RESUMO
Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.
Assuntos
Perfilação da Expressão Gênica/métodos , Imagem Óptica/métodos , Proteína Wnt1/metabolismo , Aciltransferases/metabolismo , Animais , Embrião de Galinha , Galinhas/metabolismo , Ectoderma/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoilação , Proteínas de Membrana/metabolismo , Camundongos , Crista Neural/metabolismo , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Proteína Wnt1/fisiologia , Proteína Wnt3A/metabolismo , Peixe-Zebra/metabolismoRESUMO
TRIB2, a member of the human Tribbles pseudokinase family, functions as a molecular scaffold in diverse signaling pathways. In this issue of Structure, Jamieson et al. report the first high-resolution structure of TRIB2 bound to a nanobody that offers insights into its "active-like" state.