RESUMO
Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.
Assuntos
Aminoácidos/química , Aminoácidos/genética , Aminoacil-tRNA Sintetases/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Código Genético/genética , RNA de Transferência/genética , Aminoácidos/metabolismo , Animais , Dependovirus/genética , Methanosarcina/genética , Camundongos , Estrutura Molecular , RNA de Transferência/metabolismoRESUMO
We investigated the link between cell movement and plasma membrane recycling using a fast-acting, temperature-sensitive mutant of the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells become almost paralysed within minutes at the restrictive temperature. However, they can still sense cyclic-AMP (cAMP) gradients and polymerise actin up-gradient, but form only abortive pseudopodia, which cannot expand. They also relay a cAMP signal normally, suggesting that cAMP is released by a non-exocytic mechanism. To investigate why SecA is required for motility, we examined membrane trafficking in the mutant. Plasma membrane circulation is rapidly inhibited at the restrictive temperature and the cells acquire a prominent vesicle. Organelle-specific markers show that this is an undischarged contractile vacuole, and we found the cells are correspondingly osmo-sensitive. Electron microscopy shows that many smaller vesicles, probably originating from the plasma membrane, also accumulate at the restrictive temperature. Consistent with this, the surface area of mutant cells shrinks. We suggest that SecA mutant cells cannot move at the restrictive temperature because their block in exocytosis results in a net uptake of plasma membrane, reducing its area, and so restricting pseudopodial expansion. This demonstrates the importance of proper surface area regulation in cell movement.
