RESUMO
α2ß1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2ß1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2ß1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/genética , Membrana Celular/metabolismo , Integrina alfa2beta1/metabolismo , Cinesinas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Reguladoras de Apoptose , Colágeno/metabolismo , Citoesqueleto/genética , Endocitose/genética , Feminino , Testes Genéticos/métodos , Células HeLa , Humanos , Integrina alfa2beta1/genética , Cinesinas/genética , Microscopia de Fluorescência , Metástase Neoplásica , Ligação Proteica/genética , Transporte Proteico/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (ß1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.