Subcellular Localization with Green Fluorescent Protein
(. . . make all of your mitochondria and microfilaments glow-in-the-dark!)
 
What is Green Fluorescent Protein?
 
   Green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoriaisa reporter molecule that can be used to monitor gene expression and protein localization.  GFP fluoresces bright green when exposed to ultraviolet light making it easily detectable by microscopy, flourometry, or flow cytometry.  Therefore, when the GFP molecule is ligated to the promoter of your gene of interest, anywhere 'your gene' is expressed fluoresces green when exposed to ultraviolet light.  Some other perks of GFP fluorescence are that the GFP protein is stable, species independent, and can be monitored in fixed cells as well as non invasively in living cells.

So How Can We Make Our Mitochondria and Microfilaments Glow-In-the-Dark?

     Clontech  has developed several optimized GFP variants, such as enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP), with which they have constructed mammalian expression vectors.  Three new Living ColorsTM Vectors-pEGFP-Actin, pEGFP-Tub, and pEYFP-Mito- are now commercially available for visualizing actin and microtubule dynamics in living cells, as well as labeling mitochondria with bright yellow-green fluorescence.
 

pEGFP-Actin

    Actin is the most abundant protein in most eucaryotic cells.  Most animal cells contain a dense network of actin filaments and associated proteins just beneath the plasma membrane, which gives mechanical strength to the surface of the cell and enables the cell to change shape and move.  Actin polymerization and depolymerization drives many of the surface movements of cells.
    pEGFP-Actin Vector encodes human cytoplasmic beta-actin fused to the C-terminus of EGFP. pEGFP-Actin can be transfected into mammalian cells using any standard transfection method.  Fluorescent actin filaments are produced when the expressed fusion protein is directly incorporated into growing actin polymers.  As a result, the EGFP-Actin protein enables one to visualize actin polymerization and depolymerization as it occurs within the cell.
 

 
 
  Figure 1.  This image is from Clontech <http://www.clontech.com> displaying the expression of the EGFP-actin fusion protein in MDCK cells.
 
 
 
pEGFP-Tub
 
    Tubulin is the protein component of microtubules.  Generally, microtubules exist as single filaments within a cell which radiate from a position close to the nucleus outward throughout the cell.  Microtubules function as a system of fibers along which vesicles and other membrane-bounded organelles can travel throughout the cell, and they regulate cell shape, cell movement, and the plane of cell division.  Similar to actin polymerization, microtubule assembly and disassembly plays an important role in many cellular processes.
    pEGFP-Tub Vector encodes human alpha-tubulin fused to the C-terminus of EGFP.  pEGFP-Tub can be transfected into mammalian cells using any standard transfection method.  The fusion protein incorporates directly into microtubules and therefore allows the observation of microtubule polymerization and depolymerization in living cells.
 
 
 
 
 
Figure 2.   This image is from Clontech illustratinf the EGFP-tub protein incorporated into the microtubules of MDCK cells.
 
 

pEGFP-Mito

    The mitochondrion is the organelle of the cell which carries out most cellular oxidations and produces the bulk of the animal cells ATP.  Mitochondria occupy a substantial fraction of the cytoplasm of most eucaryotic cells, although orientation and distribution of mitochondria varies in different types of cells.  In some cells, mitochondria form long moving filaments of chains, whereas in other cells, mitochondria remain fixed in one position where they provide ATP directly to a site of unusually high ATP consumption, for example wedged between adjacent myofibrils in a cardiac muscle cell.
    pEYFP-Mito Vector encodes for subunit VIII of the human cytochrome c oxidase fused to the N-terminus of EYFP.  pEYFP-Mito can be transfected into mammalian cells using any standard tranfection method.  The expressed fusion protein effectively translocates EYFP into the mitochondria causing the mitochondria to fluoresce a bright yellow-green.  Fluorescence can be observed in both living or fixed cells.
 

  

Figure 3.  This figure is from Clontech demonstrating the expression of the EYFP-mito fusion protein targeted to the mitochondrion in CHO-K1 cells.

 
 
Works Cited
 
 

Alberts, Bruce [et al.].  The Cell. 2nd ed. New York:Garland Publishing,Inc.,1989.

1999 March 31.  Clontech Living Colors User Manual: Introduction. <http://www.clontech.com/clontech/Manuals/GFP/Intro.html>  Accessed 1999 Feb 13.

1998 Dec 28.  Clontech pEYFP-Mito Vector Information. <http://www.clontech.com/clontech/Vectors/pEYFP-Mito.html>  Accessed 1999 Feb 13.

1998 Oct 7.  Clontech Living Colors(TM) Subcellular Localization Vectors. <http://www.clontech.com/archive/OCT98UPD/LivingColors.html> Accessed 1999 Feb 11.

1998 Dec 21.  Clontech pEGFP-Tub Vector Information. <http:www.clontech.com/clontech/Vectors/pEGFP-Tub.html>  Accessed 1999 Feb 13.
 
 

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