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In vivo real-time imaging of nuclear-cytoplasmic dynamics Jason Gregorin Discovered in 1960s by Osamu Shimomura, Martin Chalfie, and Roger Tsien. Nobel Prize awarded in 2008 Gene for GFP successfully cloned in the early 90s Comes from the jellyfish Aequorea victoria 238 amino acids 11 β strands forming a β barrel 1 central alpha helix Fluorophore is p-hydroxybenzylideneimidozolidinone Fluorophore forms as post-translational modification from internal cyclisation and oxidation Residues involved: Ser65-Tyr66-Gly67 Similar in function to GFP Isolated from the Discosoma coral Emits a longer wavelength producing a red fluorescent color Used to contrast with GFP Nuclear-cytoplasmic dynamics ◦ Requires the use of fluorescent labeling of the nucleus and cytoplasm ◦ GFP is linked with the histone protein H2B This labels the nucleus green ◦ RFP is expressed normally in cytoplasm ◦ Various microscopy methods used ◦ In vitro/In vivo Skin fold chambers, exteriorization of organs, subcutaneous windows, non-invasive whole body imaging Further understanding of nuclear and cytoplasmic ratios, shape changes, cell cycle in living cells Further knowledge of cancer mechanisms on the inter and intra cell level GFP/RFP labelled mouse mammary cancer tissue Mitosis,in vitro, 5 minute intervals Apoptosis Induced with staurosporine A= no treatment B-G 2 hour intervals GFP/RFP cancer cells introduced to mice Deformation of cancer cells leads to arrest in capillaries Allowed for determination of cancer “flow rate” This demonstrates genetic exchange in cancer cells (human pancreas cells used). Possibility for better understanding the mechanisms for creating highly metastatic cells Pre-treatment with cyclophosphamide. A) Pretreated mouse B) Normal, non-treated mouse Though cyclophosphamide is typically used as an effective cancer drug, use at improper times may have side effects allowing easier spread Left- Mouse mammary tumor Right- 12 hours after treating with doxorubicin HOFFMAN, R. (2008). In vivo real-time imaging of nuclear-cytoplasmic dynamics of dormancy, proliferation and death of cancer cells. APMIS, 116(7/8), 716-729. doi:10.1111/j.16000463.2008.01036.x. Haldar, S., & Chattopadhyay, A. (2009). Green fluorescent protein: a molecular lantern that illuminates the cellular interior. Journal of Biosciences, 34(2), 169-172. Retrieved from Academic Search Complete database.
