Quantitative microscopy reveals dynamics and fate of clustered IRE1α

Abstract

The endoplasmic reticulum (ER) membrane-resident stress sensor inositol-requiring enzyme 1 (IRE1) governs the most evolutionarily conserved branch of the unfolded protein response. Upon sensing an accumulation of unfolded proteins in the ER lumen, IRE1 activates its cytoplasmic kinase and ribonuclease domains to transduce the signal. IRE1 activity correlates with its assembly into large clusters, yet the biophysical characteristics of IRE1 clusters remain poorly characterized. We combined superresolution microscopy, single-particle tracking, fluorescence recovery, and photoconversion to examine IRE1 clustering quantitatively in living human and mouse cells. Our results revealed that: 1) In contrast to qualitative impressions gleaned from microscopic images, IRE1 clusters comprise only a small fraction (∼5%) of the total IRE1 in the cell; 2) IRE1 clusters have complex topologies that display features of higher-order organization; 3) IRE1 clusters contain a diffusionally constrained core, indicating that they are not phase-separated liquid condensates; 4) IRE1 molecules in clusters remain diffusionally accessible to the free pool of IRE1 molecules in the general ER network; 5) when IRE1 clusters disappear at later time points of ER stress as IRE1 signaling attenuates, their constituent molecules are released back into the ER network and not degraded; 6) IRE1 cluster assembly and disassembly are mechanistically distinct; and 7) IRE1 clusters’ mobility is nearly independent of cluster size. Taken together, these insights define the clusters as dynamic assemblies with unique properties. The analysis tools developed for this study will be widely applicable to investigations of clustering behaviors in other signaling proteins.

New insights on human IRE1 tetramer structures based on molecular modeling

“…The calculated PCA and NMA dominant large scale motions in all investigated systems provide a molecular level validation of IRE1 RNase activation and IRE1 clustering as a dynamic process 3 , 6 . In accordance with experimental studies 4 the current simulations corroborate the dynamics of the IRE1 tetramer, in contrast with the static picture provided by crystal structures. The IRE1 tetramer dynamics furthermore provide insight into the mechanistic assembly and disassembly of the even larger oligomers repeatedly observed in cells, as being a dynamic process rather than locked arrangements of IRE1 oligomers.…”

“…The cytosolic domain, in turn, is composed of two catalytic domains: a kinase and an RNase domain 2 . Several experimental studies such as X-ray crystallography 3 , live cell microscopy 4 , kinetic studies of RNA cleavage 5 , Western blots, microscopy and image analysis 6 , and in vitro cleavage and splicing assays 7 have provide mechanistic insights on IRE1 activation 3 , 6 , 8 : IRE1 forms dimers, tetramers, and larger order oligomers. Upon IRE1 activation, the protein dimerizes into a face-to-face dimer (Supplementary Fig.…”

The Structure, Activation and Signaling of IRE1 and Its Role in Determining Cell Fate

“…When the ER stress attenuates, disassembly occurs, and the constituent molecules of IRE1 clusters are released back into the ER network in a distinct manner than that of cluster assemble. Whilst the actual role of IRE1 clusters remains elusive, it is speculated that they act as temporary storage compartments for hyperactivated IRE1 or excessive IRE1 molecules, behaving as some sort of buffering mechanism against overactivation of IRE1; alternatively, they may target different mRNA substrates than single IRE1 subunits, with the former ones selectively inducing XBP1 mRNA processing and the latter―RIDD [ 9 , 80 ]. This is in accordance with the recent finding that IRE1 clustering and RNase activity are in fact functionally independent of each other.…”

“…IRE1 consists of two domains: The N-terminal, ER luminal domain, which senses unfolded proteins, and C-terminal cytoplasmic region, which initiates UPR via serine/threonine kinase and endoribonuclease (RNase) domains [ 8 ]. The latter becomes activated via conformational change, autophosphorylation, and higher-order assembly [ 9 ]. Like PERK, IRE1 is capable of inducing cell fate by two distinct routes: The first by induction of adaptive cellular response by unconventional splicing of the transcription factor X-box binding protein 1 (XBP1) mRNA or Regulated IRE1-Dependent Decay (RIDD) posttranscriptional modifications, and the second by activation of the pro-apoptotic c-Jun N-terminal kinase (JNK), which takes place under prolonged or severe stress conditions [ 10 , 11 ].…”

Dynamics and clustering of IRE1α during ER stress

“…The UPR relies on three transmembrane signaling proteins, IRE1, PERK, and ATF6, each of which senses protein misfolding in the ER through a luminal domain and transmits to the cytoplasm and nucleus in order to induce the expression of distinct subsets of ER chaperones and other factors that ameliorate ER stress (3). In PNAS, Belyy et al (4) employ advanced quantitative imaging strategies to reveal the intricacies of the regulation of IRE1 during ER stress in the cellular context.…”