INVESTIGADORES
MAYORGA Luis Segundo
congresos y reuniones científicas
Título:
Perfringolysin O: a useful tool to study exocytosis
Autor/es:
POCOGNONI CA, MAYORGA LS, BELMONTE SA.
Lugar:
Potrero de los Funes
Reunión:
Congreso; SAIB; 2011
Institución organizadora:
SAIB
Resumen:
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