RESUMO
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
In the normal breast, hepatocyte growth factor (HGF) is primarily expressed by stromal cells, and stimulates in a paracrine manner epithelial cells expressing the HGF receptor (Met). In invasive human breast carcinomas, HGF and Met are frequently overexpressed, possibly establishing an autocrine HGF/Met loop that promotes tumour cell invasion. However, the mechanisms leading to autocrine HGF expression in carcinoma cells are not known. We previously demonstrated a cooperative effect between c-Src and Stat3 in the activation of HGF transcription in mammary carcinoma cells. The present report defines a novel Stat3 consensus site at nt -95 in the HGF promoter that is highly conserved in human and mouse, and is required for c-Src and Stat3 to activate HGF transcription in breast epithelial cells. DNA-protein binding studies demonstrated high affinity binding of a Stat3-containing complex to the nt -95 site. Endogenous Stat3 binding to this region of the HGF promoter in carcinoma cells expressing HGF was demonstrated using a chromatin immunoprecipitation assay. In addition, coexpression of Stat3 and activated c-Src caused increased expression of endogenous HGF mRNA and protein and marked cell scattering in breast epithelial cells. Our results delineate a novel c-Src/Stat3-dependent mechanism that regulates HGF promoter activity, and is linked to transformation of mammary epithelial cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Neoplasias Mamárias Experimentais/genética , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Fator de Transcrição STAT3/fisiologia , Transcrição Gênica , Animais , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Genes Dominantes , Fator de Crescimento de Hepatócito/metabolismo , Luciferases/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
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.