Mechanisms of homeostatic re-adjustment in the Endoplasmic Reticulum

Il 16/09/2016 ore 14.30 - 15.30

Sala Conferenze CNR, Via P. Castellino,111 80131 Napoli

Dr. Eelco van Anken from San Raffaele Scientific Institute in Milan will give a seminar on homeostatic re-adjustment in the Endoplasmic Reticulum.

The Unfolded Protein Response (UPR) is a pro-survival homeostatic response that enables cells to cope with endoplasmic reticulum (ER) stress. Yet, if ER homeostasis cannot be restored, the UPR transducers IRE1 and PERK turn pro-apoptotic. Inducible overexpression of the IgM heavy chain (µs) provokes a full-blown UPR in HeLa cells but no loss of cell viability, except upon ablation of the UPR transducer ATF6. Further ablation of IRE1 worsened cell fate, while reconstitution with GFP-tagged IRE1 expressed at near-endogenous levels restored viability. IRE1 signals ER-stress through the processing of XBP1 mRNA, such that it encodes a transcription factor that drives expression of ER components. Sustained IRE1 activation, however, leads to its RNase activity to be unleashed towards other mRNAs as well.

We found that IRE1-GFP formed clusters during acute µs–driven ER stress, but it relocalized diffusely throughout the ER under chronic µs–driven ER stress. IRE1-GFP clusters persisted instead when ER homeostatic readjustment was unsuccessful, e.g. due to ATF6 ablation. Through advanced microscopy techniques we obtained evidence that life/death decisions are taken through concentration of otherwise pro-survival IRE1 dimers into pro-apoptotic clusters, where IRE1 dimers are at a concentration that sustains cross-autophosphorylation and ensuing pro-apoptotic enhanced RNase activity. Interestingly, upon removal of the CH1 domain from µs no classical UPR is triggered. Instead, µ?CH1 is retained in endoplasmic reticulum (ER) derived dilated structures, called Russell Bodies (RBs).

To appreciate this phenomenon better, we developed a Halo-tag based Correlative Light and Electron Microscopy (CLEM) approach and showed that RBs are in communication with ER cisternae through a sphincter-like structure. By use of pulse-chase CLEM, Dr van Anken's lab  followed RB formation with time and found that early after the onset of μΔCH1-Halo synthesis, it is diffusely dispersed throughout the ER, whereas later it exclusively relocates to RBs, leaving the remainder of the ER void of μΔCH1-Halo, which may explain why the UPR is not activated by µ?CH1.

Organizzato da:
IBP, Cnr

Referente organizzativo:
Alberto Luini
CNR - Istituto di biochimica delle proteine
Via P. Castellino, 111 80131 Napoili
a.luini@ibp.cnr.it
081/6132535

Modalità di accesso: ingresso libero