In vivo real-time imaging of nuclear-cytoplasmic dynamics Jason Gregorin

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Transcript of In vivo real-time imaging of nuclear-cytoplasmic dynamics Jason Gregorin

Green Fluorescent Protein:

Green Fluorescent Protein:In vivo real-time imaging of nuclear-cytoplasmic dynamicsJason GregorinDiscovered in 1960s by Osamu Shimomura, Martin Chalfie, and Roger Tsien.Nobel Prize awarded in 2008Gene for GFP successfully cloned in the early 90sComes from the jellyfish Aequorea victoriaBackground238 amino acids11 strands forming a barrel1 central alpha helixFluorophore is p-hydroxybenzylideneimidozolidinoneFluorophore forms as post-translational modification from internal cyclisation and oxidationResidues involved: Ser65-Tyr66-Gly67

Basic StructureLeft: Aequorea victoriaRight: GFP structure

Forming of Fluorophore

Similar in function to GFPIsolated from the Discosoma coralEmits a longer wavelength producing a red fluorescent colorUsed to contrast with GFPRed Fluorescent ProteinNuclear-cytoplasmic dynamicsRequires the use of fluorescent labeling of the nucleus and cytoplasmGFP is linked with the histone protein H2BThis labels the nucleus greenRFP is expressed normally in cytoplasmVarious microscopy methods usedIn vitro/In vivoSkin fold chambers, exteriorization of organs, subcutaneous windows, non-invasive whole body imaging

In vivo real-time techniqueFurther understanding of nuclear and cytoplasmic ratios, shape changes, cell cycle in living cellsFurther knowledge of cancer mechanisms on the inter and intra cell level

Purpose

GFP/RFP labelled mouse mammary cancer tissueMitosis,in vitro, 5 minute intervals

ApoptosisInduced with staurosporineA= no treatmentB-G 2 hour intervals

GFP/RFP cancer cells introduced to miceDeformation of cancer cells leads to arrest in capillariesAllowed for determination of cancer flow rateCancer Circulation

Genetic Exchange in Cancer Cells

This demonstrates genetic exchange in cancer cells (human pancreas cells used). Possibility for better understanding the mechanisms for creating highly metastatic cellsDiscovery of Harmful Side Effects

Pre-treatment with cyclophosphamide. A) Pre-treated mouseB) Normal, non-treated mouseThough cyclophosphamide is typically used as an effective cancer drug, use at improper times may have side effects allowing easier spreadLeft- Mouse mammary tumorRight- 12 hours after treating with doxorubicinThe Future of Testing for the Cure

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.1600-0463.2008.01036.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.References