RESUMO
Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.
Assuntos
Mutação/genética , Sistema Nervoso/embriologia , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Epistasia Genética , Feminino , Testes Genéticos , Masculino , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Dados de Sequência Molecular , Atividade Motora , Placa Motora/patologia , Neurônios Motores/patologia , Sistema Nervoso/patologia , Junção Neuromuscular/patologia , Fenótipo , Polimerização , Multimerização Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/metabolismo , Sinapses/patologia , Tubulina (Proteína)/químicaRESUMO
RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by â¼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from â¼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.
Assuntos
Fármacos Anti-HIV/farmacologia , DNA Polimerase Dirigida por RNA/química , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/química , Ribonuclease H/fisiologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/enzimologia , Sequência de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Pegada de DNA , Transcriptase Reversa do HIV/antagonistas & inibidores , Hidrazonas/síntese química , Hidrazonas/farmacologia , Indicadores e Reagentes , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Magnésio/farmacologia , Manganês/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/enzimologia , Naftiridinas/síntese química , Naftiridinas/farmacologia , Plasmídeos/genéticaRESUMO
Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.