Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly perme abilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10(5)-10(6) molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensit ive to actin disruption or to inhibition of protein synthesis. Resealed cel ls were viable for days and retained the capacity to endocytose and to prol iferate. The active domains of large clostridial toxins were introduced int o three different cell lines. The domains were derived from Clostridium dif ficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate sma ll G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylat es actin. After delivery with SLO, all three toxins disrupted the actin cyt oskeleton to cause rounding up of the cells. Glucosylation assays demonstra ted that C-proteins Rho and Ras were retained in the permeabilized cells an d were modified by the respective toxins. Inactivation of these C-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribos ylation of actin by the C. botulinum C2-toxin resulted in enhanced secretio n in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.