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A 66-year-old female with a history of sinusitis presented with persistent diplopia and worsening eyelid swelling. Examination revealed bilateral vision loss, cranial nerve palsies, left-sided chemosis, proptosis, and edema. Initial imaging showed sphenoid sinus opacification, bilateral ethmoid disease, lack of filling of bilateral cavernous sinuses, and partial thrombosis of the left superior ophthalmic vein. The sinus findings were evaluated with endoscopic sinus surgery, which was unremarkable. Subsequent orbital imaging suggested the lesions were in keeping with tumor thrombus as opposed to bland thrombus. CT scans revealed a large mass in the right axilla, which was biopsied and confirmed the diagnosis of diffuse large B-cell lymphoma (DLBCL). The patient was treated with anticoagulation, steroids, and chemotherapy resulting in marked improvement. Our report highlights a rare case of tumor thrombus from DLBCL causing bilateral cavernous sinus thrombosis and, to the best of our knowledge, the first documented case of superior ophthalmic vein thrombosis from tumor thrombus.
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The most common causes of vision loss in neurofibromatosis 1 (NF1) patients are sequelae from tumors such as optic pathway glioma, plexiform neurofibroma, or secondary glaucoma. Here we report the case of a six-year-old female with anisometropic amblyopia resulting from an isolated unilateral macro-ophthalmia with a known history of NF1. Our patient progressed to light perception vision in the left eye due to a non-neoplastic cause associated with NF1 with at least two years of documented unilateral macro-ophthalmia without any ophthalmology referral or evaluation. This case aims to highlight the importance of early and deliberate ophthalmologic examination in all patients with neurofibromatosis 1 to assess for appropriate visual development and early intervention.
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Mpox (initially reported as monkeypox virus Clade IIb) ravaged the non-endemic world in 2022 with dermatological and systemic manifestations. The rapid propagation of this virus shed light on the scarcity of information for a virus that was first reported in 1958. We present the first probable neonatal case of mpox with ocular involvement. Ophthalmologists may be the first to diagnose mpox or be a part of the multidisciplinary team required for adequate work-up and treatment to prevent life-long sequelae in the neonatal population.
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Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common cellular pathway. To further understand the in vivo role of one of these human genes (FANCG), we generated a targeted disruption of murine Fancg and bred mice homozygous for the targeted allele. Similar to the phenotype of the previously described Fancc(-/-) and Fanca(-/-) mice, the Fancg(-/-) mice had normal viability and no gross developmental abnormalities. Primary splenic lymphocytes, bone marrow progenitor cells, and murine embryo fibroblasts from the Fancg(-/-) mice demonstrated spontaneous chromosome breakage and increased sensitivity to mitomycin C and, to a lesser extent, ionizing radiation. Fancg(-/-) lymphocytes had a defect in the FA pathway, based on their failure to activate the monoubiquitination of the downstream Fancd2 protein in response to IR. Finally, Fancg(-/-) mice had decreased fertility and abnormal gonadal histology. In conclusion, disruption of the Fancg gene confirms the role of Fancg in the FA pathway. The Fancg(-/-) mouse may be useful as an animal model for future gene therapy and cancer susceptibility studies.
Assuntos
Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Alelos , Animais , Quebra Cromossômica , Modelos Animais de Doenças , Proteína do Grupo de Complementação G da Anemia de Fanconi , Células-Tronco Hematopoéticas/ultraestrutura , Homozigoto , Humanos , Immunoblotting , Infertilidade/genética , Camundongos , Camundongos Knockout , Mitomicina/farmacologia , Mutagênese , Fenótipo , Baço/ultraestruturaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Proteínas de Insetos/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Transdução de Sinais , Animais , Citoplasma/metabolismo , Drosophila/embriologia , Complexo Dinactina , Dineínas/genética , Metáfase , Proteínas Associadas aos Microtúbulos/genética , Mitose/fisiologia , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células-Tronco/metabolismoRESUMO
OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.
Assuntos
Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Adolescente , Alelos , Criança , Pré-Escolar , DNA Complementar/análise , DNA Complementar/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi , Feminino , Heterozigoto , Humanos , Masculino , MutaçãoRESUMO
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.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Dineínas/química , Dineínas/metabolismo , Dobramento de Proteína , Animais , Aspergillus nidulans/enzimologia , Cromatografia em Gel , Dicroísmo Circular , Sequência Conservada , Citoplasma/enzimologia , Dimerização , Drosophila melanogaster/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Guanidina , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Conformação Proteica , Desnaturação Proteica , Espectrometria de Fluorescência , Termodinâmica , UltracentrifugaçãoRESUMO
A plethora of cytoplasmic motors contribute to the directed transport of a wide range of cellular organelles and molecules. Recent studies have advanced our understanding of cargo attachment to motor molecules and the regulation of intracellular transport.
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Cinesinas/metabolismo , Neurônios/metabolismo , Animais , Transporte BiológicoRESUMO
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.
Assuntos
Dineínas/fisiologia , Cinetocoros/fisiologia , Microtúbulos/fisiologia , Espermatócitos/citologia , Animais , Ciclo Celular/fisiologia , Dineínas/análise , Gafanhotos , Cinetocoros/ultraestrutura , Masculino , Metáfase , Microtúbulos/ultraestrutura , Espermatócitos/fisiologia , Espermatócitos/ultraestrutura , Estresse MecânicoRESUMO
The microtubule motor cytoplasmic dynein performs multiple cellular functions; however, the regulation and targeting of the motor to different cargoes is not well understood. A biochemical interaction between the dynein intermediate chain subunit and the p150-Glued component of the dynein regulatory complex, dynactin, has supported the hypothesis that the intermediate chain is a key modulator of dynein attachment to cellular cargoes. In this report, we identify multiple intermediate chain polypeptides that cosediment with the 19S dynein complex and two differentially expressed transcripts derived from the single cytoplasmic dynein intermediate chain (Cdic) gene that differ in the 3' untranslated region sequence. These results support previous observations of multiple Cdic gene products that may contribute to the specialization of dynein function. Most significantly, we provide genetic evidence that the interaction between the dynein intermediate chain and p150-Glued is functionally relevant. We use a genomic Cdic transgene to show that extra copies of the dynein intermediate chain gene act to suppress the rough eye phenotype of the mutant Glued(1), a mutation in the p150-Glued subunit of dynactin. Furthermore, we show that the interaction between the dynein intermediate chain and p150-Glued is dependent on the dosage of the Cdic gene. This result suggests that the dynein intermediate chain may be a limiting component in the assembly of the dynein complex and that the regulation of the interaction between the dynein intermediate chain and dynactin is critical for dynein function.
Assuntos
Citoplasma/metabolismo , Dineínas/genética , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Mecanismo Genético de Compensação de Dose , Drosophila/genética , Complexo Dinactina , Anormalidades do Olho/genética , Feminino , Dosagem de Genes , Técnicas Genéticas , Proteínas Associadas aos Microtúbulos/genética , Mutação , Subunidades Proteicas , Transcrição Gênica , Cromossomo XRESUMO
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.
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Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Ubiquitina-Proteína Ligases , Animais , Encéfalo/citologia , Proteínas de Ciclo Celular/genética , Drosophila/genética , Imunofluorescência , Proteínas de Insetos/genética , Larva/citologia , Microscopia Confocal , Mitose/fisiologia , Mutação , Peptídeo Sintases/genética , Poliploidia , Proteínas Ligases SKP Culina F-BoxRESUMO
Skin necrosis and priapism are unusual complications of warfarin therapy. We report a teenager with warfarin-associated skin necrosis and priapism who was subsequently found to be a compound heterozygote for protein C deficiency and a heterozygote for the factor V Leiden mutation.
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Anticoagulantes/efeitos adversos , Toxidermias/etiologia , Priapismo/induzido quimicamente , Varfarina/efeitos adversos , Adolescente , Toxidermias/complicações , Toxidermias/patologia , Fator V/genética , Humanos , Masculino , Necrose , Priapismo/complicações , Deficiência de Proteína C/complicações , Trombofilia/genéticaRESUMO
Localization of bicoid messenger RNA to the anterior cortex of the developing oocyte is essential for correct anterior-posterior patterning of the Drosophila embryo. It now seems that the Swallow protein functions as an adaptor, bridging bicoid mRNA to dynein, a molecular motor that would transport the complex anteriorly along microtubules.
Assuntos
Proteínas de Drosophila , Dineínas/genética , Dineínas/metabolismo , Proteínas de Homeodomínio/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genética , Animais , Drosophila , Proteínas de Homeodomínio/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismoRESUMO
OBJECTIVES: To evaluate safety, efficacy, and outcome after combination thrombolytic and anticoagulant therapy. STUDY DESIGN: An open nonrandomized clinical protocol with prospective standardized monitoring and data collection. Children with a documented first episode of deep vein thrombosis were treated with urokinase 4400 U/kg load and per hour with unfractionated heparin at 10 U/kg/h. At 48 hours heparin infusions were increased to achieve a therapeutic level for 5 days. Children were given therapeutic warfarin for at least 3 months. Outcome was assessed at 48 hours and > or =1 year with history, physical examination, high-resolution imaging, and Doppler ultrasonography +/- impedance and photo plethysmography. RESULTS: Thirty-two children were treated. There was 1 thrombotic death, 1 nonfatal thrombus progression, and 1 pulmonary embolism. At 48 hours half of the children showed substantial clot lysis, and on follow-up these children had complete resolution and had no symptoms. Three children with poor early clot lysis had recurrent thromboemboli, pulmonary embolism, or both, 2 had limb pain and swelling, and 2 had asymptomatic swelling. Two children had minor bleeding, whereas systemic reactions were common. CONCLUSIONS: Combination therapy in children (urokinase and unfractionated heparin) was safe and efficacious. A prospective, randomized, controlled study in children is needed.
Assuntos
Anticoagulantes/uso terapêutico , Fibrinolíticos/uso terapêutico , Terapia Trombolítica , Trombose Venosa/tratamento farmacológico , Adolescente , Anticoagulantes/efeitos adversos , Criança , Pré-Escolar , Quimioterapia Combinada , Feminino , Fibrinolíticos/efeitos adversos , Seguimentos , Humanos , Lactente , Masculino , Pletismografia/métodos , Estudos Prospectivos , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico por imagem , Embolia Pulmonar/tratamento farmacológico , Terapia Trombolítica/efeitos adversos , Terapia Trombolítica/estatística & dados numéricos , Resultado do Tratamento , Ultrassonografia , Trombose Venosa/sangue , Trombose Venosa/diagnóstico por imagemRESUMO
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.
Assuntos
Axônios/metabolismo , Drosophila/genética , Dineínas/genética , Cinesinas/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Axônios/ultraestrutura , Citoplasma/química , Drosophila/embriologia , Drosophila/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Cinesinas/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fenótipo , Testes de PrecipitinaRESUMO
Several regions of the brain, including the horizontal limb of the diagonal band of Broca (HDB), contain neurons that are responsive to changes in local temperature. These neurons are hypothesized to participate in thermoregulation and sleep-wake control. The HDB contains a large number of gamma-aminobutyric acid (GABA) terminals, and it has many neurons that utilize GABA as a neurotransmitter. Therefore, in this study we characterized the in vitro effects of the GABAA receptor agonist muscimol (0.5, 0.25, 0.1 and 0.0625 microM doses) and the GABAA receptor antagonist bicuculline (3.0 and 1.0 microM doses) on the firing rate and thermosensitivity of HDB neurons. Of the 51 neurons recorded in a submerged slice chamber, 53% were warm sensitive, 45% were temperature insensitive and 2% were cold sensitive. All neurons exposed to bath applied muscimol exhibited reductions in both firing rate and thermosensitivity. Muscimol induced reductions were maintained for at least 20 min after washout. Neurons exposed to bicuculline had no change in firing rate or thermosensitivity. However, after bicuculline washout there were reductions in both firing rate and thermosensitivity. These findings support the hypothesis that GABAA receptor induced inhibition of HDB thermosensitive neurons can modulate both thermoregulation and sleep-wake control.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Feixe Diagonal de Broca/citologia , Neurônios/química , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Animais , Bicuculina/farmacologia , Feixe Diagonal de Broca/anatomia & histologia , Feixe Diagonal de Broca/fisiologia , Eletrofisiologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Muscimol/farmacologia , Área Pré-Óptica/citologia , Ratos , Ratos Sprague-Dawley , Sono/fisiologia , Temperatura , Ácido gama-Aminobutírico/fisiologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Proteínas Contráteis/metabolismo , Drosophila melanogaster/fisiologia , Proteínas dos Microfilamentos/metabolismo , Oogênese/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Adesão Celular , Membrana Celular/fisiologia , Tamanho Celular , Clonagem Molecular , Proteínas Contráteis/química , Proteínas Contráteis/genética , Citoplasma/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Fertilidade , Filaminas , Genes de Insetos/genética , Genes de Insetos/fisiologia , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Mutação/genética , Ovário/anormalidades , Ovário/citologia , Ovário/metabolismo , Ovário/ultraestrutura , Peptídeos/química , Peptídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Centrossomo/metabolismo , Citoplasma/metabolismo , Drosophila melanogaster/embriologia , Dineínas/metabolismo , Mitose , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Núcleo Celular/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Dineínas/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Células Gigantes/citologia , Células Gigantes/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Interfase , Masculino , Microtúbulos/metabolismo , Movimento , Mutação , Membrana Nuclear/metabolismo , Fuso Acromático/metabolismoRESUMO
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.
Assuntos
Citoplasma/enzimologia , Dineínas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Dineínas/química , Epitopos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Paclitaxel/farmacologia , Peptídeos/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
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.