport. In addition, insights were gained into how and why organelles are transported cooperatively along the microtubule and actin cytoskeletons, and into the importance of motor-mediated transport in the organization of the cytoskeleton itself.

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150glued, binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150glued, we replaced the wild-type p150glued in Drosophila melanogaster S2 cells with mutant ΔN-p150 lacking residues 1–200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150glued with ΔN-p150glued has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150glued has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.

The role of microtubule movement in bidirectional organelle transport

We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.

Initial Neurite Outgrowth in Drosophila Neurons Is Driven by Kinesin-Powered Microtubule Sliding

Remarkably, forces within a neuron can extend its axon to a target that could be meters away. The two main cytoskeleton components in neurons are microtubules, which are mostly bundled along the axon shaft, and actin filaments, which are highly enriched in a structure at the axon distal tip, the growth cone. Neurite extension has been thought to be driven by a combination of two forces: pushing via microtubule assembly, and/or pulling by an actin-driven mechanism in the growth cone. Here we show that a novel mechanism, sliding of microtubules against each other by the microtubule motor kinesin-1, provides the mechanical forces necessary for initial neurite extension in Drosophila neurons. Neither actin filaments in the growth cone nor tubulin polymerization is required for initial outgrowth. Microtubule sliding in neurons is developmentally regulated and is suppressed during neuronal maturation. As kinesin-1 is highly evolutionarily conserved from Drosophila to humans, it is likely that kinesin-1-powered microtubule sliding plays an important role in neurite extension in many types of neurons across species.