Looking at the high-resolution structure of IRE1 inside cells using sub-tomogram averaging
EMSL Project ID
50747
Abstract
The primary objective of our proposal is to image IRE1 foci using high-resolution cellular electron cryo-tomography (ECT). IRE1 is a member of the unfolded protein response (UPR). The UPR is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. Therefore, the UPR plays a role in both health and diseases. Upon sensing unfolded protein accumulation during ER stress, the UPR either reestablishes homeostasis or initiates apoptosis when stress cannot be resolved. Both the luminal and cytoplasmic domains of IRE1 have been crystalized as dimer or higher-order oligomers in vitro. When tagged with GFP in live cells, IRE1 forms large dynamic oligomers termed foci upon UPR induction and the dynamics of foci formation roughly correlate to IRE1 activation time course. Understanding how these clusters form in the ER membrane and activate downstream signaling effectors is a major open question, which we can now address with new available imaging technology for cellular ECT. Our aim is to visualize in situ the structural basis of activation of the IRE1 stress sensor by solving its high-resolution structure inside the cell using a combination of cryogenic correlated light and electron microscopy (cryo-CLEM) and sub-tomogram averaging. We aim to generate a map of oligomerized IRE1 inside the cell at 8 Å resolution to resolve alpha helices and confidently dock the already solved crystal structures.
Project Details
Start Date
2019-04-15
End Date
2019-12-05
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members