A song made famous by Frank Sinatra, That’s Life, talks about some important facts/lessons about life that also connect to science.
The song talks says life is all about the highs and lows about life. Science, as I found out, is also all about the highs… and the lows. Things do not always go according to plan. The deletion that you wanted to make, does not delete. Sometimes, you may try a mutagenesis reaction multiple times with no luck, but that is okay. Science takes time, and eventually, it will come back around and you may get a result, so don’t quit.
Over the summer, I attempted to delete Sec24D from SW1353 (a chrondrosarcoma cell line), and SAOS-2 (a osteosarcoma cell line) with no luck. Additionally, I’ve attempted several mutagenesis reactions with minimal successes. These were the lows. However, I did have some highs. Other projects and experiments went smoothly, and I am happy for all experiences.
I am also thankful for my successes, and “failures”. There is more to say about what I did over the summer, but I would like to save it for my poster presentation that will be held on July 27, 2018, and future blogs.
As a teaser for the poster presentation, I will post a draft (science is all about sharing current knowledge about a specific topic, so please understand this is subject to change) of my project’s abstract.
What role does SEC24D O-GlcNAcylation play in collagen secretion?
The COPII complex mediates traffic from the endoplasmic reticulum (ER) with five major components: SAR1, SEC13, SEC23, SEC24, and SEC31. Posttranslational modification plays an important role in the regulation of the COPII complex. Our lab and others have found that O-linked β-N-acetylglucosamine (O-GlcNAc), a single sugar modification added to serine and threonine of intracellular proteins, decorates many COPII components. I mainly focused on O-GlcNAcylation of SEC24D, a subunit of the COPII complex, because in humans, SEC24D mutations cause a subtype of osteogenesis imperfecta, a collagen trafficking disorder. I wanted to answer the question, “What role does SEC24D O-GlcNAcylation play in collagen secretion?” I hypothesized that SEC 24D O-GlcNAcylation is necessary for normal collagen secretion. To test this hypothesis, I am deleting SEC24D in osteosarcoma cells using CRISPR-CAS9 and creating unglycosylatable SEC24D mutants to reintroduce into the SEC 24D deleted cell line. After reintroducing the mutants, I will look at collagen secretion using immunofluorescence microscopy (IF) to determine the role of SEC24D O-GlcNAcylation in collagen secretion. We expect the IF to show more collagen in the ER of the mutants than the wild type, showing SEC24D O-GlcNAcylation is important in collagen secretion.