Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.     

Nudel modulates kinetochore association and function of cytoplasmic dynein in M phase

The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly ( approximately 78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.     

Human Nudel and NudE as regulators of cytoplasmic dynein in poleward protein transport along the mitotic spindle

Emerging evidence supports the idea that a signaling pathway containing orthologs of at least mammalian NudE and Nudel, Lis1, and cytoplasmic dynein is conserved for eukaryotic nuclear migration. In mammals, this pathway has profound impact on neuronal migration during development of the central nervous system. Lis1 and dynein are also involved in other cellular functions, such as mitosis. Here we show that Nudel also participates in a subset of dynein function in M phase. Nudel was specifically phosphorylated in M phase in its serine/threonine phosphorylation motifs, probably by Cdc2 and also Erk1 and -2. A fraction of Nudel bound to centrosomes strongly in interphase and localized to mitotic spindles in early M phase. By using mutants incapable of or simulating phosphorylation, we confirmed that phosphorylation of Nudel regulated the cell-cycle-dependent distribution, possibly by increasing its dissociation rate at the microtubule-organizing center. Moreover, phosphorylated Nudel or the phosphorylation-mimicking mutant bound Lis1 more efficiently. We further demonstrated that a Nudel mutant incapable of binding to Lis1 impaired the poleward movement of dynein and hence the dynein-mediated transport of kinetochore proteins to spindle poles along microtubules, a process contributing to inactivation of the spindle checkpoint in mitosis. These results point to the importance of Nudel-Lis1 interaction for the dynein activity in M phase and to a possible role of Nudel phosphorylation as facilitating such interaction. In addition, comparative studies suggest that NudE is also functionally related to its paralog, Nudel.     

Nudel contributes to microtubule anchoring at the mother centriole and is involved in both dynein-dependent and -independent centrosomal protein assembly

The centrosome is the major microtubule-organizing center in animal cells. Although the cytoplasmic dynein regulator Nudel interacts with centrosomes, its role herein remains unclear. Here, we show that in Cos7 cells Nudel is a mother centriole protein with rapid turnover independent of dynein activity. During centriole duplication, Nudel targets to the new mother centriole later than ninein but earlier than dynactin. Its centrosome localization requires a C-terminal region that is essential for associations with dynein, dynactin, pericentriolar material (PCM)-1, pericentrin, and gamma-tubulin. Overexpression of a mutant Nudel lacking this region, a treatment previously shown to inactivate dynein, dislocates centrosomal Lis1, dynactin, and PCM-1, with little influence on pericentrin and gamma-tubulin in Cos7 and HeLa cells. Silencing Nudel in HeLa cells markedly decreases centrosomal targeting of all the aforementioned proteins. Silencing Nudel also represses centrosomal MT nucleation and anchoring. Furthermore, Nudel can interact with pericentrin independently of dynein. Our current results suggest that Nudel plays a role in both dynein-mediated centripetal transport of dynactin, Lis1, and PCM-1 as well as in dynein-independent centrosomal targeting of pericentrin and gamma-tubulin. Moreover, Nudel seems to tether dynactin and dynein to the mother centriole for MT anchoring.     

Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.     

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.     

Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle- dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.     

Nudel Promotes Axonal Lysosome Clearance and Endo-lysosome Formation via Dynein-Mediated Transport

Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end-directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti-Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo-lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N-terminal Nudel, anti-dynein antibody or a p150^Glued mutant capable of abrogating the dynein–dynactin association. These results indicate a critical role of Nudel in dynein-mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.     

Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality

Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.     

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.     

Extragenic Suppressors of a Dynein Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed ``motor.' Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.     

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.     

Lis1, the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation.

Lissencephaly is a severe congenital brain malformation resulting from incomplete neuronal migration. One causal gene, LIS1, is homologous to nudF, a gene required for nuclear migration in A. nidulans. We have characterized the Drosophila homolog of LIS1 (Lis1) and show that Lis1 is essential for fly development. Analysis of ovarian Lis1 mutant clones demonstrates that Lis1 is required in the germline for synchronized germline cell division, fusome integrity and oocyte differentiation. Abnormal packaging of the cysts was observed in Lis1 mutant clones. Our results indicate that LIS1 is important for cell division and differentiation and the function of the membrane cytoskeleton. They support the notion that LIS1 functions with the dynein complex to regulate nuclear migration or cell migration.     

UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration

Nuclei migrate during many events, including fertilization, establishment of polarity, differentiation, and cell division. The Caenorhabditis elegans KASH protein UNC-83 localizes to the outer nuclear membrane where it recruits kinesin-1 to provide the major motor activity required for nuclear migration in embryonic hyp7 cells. Here we show that UNC-83 also recruits two dynein-regulating complexes to the cytoplasmic face of the nucleus that play a regulatory role. One consists of the NudE homolog NUD-2 and the NudF/Lis1/Pac1 homolog LIS-1, and the other includes dynein light chain DLC-1, the BicaudalD homolog BICD-1, and the Egalitarian homologue EGAL-1. Genetic disruption of any member of these two complexes caused nuclear migration defects that were enhanced in some double mutant animals, suggesting that BICD-1 and EGAL-1 function in parallel to NUD-2. Dynein heavy chain mutant animals also had a nuclear migration defect, suggesting these complexes function through dynein. Deletion analysis indicated that independent domains of UNC-83 interact with kinesin and dynein. These data suggest a model where UNC-83 acts as the cargo-specific adaptor between the outer nuclear membrane and the microtubule motors kinesin-1 and dynein. Kinesin-1 functions as the major force generator during nuclear migration, while dynein is involved in regulation of bidirectional transport of the nucleus.     

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.     

Misfolded Gβ is recruited to cytoplasmic dynein by Nudel for efficient clearance

The Gβγ heterodimer is an important signal transducer. Gβ, however, is prone to misfolding due to its requirement for Gγ and chaperones for proper folding. How cells dispose of misfolded Gβ (mfGβ) is not clear. Here, we showed that mfGβ was able to be polyubiquitinated and subsequently degraded by the proteasome. It was sequestered in aggresomes after the inhibition of the proteasome activity with MG132. Sustained activation of Gβγ signaling further elevated cellular levels of the ubiquitinated Gβ. Moreover, Nudel, a regulator of cytoplasmic dynein, the microtubule minus end-directed motor, directly interacted with both the unubiquitinated and ubiquitinated mfGβ. Increasing the levels of both mfGβ and Nudel promoted the association of Gβ with both Nudel and dynein, resulting in robust aggresome formation in a dynein-dependent manner. Depletion of Nudel by RNAi reduced the dynein-associated mfGβ, impaired the MG132-induced aggresome formation, and markedly prolonged the half-life of nascent Gβ. Therefore, cytosolic mfGβ is recruited to dynein by Nudel and transported to the centrosome for rapid sequestration and degradation. Such a process not only eliminates mfGβ efficiently for the control of protein quality, but may also help to terminate the Gβγ signaling.     

Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.     

Interaction mapping of a dynein heavy chain. Identification of dimerization and intermediate-chain binding domains.

Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an approximately 150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed.     

Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1

Whereas total loss of Lis1 is lethal, disruption of one allele of the Lis1 gene results in brain abnormalities, indicating that developing neurons are particularly sensitive to a reduction in Lis1 dosage. Here we show that Lis1 is enriched in neurons relative to levels in other cell types, and that Lis1 interacts with the microtubule motor cytoplasmic dynein. Production of more Lis1 in non-neuronal cells increases retrograde movement of cytoplasmic dynein and leads to peripheral accumulation of microtubules. These changes may reflect neuron-like dynein behaviours induced by abundant Lis1. Lis1 deficiency produces the opposite phenotype. Our results indicate that abundance of Lis1 in neurons may stimulate specific dynein functions that function in neuronal migration and axon growth.