So How Can We Make Our Mitochondria and Microfilaments Glow-In-the-Dark?
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
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.
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
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.
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.
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.
Accessed 1999 Feb 13.
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