The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.     

Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.     

Cytoplasmic dynein and microtubule transport in the axon: The action connection

The neuron uses two families of microtubule-based motors for fast axonal transport, kinesin, and cytoplasmic dynein. Cytoplasmic dynein moves membranous organelles from the distal regions of the axon to the cell body. Because dynein is synthesized in the cell body, it must first be delivered to the axon tip. It has recently been shown that cytoplasmic dynein is moved from the cell body along the axon by two different mechanisms. A small amount is associated with fast anterograde transport, the membranous organelles moved by kinesin. Most of the dynein is transported in slow component b, the actin-based transport compartment. Dynactin, a protein complex that binds dynein, is also transported in slow component b. The dynein in slow component b binds to microtubules in an ATP-dependent manner in vitro, suggesting that this dynein is enzymatically active. The finding that functionally active dynein, and dynactin, are associated with the actin-based transport compartment suggests a mechanism whereby dynein anchored to the actin cytoskeleton via dynactin provides the motive force for microtubule movement in the axon.     

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.     

An Alpha Tubulin Mutation Suppresses Nuclear Migration Mutations in Aspergillus Nidulans

Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs(-)) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein β-subunit-like protein. Hs(-) mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs(-) phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs(-) mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs(-) nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs(-) nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.     

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.     

Long-Distance Movement of Aspergillus nidulans Early Endosomes on Microtubule Tracks

In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabA^Rab5 localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4-64 shortly after dye internalization. RabA^Rab5 membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1^ts mutation results in accumulation of RabA^Rab5 endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.     

Formation of spindle poles by dynein/dynactin-dependent transport of NuMA

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.     

Neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro-10 and ro-11. ro-10 and ro-11 are non-essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro-10 and ro-11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin-related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro-10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro-11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

Nucleotide-dependent behavior of single molecules of cytoplasmic dynein on microtubules in vitro

We visualized the nucleotide-dependent behavior of single molecules of mammalian native cytoplasmic dynein using fragments of dynactin p150 with or without its N-terminal microtubule binding domain. The results indicate that the binding affinity of dynein for microtubules is high in AMP-PNP, middle in ADP or no nucleotide, and low in ADP·Pi conditions. It is also demonstrated that the microtubule binding domain of dynactin p150 maintains the association of dynein with microtubules without altering the motile property of dynein in the weak binding state. In addition, we observed bidirectional movement of dynein in the presence of ATP as well as in ADP/Vi condition, suggesting that the bidirectional movement is driven by diffusion rather than active transport.     

The p25 subunit of the dynactin complex is required for dynein-early endosome interaction

Cytoplasmic dynein transports various cellular cargoes including early endosomes, but how dynein is linked to early endosomes is unclear. We find that the Aspergillus nidulans orthologue of the p25 subunit of dynactin is critical for dynein-mediated early endosome movement but not for dynein-mediated nuclear distribution. In the absence of NUDF/LIS1, p25 deletion abolished the localization of dynein-dynactin to the hyphal tip where early endosomes abnormally accumulate but did not prevent dynein-dynactin localization to microtubule plus ends. Within the dynactin complex, p25 locates at the pointed end of the Arp1 filament with Arp11 and p62, and our data suggest that Arp11 but not p62 is important for p25-dynactin association. Loss of either Arp1 or p25 significantly weakened the physical interaction between dynein and early endosomes, although loss of p25 did not apparently affect the integrity of the Arp1 filament. These results indicate that p25, in conjunction with the rest of the dynactin complex, is important for dynein-early endosome interaction.     

CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).     

Dynein-Driven Mitotic Spindle Positioning Restricted to Anaphase by She1p Inhibition of Dynactin Recruitment

Dynein is a minus-end–directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1Δ cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans

The multisubunit microtubule motor, cytoplasmic dynein, targets to various subcellular locations in eukaryotic cells for various functions. The cytoplasmic dynein heavy chain (HC) contains the microtubule binding and ATP binding sites for motor function, whereas the intermediate chain (IC) is implicated in the in vivo targeting of the HC. Concerning any targeting event, it is not known whether the IC has to form a complex with the HC for targeting or whether the IC can target to a site independently of the HC. In the filamentous fungus Aspergillus nidulans, the dynein HC is localized to the ends of microtubules near the hyphal tip. In this study, we demonstrate that our newly identified dynein IC in A. nidulans is also localized to microtubule ends and is required for HC's localization to microtubule ends in living cells. With the combination of two reagents, an HC loss-of function mutant and the green fluorescent protein (GFP)-fused IC that retains its function, we show that the IC's localization to microtubule ends also requires HC, suggesting that cytoplasmic dynein HC-IC complex formation is important for microtubule end targeting. In addition, we show that the HC localization is not apparently altered in the deletion mutant of NUDF, a LIS1-like protein that interacts directly with the ATP-binding domain of the HC. Our study suggests that, although HC-IC association is important for the targeting of dynein to microtubule ends, other essential components, such as NUDF, may interact with the targeted dynein complex to produce full motor activities in vivo.     

Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end- directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC- binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.     

Dynactin is required for transport initiation from the distal axon

Dynactin is a required cofactor for the minus-end-directed microtubule motor cytoplasmic dynein. Mutations within the highly conserved CAP-Gly domain of dynactin cause neurodegenerative disease. Here, we show that the CAP-Gly domain is necessary to enrich dynactin at the distal end of primary neurons. While the CAP-Gly domain is not required for sustained transport along the axon, we find that the distal accumulation facilitates the efficient initiation of retrograde vesicular transport from the neurite tip. Neurodegenerative disease mutations in the CAP-Gly domain prevent the distal enrichment of dynactin thereby inhibiting the initiation of retrograde transport. Thus, we propose a model in which distal dynactin is a key mediator in promoting the interaction among the microtubule, dynein motor, and cargo for the efficient initiation of transport. Mutations in?the CAP-Gly domain disrupt the formation of the?motor-cargo complex, highlighting the specific defects in axonal transport that may lead to neurodegeneration.     

Roles of NUDE and NUDF proteins of Aspergillus nidulans: insights from intracellular localization and overexpression effects

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes ("comets") that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudE gene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditional nudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF and nudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between the nudF and apsA genes suggests a role for NUDF at the cell cortex.     

Cytoplasmic dynein/dynactin mediates the assembly of aggresomes

Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.