Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein.

Cytoplasmic dynein is a multisubunit ATPase that transforms chemical energy into motion along microtubules. LC8, a 10 kDa light chain subunit of the dynein complex, is highly conserved with 94% sequence identity between Drosophila and human. The precise function of this protein is unknown, but its ubiquitous expression and conservation suggest a critical role in the function of the dynein motor complex. We have overexpressed LC8 from Drosophila melanogaster and characterized its dimerization and folding using analytical ultracentrifugation, size-exclusion chromatography, circular dichroism, and fluorescence spectroscopy. Sedimentation equilibrium measurements of LC8 at pH 7 reveal a reversible monomer-dimer equilibrium with a dissociation constant of 12 microM at 4 degrees C. At lower pH, LC8 dissociates to a monomer, with a transition midpoint at pH 4.8. Far-UV CD and fluorescence spectra demonstrate that pH-dissociated LC8 retains native secondary and tertiary structures, while the diminished near-UV CD signal shows loss of quaternary structure. The observation that dimeric LC8 dissociates at low pH can be explained by titration of a histidine pair in the dimer interface. Equilibrium denaturation experiments with a protein concentration range spanning almost 2 orders of magnitude indicate that unfolding of LC8 dimer is a two-stage process, in which global unfolding is preceded by dissociation to a folded monomer. The nativelike tertiary structure of the monomer suggests a role for the monomer-dimer equilibrium of LC8 in dynein function.

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension.

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores. We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.

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.

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport.

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila.

Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immunofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein.

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.

ZW10 helps recruit dynactin and dynein to the kinetochore.

Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, our results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset.

The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila.

During animal development cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In the fruit fly, Drosophila melanogaster, the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast which undergoes 4 synchronous divisions with incomplete cytokinesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, we show that cytoplasmic dynein is required at two stages of oogenesis. Early in oogenesis, dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The localization of dynein in mitotic cysts suggests spindle orientation is mediated by the microtubule motor cytoplasmic dynein. Later in oogenesis, dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the cytoplasmic dynein motor in cellular determination and differentiation.

Cytoplasmic dynein function is essential in Drosophila melanogaster.

The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.

Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.

The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte.

A cytoplasmic dynein motor in Drosophila: identification and localization during embryogenesis.

We have characterized a cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos. A prominent high molecular weight (HMW) polypeptide (> 400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system.

Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.

A family of dynein genes in Drosophila melanogaster.

We report the identification and initial characterization of seven Drosophila dynein heavy chain genes. Each gene is single copy and maps to a unique genomic location. Sequence analysis of partial clones reveals that each encodes a highly conserved portion of the putative dynein hydrolytic ATP-binding site in dyneins that includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from a Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, that shows extensive amino acid identity to cytoplasmic dynein isoforms from other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes for all seven of the dynein-related sequences hybridize to transcripts that are of the appropriate size, approximately 14 kilobases, to encode the characteristic high molecular weight dynein heavy chain polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes. Transcripts from five of the six remaining genes are also detected in much lesser amounts in tissues other than testes. All but one of the dynein transcripts are expressed at comparable levels in testes suggesting their participation in flagellar axoneme assembly and motility.

A fertility region on the Y chromosome of Drosophila melanogaster encodes a dynein microtubule motor.

A clone encoding a portion of the highly conserved ATP-binding domain of a dynein heavy-chain polypeptide was mapped to a region of the Drosophila melanogaster Y chromosome. Dyneins are large multisubunit enzymes that utilize the hydrolysis of ATP to move along microtubules. They were first identified as the motors that provide the force for flagellar and ciliary bending. Seven different dynein heavy-chain genes have been identified in D. melanogaster by PCR. In the present study, we demonstrate that one of the dynein genes, Dhc-Yh3, is located in Y chromosome region h3, which is contained within kl-5, a locus required for male fertility. The PCR clone derived from Dhc-Yh3 is 85% identical to the corresponding region of the beta heavy chain of sea urchin flagellar dynein but only 53% identical to a cytoplasmic dynein heavy chain from Drosophila. In situ hybridization to Drosophila testes shows Dhc-Yh3 is expressed in wild-type males but not in males missing the kl-5 region. These results are consistent with the hypothesis that the Y chromosome is needed for male fertility because it contains conventional genes that function during spermiogenesis.

Cytoplasmic dynein is localized to kinetochores during mitosis.

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.

Poleward force at the kinetochore in metaphase depends on the number of kinetochore microtubules.

To examine the dependence of poleward force at a kinetochore on the number of kinetochore microtubules (kMTs), we altered the normal balance in the number of microtubules at opposing homologous kinetochores in meiosis I grasshopper spermatocytes at metaphase with a focused laser microbeam. Observations were made with light and electron microscopy. Irradiations that partially damaged one homologous kinetochore caused the bivalent chromosome to shift to a new equilibrium position closer to the pole to which the unirradiated kinetochore was tethered; the greater the dose of irradiation, the farther the chromosome moved. The number of kMTs on the irradiated kinetochore decreased with severity of irradiation, while the number of kMTs on the unirradiated kinetochore remained constant and independent of chromosome-to-pole distance. Assuming a balance of forces on the chromosome at congression equilibrium, our results demonstrate that the net poleward force on a chromosome depends on the number of kMTs and the distance from the pole. In contrast, the velocity of chromosome movement showed little dependence on the number of kMTs. Possible mechanisms which explain the relationship between the poleward force at a kinetochore, the number of kinetochore microtubules, and the lengths of the kinetochore fibers at congression equilibrium include a "traction fiber model" in which poleward force producers are distributed along the length of the kinetochore fibers, or a "kinetochore motor-polar ejection model" in which force producers located at or near the kinetochore pull the chromosomes poleward along the kMTs and against an ejection force that is produced by the polar microtubule array and increases in strength toward the pole.

Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.