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

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) Pre- treated 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), doi: /j x.  Haldar, S., & Chattopadhyay, A. (2009). Green fluorescent protein: a molecular lantern that illuminates the cellular interior. Journal of Biosciences, 34(2), Retrieved from Academic Search Complete database.