ZW10 function in mitotic checkpoint control, dynein targeting and membrane trafficking: is dynein the unifying theme?

ZW10 was initially identified as a mitotic checkpoint protein involved in chromosome segregation. It was subsequently implicated in targeting cytoplasmic dynein and dynactin to mitotic kinetochores, though the relationship between these functions remains incompletely understood. Recent studies have revealed that ZW10 performs important functions in nondividing cells as well. These include cytoplasmic dynein targeting to Golgi and other membranes, but also SNARE-mediated ER-Golgi trafficking. Identifying a unifying function for ZW10 in these diverse contexts has been elusive, but likely involves cytoplasmic dynein, as discussed here.

Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end-directed movements. Golgi membrane-associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.

A role for cytoplasmic dynein and LIS1 in directed cell movement.

Cytoplasmic dynein has been implicated in numerous aspects of intracellular movement. We recently found dynein inhibitors to interfere with the reorientation of the microtubule cytoskeleton during healing of wounded NIH3T3 cell monolayers. We now find that dynein and its regulators dynactin and LIS1 localize to the leading cell cortex during this process. In the presence of serum, bright diffuse staining was observed in regions of active ruffling. This pattern was abolished by cytochalasin D, and was not observed in cells treated with lysophosphatidic acid, conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin containing structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant negative dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement.

Differential regulation of dynein-driven melanosome movement.

Cytoplasmic dyneins are multisubunit minus-end-directed microtubule motors. Different isoforms of dynein are thought to provide a means for independent movement of different organelles. We investigated the differential regulation of dynein-driven transport of pigment organelles (melanosomes) in Xenopus melanophores. Aggregation of melanosomes to the cell center does not change the localization of mitochondria, nor does dispersion of melanosomes cause a change in the perinuclear localization of the Golgi complex, indicating that melanosomes bear a dedicated form of dynein. We examined the subcellular fractionation behavior of dynein light intermediate chains (LIC) and identified at least three forms immunologically, only one of which fractionated with melanosomes. Melanosome aggregation was specifically blocked after injection of an antibody recognizing this LIC. Our data indicate that melanosome-associated dynein is regulated independently of bulk cytoplasmic dynein and involves a subfraction of dynein with a distinct subunit composition.

LIS1, CLIP-170's key to the dynein/dynactin pathway.

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.

Dynein at the cortex.

Cytoplasmic dynein is a minus end directed microtubule motor protein with numerous functions during interphase and mitosis. Recent evidence has identified several roles mediated by a fraction of cytoplasmic dynein associated with the cell cortex. So far, these include nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing, but others are likely. The possibility that a cortically bound form of dynein might represent its most ancient evolutionary state is discussed.