Green Fluorescent Protein:

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

Background

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

Basic Structure

Left: Aequorea victoriaRight: GFP structure

Forming of Fluorophore

Similar in function to GFP

Isolated from the Discosoma coral

Emits a longer wavelength producing a red

fluorescent color

Used to contrast with GFP

Red Fluorescent Protein

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

In vivo real-time technique

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

Purpose

GFP/RFP labelled mouse mammary cancer tissue

Mitosis,in vitro, 5 minute intervals

ApoptosisInduced 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”

Cancer 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 cells

Discovery of Harmful Side Effects

Pre-treatment with cyclophosphamide. A) Pre-treated mouseB) 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

The 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