Thomas S. Hays

Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein

We describe the dynamics of kinetochore dynein–dynactin in living Drosophila embryos and examine the effect of mutant dynein on the metaphase checkpoint. A functional conjugate of dynamitin with green fluorescent protein accumulates rapidly at prometaphase kinetochores, and subsequently migrates off kinetochores towards the poles during late prometaphase and metaphase. This behaviour is seen for several metaphase checkpoint proteins, including Rough deal (Rod). In neuroblasts, hypomorphic dynein mutants accumulate in metaphase and block the normal redistribution of Rod from kinetochores to microtubules. By transporting checkpoint proteins away from correctly attached kinetochores, dynein might contribute to shutting off the metaphase checkpoint, allowing anaphase to ensue.

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.

The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization

Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.

The SCF ubiquitin ligase protein Slimb regulates centrosome duplication in Drosophila

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.

In Vivo Dynamics of the Rough Deal Checkpoint Protein during Drosophila Mitosis

Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint and for recruitment of dynein/dynactin to the kinetochore. The Rod complex, like most classical metaphase checkpoint components, forms part of the outer domain of unattached kinetochores. Here we analyze the dynamics of a GFP-Rod chimera in living syncytial Drosophila embryos. Uniquely among checkpoint proteins, GFP-Rod robustly streams from kinetochores along microtubules, from the time of chromosome attachment until anaphase onset. Prometaphase and metaphase kinetochores continuously recruit new Rod, thus feeding the current. Rod flux from kinetochores appears to require biorientation but not tension because it continues in the presence of taxol. As with Mad2, kinetochore- and spindle-associated Rod rapidly turns over with free cytosolic Rod, both during normal mitosis and after colchicine treatment, with a t1/2 of 25-45 s. GFP-Rod coimmunoprecipitates with dynein/dynactin, and in the absence of microtubules both Rod and dynactin accumulate on kinetochores. Nevertheless, Rod and dynein/dynactin behavior are distinguishable. We propose that the Rod complex is a major component of the fibrous corona and that the recruitment of Rod during metaphase is required to replenish kinetochore dynein after checkpoint conditions have been satisfied but before anaphase onset.

Dynein-mediated apical localization of crumbs transcripts is required for Crumbs activity in epithelial polarity

Asymmetrical localization of transcripts coupled with localized translation constitutes an important mechanism widely deployed to regulate gene activity in a spatial manner. The conserved transmembrane protein Crumbs (Crb) is an important regulator of epithelial polarity. However, it remains unclear how Crb is targeted to the apical domain. Here, we show that the cytoplasmic dynein complex transports both Crb protein and transcripts to the apical domain of Drosophila melanogaster follicular cells (FCs). The crb 3′ untranslated region (UTR) is necessary and sufficient for the apical localization of its transcript and this apical transcript localization is crucial for crb function. In crb mutant FCs, Crb protein derived from transgenes lacking the 3′ UTR does not effectively localize to the apical domain and does not effectively restore normal epithelial polarity. We propose that dynein-mediated messenger RNA transport coupled with a localized translation mechanism is involved in localizing Crb to the apical domain to mediate epithelial apicobasal polarity and that this mechanism might be widely used to regulate cellular polarity.

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

Amyloid precursor protein (APP) vesicle movement by kinesin-1 and cytoplasmic dynein exhibits kinesin-1–dependent velocity. Our data also suggest that kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and that their direction and velocity are controlled at least in part by dynein IC.

The Intermediate Chain of Cytoplasmic Dynein Is Partially Disordered and Gains Structure upon Binding to Light-Chain LC8 †

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Dynein Light Intermediate Chain: An Essential Subunit That Contributes to Spindle Checkpoint Inactivation

The dynein light intermediate chain (LIC) is a subunit unique to the cytoplasmic form of dynein, but how it contributes to dynein function is not fully understood. Previous work has established that the LIC homodimer binds directly to the dynein heavy chain and may mediate the attachment of dynein to centrosomes and other cargoes. Here, we report our characterization of the LIC in Drosophila. Unlike vertebrates, in which two Lic genes encode multiple subunit isoforms, the Drosophila LIC is encoded by a single gene. We determined that the single LIC polypeptide is phosphorylated, and that different phosphoisoforms can assemble into the dynein motor complex. Our mutational analyses demonstrate that, similar to other dynein subunits, the Drosophila LIC is required for zygotic development, germline specification of the oocyte, and mitotic cell division. We show that RNA interference depletion of LIC in Drosophila S2 cells does not block the recruitment of a dynein complex to kinetochores, but it does delay inactivation of Mad2 signaling and mitotic progression. Our observations suggest the LIC contributes to a broad range of dynein functions.