MAYORGA Luis Segundo
congresos y reuniones científicas
Perfringolysin O: a useful tool to study exocytosis
POCOGNONI CA, MAYORGA LS, BELMONTE SA.
Potrero de los Funes
Congreso; SAIB; 2011
The acrosome overlies the nucleus of the spermatozoon. It undergoes calcium-dependent exocytosis termed acrosomal exocytosis (AE) required for fertilization. Because spermatozoa are transcriptional and translationally inactive overexpresion or interference RNA cannot be used. To study the role of different proteins in AE we took advantage of perfringolysin O (PFO) properties. This cytolysin assembles to membranes creating 25 nm aqueous pores. We measured pore forming activity of native PFO using eosin or propidium iodide. The latter fluorescent dye was used to analyze the dynamics of this process in real time. Doseresponse curves using different incubation times demonstrated that 10 min incubation at 37ºC were enough for the cytolysin activity. Western blot experiments showed that PFO binds to sperm membranes in a cholesterol-dependent manner. We chose a 25 nM concentration to validate the use of the toxin for exocytosis studies. Calcium or Rab3A added to permeabilized sperm succeeded in inducing exocytosis while calcium-triggered exocytosis were abrogated by loading the permeabilized cells with synaptotagmin VI C2B domain, RIM-Rab3-GTP binding domain or antibodies anti Rab3A, complexin or Epac before adding calcium. Furthermore, we tested PFOC459A where the Cys459 was mutated to Ala and does not require thiol activation. Both toxins were valuable tools for exocytosis research