RESUMO
Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist, crippling mitochondrial mutations are exceedingly rare. Recent studies have demonstrated strong purifying selection in the mouse female germline. However, the mechanisms underlying positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila melanogaster oogenesis, finding that mtDNA replication commenced before oocyte determination during the late germarium stage and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA allele, mt:CoI(T300I), which resulted in reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of the mt:CoI(T300I) allele in heteroplasmic flies was decreased, both during oogenesis and over multiple generations, at the restrictive temperature. Furthermore, we determined that selection against mt:CoI(T300I) overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for the selective amplification of wild-type mtDNA, which may be evolutionarily conserved to limit the transmission of deleterious mutations.
Assuntos
Replicação do DNA/fisiologia , DNA Mitocondrial/genética , DNA Mitocondrial/fisiologia , Drosophila melanogaster/fisiologia , Evolução Molecular , Oogênese/fisiologia , Seleção Genética/fisiologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona , Drosophila melanogaster/genética , Feminino , Genes Letais/genética , Modelos Genéticos , Oogênese/genética , Seleção Genética/genética , Temperatura , Fatores de TempoRESUMO
Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-κB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFκB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Hematopoese/genética , Vesículas Transportadoras/genética , Proteínas de Transporte Vesicular/fisiologia , Animais , Animais Geneticamente Modificados , Autofagia/genética , Proteína Beclina-1 , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Epistasia Genética/fisiologia , Hematopoese/fisiologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Transporte Proteico/genética , Via Secretória/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoAssuntos
Morte Celular/fisiologia , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Humanos , Membranas Mitocondriais/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34(-/-) animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.
