Myofibrillogenesis visualized in living embryonic cardiomyocytes

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, α-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing α-actinin. The GFP–α-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of α-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils—i.e., the precursors of mature myofibrils.

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 permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.