Assuntos
Pneumopatias/genética , Linfopenia/genética , Doenças da Imunodeficiência Primária/genética , Proteínas rac de Ligação ao GTP/genética , Adulto , Linfócitos B/imunologia , Progressão da Doença , Proteínas Ativadoras de GTPase/metabolismo , Mutação com Ganho de Função , Doença Enxerto-Hospedeiro/tratamento farmacológico , Guanosina Trifosfato/metabolismo , Transplante de Células-Tronco Hematopoéticas , Heterozigoto , Humanos , Memória Imunológica/imunologia , Imunossupressores/uso terapêutico , Lactente , Pneumopatias/imunologia , Pneumopatias/fisiopatologia , Pneumopatias/cirurgia , Transplante de Pulmão , Linfopenia/imunologia , Masculino , Simulação de Acoplamento Molecular , Neutrófilos , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/terapia , Recidiva , Infecções Respiratórias/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/ultraestrutura , Proteína RAC2 de Ligação ao GTPRESUMO
Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived from Avena sativa (oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. The LOV domain incorporates a flavin as the photon-absorbing cofactor and can be activated by light in a reversible and repeatable fashion. In cultured cells, global illumination with blue light rapidly activated Rac and led to cell spreading and membrane ruffling. Localized and pulsed illumination generated a gradient of Rac activity and induced directional migration. In this chapter, we will describe the techniques in detail and present some examples of applications of using photoactivatable Rac (PA-Rac) in living cells.
Assuntos
Técnicas Citológicas/métodos , Microscopia de Fluorescência/métodos , Optogenética/métodos , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/ultraestrutura , Animais , Avena/química , Células HeLa , Humanos , Luz , Iluminação , Camundongos , Microscopia de Fluorescência/instrumentação , Fototropinas/análise , Fototropinas/genética , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Hemocyte development in the Drosophila embryo is a genetic model to study blood cell differentiation, cell migration and phagocytosis. Macrophages, which make up the majority of embryonic hemocytes, migrate extensively as individual cells on basement membrane-covered surfaces. The molecular mechanisms that contribute to this migration process are currently not well understood. We report the generation, by P element replacement, of two Gal4 lines that drive expression of UAS-controlled target genes during early (gcm-Gal4) or late (Coll-Gal4) stages of macrophage migration. gcm-Gal4 is used for live imaging analysis showing that macrophages extend large, dynamic lamellipodia as their main protrusions as well as filopodia. We use both Gal4 lines to express dominantnegative and constitutively active isoforms of the Rho GTPases Rac1, Cdc42, Rho1 and RhoL in macrophages, and complement these experiments by analyzing embryos mutant for Rho GTPases. Our findings suggest that Rac1 and Rac2 act redundantly in controlling migration and lamellipodia formation in Drosophila macrophages, and that the third Drosophila Rac gene, Mtl, makes no significant contribution to macrophage migration. Cdc42 appears not to be required within macrophages but in other tissues of the embryo to guide macrophages to the ventral trunk region. No evidence was found for a requirement of Rho1 or RhoL in macrophage migration. Finally, to estimate the number of genes whose zygotic expression is required for macrophage migration we analyzed 208 chromosomal deletions that cover most of the Drosophila genome. We find eight deletions that cause defects in macrophage migration suggesting the existence of approximately ten zygotic genes essential for macrophage migration.
