RESUMO
MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-ß, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Proteínas dos Microfilamentos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias Gástricas/genética , Proteína cdc42 de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/patologia , Proteína 7 com Repetições F-Box-WD/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica , Transdução de Sinais , Neoplasias Gástricas/patologia , Ativação Transcricional/genética , Peixe-ZebraRESUMO
The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single proteins in planar supported biomimetic membranes. This chapter describes, in detail, the construction of a simple single molecule microscope, which includes: (1) a parts list, (2) temperature control, (3) an alignment procedure, (4) a calibration procedure, and (5) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported bilayers on hydrophilically treated borosilicate and quartz. These include (1) POPC bilayers, (2) POPC/PEG-PE cushioned bilayers, (3) POPC/PEG-PE cushioned bilayers on BSA passivated substrates, and (4) a cushioned biomimetic membrane of the endoplasmic reticulum (ER). A procedure for the detergent mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. A procedure for the detergent-free incorporation of cytochrome P450 reductase (CPR) and cytochrome P450 enzymes (P450) into an ER biomimetic is also described. The final experimental section of this chapter details different procedures for data analysis including (1) quantitative analysis of mean squared displacements from individually tracked proteins, (2) gamma distribution analysis of diffusion coefficients from a small ensemble of individually tracked proteins, (3) average mean squared displacement analysis, (4) Gaussian analysis of step-size distributions, (5) Arrhenius analysis of temperature dependent data, (6) the determination of equilibrium constants from a step-size distribution, and (7) a perspective associated with the interpretation of single particle tracking data.
Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Biomimética/métodos , Detergentes/metabolismo , Difusão , Retículo Endoplasmático/metabolismo , Estudos de Avaliação como Assunto , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Polietilenoglicóis/metabolismo , Receptores de Serotonina/metabolismo , Imagem Individual de Molécula/métodosRESUMO
The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single transmembrane proteins in planar supported membrane biomimetics. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) an alignment procedure, (3) a calibration procedure, and (4) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported POPC bilayers on hydrophilically treated borosilicate and quartz, POPC/PEG-PE cushioned bilayers on hydrophilically treated surfaces, and POPC/PEG-PE cushioned bilayers on BSA passivated substrates. The procedure for the detergent-mediated incorporation of the transmembrane protein 5HT(3A) (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. The final experimental section of this chapter details different procedures for data analysis including (1) a quantitative analysis of mean displacements from individually tracked particles, (2) a Gaussian analysis of step-size distributions, (3) the Gaussian analysis of diffusion coefficients from ensembles of transmembrane proteins, and (4) a perspective associated with the interpretation of single-particle tracking data.