Focal calcium release during acute unfolded protein response

FELIZIANI C.; HOLSTEIN D.; QUASSOLLO G.; PATON A. W.; PATON J.C.; LECHLEITER J.; BOLLO M.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

The EndoplasmicReticulum (ER) is a multi-functional organelle that plays a criticalrole in a variety of processes, where the ER Ca2+ acts as a keymessenger. Under resting conditions,the luminal Ca2+ concentration reflects a balance between activeuptake by Ca2+-ATPasesand passive efflux pathways of which the translocon can play a prominentrole).The translocon is an aqueous pore, primarily formed by the Sec61a core spanning the ER lipid bilayer, that is blocked by the ribosome on the cytosolic side and by theER chaperone, BiP, on the luminal side. We hypothesize thatduring the acute phase of the UPR (Unfolded ProteinResponse), immediately after accumulation of unfolded protein in the lumen, theCa2+ ER eflux through the translocon is increased. To test this mechanism of action, we performedcytosolic Ca2+ measurements in primary cultures of human astrocytes,expressing the Ca2+ indicator GCaMP6 tethered to the ER membrane,after induction of the UPR by Tunicamycin (Tm,glycosylation inhibitor). We observed focal release of Ca2+(microdomains) in stressed astrocytes that was significantly inhibited by transloconblockers (emetine or anisomycin). In addition, the Tm-induced Ca2+ signal was amplified by pre-treatment eitherwith AB5 cytotoxine, which specifically hydrolyses BiP, or with thetranslocon opener puromycin. The effectof these pharmacological tools was corroborated by co-immunoprecipitations that showedchanges in the interactions either between Sec61α and BiP or Sec61α and theribosomal protein S6. Importantto note that the likehood of obtained Tm-induce local Ca2+ events,increase by using either the slow chelator EGTA-AM orXestospongin C and Ryanodine (InsP3 and Ry Receptorsinhibitors, respectively). Taken together, thesedata, strongly suggest that the chaperone BiP and the ribosome are dissociatedfrom the translocon increasing Ca2+ permeability