Application of PFTs as Tools in Research

The advantages of PFTs are manifold. The effects of digitonin or saponin are normally difficult to predict, control or monitor. In contrast, the actions of PFTs are well characterized and easy to control. The toxins are easy to handle. The two agents currently used by most groups, alpha-toxin and streptolysin O, are stable and can be stored in lyophilized form tor years. No special procedures are required to 

Pores formed by PFTs are usually fairly well defined in size, so that judicious choice of toxin and of experimental conditions will generally enable one to produce stable membrane lesions ranging from approximately 8 A to 300 A in functional diameter. It becomes possible to manipulate the intracellular ionic milieu, to introduce small molecules such as nucleotides into the cells, or to apply large molecules such as antibodies to the cytoplasm. Since pure preparations of PFT are devoid of enzymatic (proteolytic) activity, they cause no direct alterations of cell constituents. In the following, simple strategies for utilization of PFTs will be outlined. 

When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 ng/ml. Most cells will become perme-abilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP. One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi ef al., 1989)). ATP depletion will always be found in cells that have been permeabilized. 

When very large pores are needed, streptolysin O is presently the most convenient toxin to apply. Streptolysin O pores are so large that cytoplasmic proteins rapidly egress from the cells however, intracellular organelles and the cytoskeleton remain, so that key cellular processes such as vesicle trafficking and movement of peptides amongst cellular compartments can be analyzed, e.g. with the use of antibodies. 

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