In the past decade there have been important advances in understanding intracellular transport by cytoplasmic dynein. The reconstitution of its motility in vitro has allowed great insight into its mechanism, and genetic and biochemical studies have identified some of its cargo-specific receptors. Loss-of function studies have firmly established the essential role of dynein adaptor proteins in a huge range of dynein-mediated processes. However, our understanding of the molecular mechanisms by which these adaptors contribute to dynein activity remains limited. Below, we discuss some of the outstanding fundamental questions regarding the functions of these dynein adaptors, and consider possible strategies towards finding their answers.
Dynein regulators are often referred to as activators because dynein-based motility in cells is repressed in their absence. However, it is possible that dynein requires regulatory proteins to repress its activity instead. several other cytoskeletal motors (for example, kinesin 1, kinesin 2, kinesin 3 and myosin V) are autoinhibited by intramolecular interactions and require external activation for motility2
. However, purified dynein seems to be fully active for processive movement, suggesting that dynein inhibition, rather than activation, requires additional factors, and that this inactive-to-active transition is a crucial part of dynein-based cargo transport. Although this has not been directly shown, it seems likely that dynein can adopt a repressed conformation in cells. Indeed, when dynein localizes to microtubule plus ends, its motility is inhibited, either through the repression of its ATPase or microtubule binding activities, or through blocking its access to the microtubule. Dynein is present on vesicles undergoing plus end-directed transport; it is therefore possible that kinesins, which move towards the opposite direction on microtubules, might simply overpower dynein, but a more energetically conservative mechanism would be dynein inhibition. Possible dynein repressors are LIS1, NUDE and NUDEL, which are perfectly positioned to control the activity of the dynein motor by interacting with the AAA1 ATPase domain. LIS1–NUDE, LIS1–NUDEL and dynactin are important for the probable inhibited state of dynein at microtubule plus ends and its subsequent activation. Dynactin forms important contacts with dynein cargos and possibly with kinesin motors as well; this could allow dynactin to transmit an activating signal to dynein when bound to cargo and to coordinate dynein and kinesin activity. Much progress has been made in understanding dynein motility in vitro
, but it is still unclear what might turn dynein on and off. in vitro
reconstitution of dynein with the ubiquitous dynein regulatory proteins (LIS1, NUDE or NUDEL and dynactin) might provide clues to this mechanism.
Another important unanswered question is how dynein links to its cargos. In the simplest model for motor-based transport in the cell, a motor diffuses through the cytoplasm and initiates cargo transport when it encounters the appropriate receptors on its cargos. At least in the case of dynein, the process of initiating cargo transport seems to be more complicated and to involve additional steps. In particular, localization of dynein and dynactin to microtubule plus ends might be an obligatory preliminary step to the initiation of some types of dynein transport. Dynein might use association with polymerizing microtubules to probe the cytoplasm for cargo (such as an organelle or the cell cortex) (). This mechanism could allow the simultaneous delivery of several motors and adaptors clustered at the micro-tubule tip. In addition, the microtubule tip could serve as a hub for the interactions of dynein and its adaptors with the many signalling molecules that are present at the plus ends of microtubules. The sequence of interactions that lead to the association of dynein with the microtubule plus end and to its eventual release and transfer to cargo is not well understood; a more precise determination of this sequence would allow a better understanding of its contribution to dynein function in the cell.
Dynein cargo binding also seems to proceed through a regulated series of interactions, perhaps reflecting a proofreading mechanism that functions to dock dynein only at specified locations. In particular, several small GTPases and their regulators interact with dynactin and Bicaudal D to facilitate dynein recruitment at specific cargos. Individual small GTPase family members act at specific subcellular compartments (for example, late endosomes or trans-Golgi vesicles), and their GTPase state and thus conformation is linked to the life cycle and direction of trafficking of these compartments. Through their nucleotide-sensitive contacts with dynein adaptors, small GTPases could provide a highly specific link between membrane vesicles and dynein. subsequent interaction of dynactin with the spectrin coat that is found on many organelles could then provide a more stable platform for dynein transport.
What strategies might best advance our understanding of how dynein functions with its regulatory proteins? Genetic methods have contributed greatly to the identification of the general dynein adaptors and specific dynein cargo receptors; however, our inventory is almost certainly incomplete. Relative to the wide array of cargos that dynein transports, only a few cargo-specific receptors for dynein have been identified and it is likely that many more dynein receptors remain to be discovered. Recent innovation in high-throughput microscopy and image analysis could facilitate the visual screens that could identify the unknown dynein receptors and regulatory factors coupling dynein to its cargos.
Although specific receptors for dynein have not been identified for most of its cargos, a better understanding of how the known dynein regulators work is an important and feasible goal. One possible path towards defining the molecular function of dynein regulators is through in vitro
reconstitution. Although dynactin was discovered through such an approach23,24
, few studies have focused on reconstituting dynein-based transport. The few dynein cargos for which receptors and their contacts with dynein and its general adaptors are known (for example, mRNA particles with Egalitarian and Bicaudal D) could provide a feasible starting point for the reconstitution of dynein cargo transport. In such a system, dynein and its adaptors could be mutated, their levels manipulated or components added sequentially, and their single molecule behaviour observed. such information would provide insight into the dynamic assembly of these complexes and the specific mechanistic contribution of each factor.
Within cells, recently developed high spatial precision fluorescence methods128–131
could help to define the composition and architecture of dynein motor supercomplexes and detect remodelling of this architecture during different stages of transport. Finally, a structural understanding of dynein and its cofactors with the resolution provided by electron microscopy or crystallography, although a challenging goal owing to the large sizes of these complexes, would provide important insight into the molecular mechanism by which they function.