You are working with two lines of adipocyte cells that you grow by cell culture

Question

You are working with two lines of adipocyte cells that you grow by cell culture (like in Bic220 lab) (A) a secretory mutant cell line (called NOGO) that does not transport GLUT4 to the plasma membrane upon insulin stimulation, and (B) the wildtype cell line that does exocytose GLUT4 to the surface of the cell upon treatment with insulin. Figure 1: GLUT4 transport to the plasma membrane is disrupted in NOGO mutant cells. Micrographs of the cellular localization of GLUT4-GFP (green) after addition of 10mM insulin to (a) NOGO mutant cells or (b) wildtype cells Scale bar represents 5um. You then conduct the following experiments to narrow down which step in the secretory pathway is defective in NOGO cells You use western blotting with phosphorylation-specific antibodies (those that only recognize phosphorylated forms of proteins) to determine the phosphorylation state of Akt and PKCA, proteins involved in the insulin signaling pathway. You find both proteins are phosphorylated in NOGO mutant cells at the same levels observed in wildtype cells Analysis using immuno-electron microscopy (similar to immunofluorescence) staining specific for GLUT4 reveals that the GLUT4 protein is enclosed within a membrane compartment in the NOGO mutant cells, identical to what you observe in wildtype cells You use fluorescent dyes to stain the ER, Golgi, lysosomes, and endosomes, and you observe that GLUT4-GFP containing compartments do not significantly co-ocalize with any of these membrane compartments in the NOGO mutants. Again, this result is consistent with what you observe in wildtype cells You observe that an unrelated integral membrane protein (IRS1) is properly inserted into the plasma membrane of NOGO mutant adipocytes, as it normally would be in wildtype cells.

Explanation / Answer

The defect appears to be recessive as all the fours pathway steps are intact i.e. like the wild type despite of the genetic mutation and change. Regulation of serine/threonine protein kinase Akt/PKB is the key step and alternation in the activity of this protein is most likely responsible for the observed defect in the NOGO mutant. Down regulating the activity of the serine/threonine protein kinase Akt/PKB by knocking out its gene will establish the claim.

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