RESUMO
Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb staining results in a close-grained fluorophore labeling with minimal linkage errors. This allowed us to perform high-quality dSTORM imaging of various targets in mammalian and yeast cells. We expect that this versatile strategy will render many more demanding cellular targets amenable to dSTORM imaging.
Assuntos
Imagem Individual de Molécula/métodos , Anticorpos de Domínio Único , Coloração e Rotulagem/métodos , Células A549 , Células HeLa , Humanos , SchizosaccharomycesRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate, which is approximately 20% during the first year and decreases to less than 6% within five years of the disease. This is due to premature dissemination accompanied by a lack of disease-specific symptoms during the initial stages. Additionally, to date there are no biomarkers for an early prognosis available.A growing number of studies indicate that epithelial to mesenchymal transition (EMT), triggered by WNT-, TGF-ß- and other signaling pathways is crucial for the initiation of the metastatic process in PDAC. Here we show, that BCL9L is up-regulated in PDAC cell lines and patient tissue compared to non-cancer controls. RNAi-induced BCL9L knockdown negatively affected proliferation, migration and invasion of pancreatic cancer cells. On a molecular basis, BCL9L depletion provoked an increment of E-cadherin protein levels, with concomitant increase of ß-catenin retention at the plasma membrane. This is linked to the induction of a strong epithelial phenotype in pancreatic cancer cells upon BCL9L knockdown even in the presence of the EMT-inducer TGF-ß. Finally, xenograft mouse models of pancreatic cancer revealed a highly significant reduction in the number of liver metastases upon BCL9L knockdown. Taken together, our findings underline the key importance of BCL9L for EMT and thus progression and metastasis of pancreatic cancer cells. Direct targeting of this protein might be a valuable approach to effectively antagonize invasion and metastasis of PDAC.
Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Transporte Proteico , Fatores de Transcrição , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima , beta Catenina/genética , beta Catenina/metabolismoRESUMO
In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.
