Polyarginine peptides, a powerful tool for delivering proteins to living cells

PAVAROTTI MA, MAYORGA LS

Potrero de los Funes

Congreso; SAIB; 2011

SAIB

Sperm must be capacitated to undergo acrosomal reaction (AR). In capacitated sperm permeabilized with SLO, we have demonstrated that SNAREs are required for AR and that these proteins are assembled in neurotoxin resistant complexes. What is the condition of SNAREs before capacitation is unknown. For this, the effect of a neurotoxin needed to be assessed during capacitation in living cells. As previously shown, proteins containing a polyarginine peptide can cross membranes. To generate a permeant neurotoxin, the light chain of tetanus toxin was crosslinked with a polyarginine peptide. The activity of this toxin was evaluated by a functional assay and immunofluorescence techniques. We corroborated that, in resting non-permeabilized capacitated sperm, SNAREs were locked in cis complexes insensitive to the neurotoxin. Upon stimulation, SNAREs became sensible to the toxin. However, when we added the permeant neurotoxin to non-capacitated sperm, SNAREs were digested, indicating that they were not protected in stable cis complexes. This observation suggested that capacitation was important for stabilizing the complexes. When sperm were incubated in the absence of albumin, a key element for capacitation in vitro, SNARE remained toxin sensitive. Our results indicate that capacitation is important for the assembly of stable SNARE complexes required for acrosomal exocytosis.