The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

Description Keeping tabs on ER stress In the endoplasmic reticulum (ER), oligomerization is a central step in modulating signaling during the unfolded protein response. Tran et al. visualized the active, oligomeric form of the ER stress sensor IRE1α residing in an ER subdomain in stressed human cells. This ER region was composed of narrow (diameter about 28 nanometers) anastomosing ER tubes with a high degree of branching and was continuous with surrounding ER structures. Within the lumen of these narrow IRE1α subdomain tubes, regular protein densities were observed, consistent with ordered oligomers of the lumenal stress–sensing domain of IRE1α. This arrangement may suggest a positive feedback mechanism involved in signaling by IRE1α. —SMH Activated forms of the unfolded protein response sensor IRE1α forms helical filaments in specialized subdomains of the endoplasmic reticulum The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno–electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.

M ost secreted and transmembrane proteins mature in the endoplasmic reticulum (ER), which is a specialized protein-folding compartment (1). To ensure proper folding, a network of quality-control pathways, termed the unfolded protein response (UPR), monitors the ER's protein-folding status and adjusts its capacity (2). The three metazoan ER-resident UPR sensors [IRE1 (3,4), PERK, and ATF6] activate in response to ER stress. UPR activation triggers corrective measures or, if the defect cannot be corrected, apoptosis (5). IRE1 activation initially provides cytoprotective outputs but then attenuates even under conditions of unmitigated ER stress (6). IRE1 attenuation predisposes the cell to apoptosis as a consequence of unopposed PERK signaling (7,8). Maladaptive UPR signaling is a hallmark of many diseases, including cancer, diabetes, and neurodegeneration (9).
Mammalian IRE1 has two paralogs (10), IRE1a and IRE1b. IRE1a is the major isoform expressed in most cell types. It is an ERtransmembrane protein bearing an ER stresssensing domain on the ER lumenal side and kinase and ribonuclease (RNase) effector domains on the cytosolic side (3,4,11). IRE1a's lumenal domain (LD) is bound by the ERlumenal chaperone BiP, which dissociates upon ER stress (12). IRE1a-LD then binds directly to accumulated unfolded proteins, which triggers its oligomerization (13,14). Oligomerization of the LD drives the juxtaposition of IRE1a's cytosolic kinase and RNase domains, which activate after trans-autophosphorylation (15). IRE1a's activated RNase domain initiates the nonconventional splicing of its substrate XBP1 mRNA (16). Spliced XBP1 mRNA encodes the transcription factor XBP1s, which up-regulates hundreds of genes to restore ER homeostasis. A second consequence of IRE1a RNase activation, termed regulated IRE1dependent mRNA decay (RIDD), is the selective degradation of mRNAs that reduces ER protein-folding burden and synergizes with XBP1s to alleviate ER stress (17).
Upon UPR induction, a fraction of IRE1 molecules oligomerize into large foci that are visible by fluorescence microscopy, which is consistent with IRE1's LD and cytosolic kinase and RNase domain dimers and higher-order oligomers that are observed in solution and crystal structures (18)(19)(20)(21)(22). Extensive mutagenesis of the interfaces predicted from the structural analyses validates their functional importance (14,18,19), and both oligomerization of the kinase and RNase domains in vitro and foci formation correlate with high enzymatic activity (22). IRE1a foci have complex morphology and dynamic behaviors (23,24) and comprise two distinct populations of IRE1a (24). A small fraction of clustered molecules rapidly exchanges with the pool of dispersed IRE1a in the ER membrane, whereas most are diffusionally constrained in the cluster core until eventual foci dissolution. The molecular principles of IRE1a's different assembly states remain a mystery.

IRE1a oligomers localize to specialized ER subdomains
We applied cryogenic correlated light and electron microscopy combined with electron cryo-tomography (cryo-CLEM-ET) to determine the ultrastructure of IRE1a foci in mammalian cells (N = 4; independent replicates). We used characterized stable cell lines that inducibly expressed fluorescently tagged human IRE1a (14,24), which faithfully recapitulated IRE1a function. We grew cells directly on electron microscopy grids, induced IRE1a expression and ER stress, and plunge-froze the samples with added nanospheres positional markers at 77 K (25).
To localize IRE1a foci, we imaged the frozen grids on a fluorescence light microscope fitted with a liquid nitrogen sample chamber (26). In initial studies with stressed cells expressing IRE1a-green fluorescent protein (GFP), we observed strong autofluorescence at 77 K (27) that hindered IRE1a foci identification ( fig. S1). To overcome this obstacle, we fused IRE1a to the brighter fluorescent protein mNeonGreen (mNG). This refinement revealed spots that emitted much higher fluorescence in the green relative to red and blue channels (Fig. 1A), which were absent in control cells that expressed IRE1a not fused to mNG (fig. S1). We used the ratio of green-to-red and green-to-blue fluorescence intensity to identify IRE1a-mNG foci reliably.
We next imaged IRE1a foci by cryo-CLEM-ET. We used nanospheres, grid features, and cell boundaries to locate the same IRE1a foci with the electron microscope that we had identified by light microscopy and then recorded tilt series. Across nine tomograms obtained from mouse embryonic fibroblasts (MEFs), IRE1a-mNG foci consistently localized to specialized regions of the ER that displayed a network of notably narrow, anastomosing tubes ( Fig. 1, B to D, and figs. S2 and S3) with an average (±SD) diameter of 28 ± 3 nm. The tubes frequently connect with each other by three-way junctions and to surrounding ER structures, forming a topologically complex, continuous membrane surface (Fig. 1D). Unlike the surrounding ER, the tubes were devoid of bound ribosomes ( fig. S4).
To obtain higher cryo-ET resolution, we used human osteosarcoma U2OS cells that expressed inducible IRE1a-mNG (24), which contained more-expansive thin regions compared with MEFs. We imaged tilt series from eight IRE1a-mNG foci and again observed thin anastomosing tubes with characteristics that closely matched those in MEF-IRE1a-mNG ( Fig. 1, E and F, and figs. S4 to S6). Thus, IRE1a foci localize to a highly specialized ER region, henceforth termed the "IRE1a subdomain."

Orthogonal methods reveal IRE1a subdomains
To further explore the IRE1a subdomains, we performed conventional and immuno-electron microscopy on human embryonic kidney (HEK) 293 cell-IRE1a-GFP (19). Electron micrographs of Epon-embedded thin sections of stressed cells-but not unstressed controls-exhibited membrane tube networks with topology that was comparable to that seen by cryo-CLEM-ET ( fig. S7). These tubes stained more strongly in their lumenal space than in the surrounding ER, which suggested a high protein density.
We next performed immunogold labeling of ultrathin HEK293-IRE1a-GFP cryosections. In nonstressed cells, gold particles that were specific to IRE1a-GFP sparsely labeled large regions of the cell with visible ER (Fig. 2, A and A′, and fig. S8). In stressed cells, clusters of gold particles labeled regions of much higher membrane complexity, which showed longitudinal and transverse cross sections of~28-nm membrane tubes (Fig. 2

IRE1a subdomains contain lumenal helical filaments
We likewise observed regular densities in the lumen of the IRE1a subdomain tubes in cryotomograms, which we interpret as oligomers of IRE1a-LD. In longitudinal sections of MEFderived tomograms, the lumenal densities resembled train tracks parallel to the membranes (Fig. 3, A and A′). Closed rings approximately concentric with the enclosing tube membrane were clearly visible where IRE1a subdomain tubes were imaged parallel to the beam (Fig. 3, B and B′, and fig. S9). The inner rings mea-sured~9 ± 0.5 nm in diameter (±SD) and were enclosed by membrane tubes~28 ± 1 nm in diameter (±SD) (Fig. 3C). In U2OS-IRE1a-mNG tomograms, the lumenal densities showed sufficient substructure to reveal two intertwined helices (Fig. 3, D to F′): Top and bottom cross sections showed equidistant parallel angled lines of opposite directionalities, whereas the middle cross section showed helical features (Fig. 3, D to G).

Subtomogram averaging resolves flexible IRE1a-LD double helices
To determine the three-dimensional structure of this density, we extracted 653 subvolumes for subtomogram averaging (fig. S10). The resulting map of averaged electron density portrayed a left-handed double helix composed of two equidistant, intertwined strands with a pitch of 17 nm in each strand (Fig. 4, A and B,  and fig. S10). This double-helical structure is reminiscent of the unit cell of the Saccharomyces cerevisiae core IRE1-LD (cLD) crystal structure [Protein Data Bank ID 2BE1 (20)], in which twofold symmetrical IRE1-cLD dimers arrange into left-handed double-helical filaments.
To compare the shape of the known IRE1a-cLD structures with the averaged map, we next endeavored to interpret the double helix in light of the yeast IRE1-cLD crystal structure in its active conformation, which forms double helices with a pitch of 38 nm, instead of the inactive dimeric human IRE1a-cLD structure. We built a human IRE1-cLD tetramer model by threading the human IRE1a-cLD sequence into the crystal structure of active yeast IRE1-cLD, as previously described (14,28). We next fit this tetramer into our double-helical map Tran   and modified the dimer:dimer interface to accommodate the helical pitch observed (Fig. 4, C and D, and fig. S11). We propagated the interface to occupy each filament of the double helix with approximately nine monomers per turn (Fig. 4, E and F). This model is consistent with the yeast cLD crystal structure but has a compressed pitch of 17 nm, and interface residues whose mutation disrupted human IRE1a foci formation and RNase activity mapped to the helical interfaces ( fig. S12) (14, 19). The modeled IRE1a-cLD helix accounts well for the averaged map, including its ribbon-like shape. We mapped the averaged subvolumes back onto the cryo-tomograms and generated a volumetric distribution for IRE1a-LD filaments within an IRE1a subdomain (Fig. 4G). The membrane tubes and helices display a range of straight (radius of curvature >175 nm) and curved segments with radii approaching 25 nm (Fig. 4H and fig. S13). This bending indicates that IRE1a-LDs and/or their oligomerization interfaces are flexible.
Infrequently, the lumenal double helices were irregularly spaced relative to tube membranes ( fig. S14). In one case, we observed helices that were not completely enclosed by membrane tubes but instead were positioned on the lumenal face of a flat ER membrane (Fig. 5, A to B′). The distance constraints that this conformation imposes can be readily accommodated by the 52-amino acid linker connecting the IRE1a-cLD to IRE1a's transmembrane domain (Fig. 5, C to G).

Discussion
The ER is formed from a single continuous membrane that is dynamically differentiated into a plethora of pleiomorphic subdomains (29)(30)(31)(32). We found that UPR activation leads to the formation of "IRE1a subdomains," which are composed of labyrinthine networks of anastomosing~28-nm membrane tubes that contain ordered IRE1a-LD double-helical filaments. Our use of cryo-CLEM-ET to determine the supramolecular arrangements of IRE1a oligomers in their native environment demonstrates the power of in situ structural biology (33).
Several independent lines of evidence support our conclusion that the observed helical densities correspond to oligomerized IRE1a-LDs: (i) We observed narrow membrane tubes in 20 out of 20 fluorescent foci analyzed, 18 of which showed protein density inside tubes of 28-nm diameter, which is consistent with the averaged helical maps. No such structures were observed in adjacent and random regions of the cell, including those emitting high auto-fluorescence; (ii) orthogonal immunogold staining revealed IRE1a localization to regions enriched with similarly narrow tubes; (iii) the double-helical architecture closely resembles the crystal structure of yeast IRE1-cLD (20); and (iv) the reconstructed volume has the same ribbon-like shape and dimensions as IRE1a-cLD oligomers.
The existence of IRE1a subdomains explains why IRE1a foci contain a mobile periphery and a nonexchangeable core (24). We surmise that the two populations represent (i) IRE1a molecules located where tubes merge with the main ER and (ii) those located deeper in the interior of the tubes. IRE1a molecules at helix ends can readily dissociate, and new IRE1a molecules can associate; they represent a mobile pool. IRE1a molecules at the foci's core that are physically trapped in arrayed helices represent a nonexchangeable pool.
The functional importance of IRE1 oligomerization is supported by mutational disruption of interfaces that affect foci formation, RNase function, and cell survival under stress (14,(18)(19)(20)(21)(22). The confinement of IRE1a in the specialized IRE1a subdomains suggests further functional consequences for the regulation of IRE1a signaling. A single unfolded protein molecule trapped inside a 100-nm-long subdomain Tran   segment has an effective concentration of 40 mM (materials and methods), which is well within the range of the affinity measured for IRE1a-unfolded protein binding in vitro (13,14). Thus, once IRE1a subdomains form under ER stress, a few activating ligand molecules would be sufficient to saturate IRE1a-LD, effectively locking IRE1a into its activated state. This effect is due to the enormous concentration IRE1a experiences upon foci formation. Without UPR activation, IRE1a-mNG is distributed over the ER surface at~10 molecules/mm 2 (24). Inside IRE1a subdomains, it is enriched to >10,000 molecules/mm 2 of subdomain membrane (materials and methods). Moreover, the complex subdomain membrane topology (34) and IRE1a-LD's helical assembly both reduce IRE1a diffusional freedom and stabilize the oligomeric state. We estimate that the local concentrations of IRE1a-cLD inside subdomain tubes and IRE1a cytosolic domains on the tubes' surface approach 4 to 5 mM and 170 to 220 mM, respectively, well exceeding the measured oligomerization affinities for purified IRE1 domains (22,35). IRE1a subdomains may also function as a diffusion barrier, in which large molecules may be excluded or diffusionally constrained (materials and methods).
The averaged human IRE1a-LD map and yeast IRE1-cLD crystal structure both revealed equidistant double left-handed helices, albeit with a 2.2-fold difference in pitch. The differences could be species specific or result from crystallization conditions. IRE1a helices are observed, although rarely, on flat ER membranes, which indicates that the IRE1a lumenal linker domains can compact to varying degrees to bridge IRE1a-cLD with the nearest membrane.
IRE1a-LD oligomers may form on flat ER membranes and subsequently deform and constrict membranes (36), perhaps using preexisting regions of high curvature (37), to form the tubes of regular diameter. Alternatively, in 2 out of 20 tomograms, IRE1a-mNG foci localized to irregular, thin membrane tubes without ordered lumenal filaments ( fig. S14), which possibly reflects an intermediate assembly state that captured IRE1a's localization preceding LD helix formation.
Our discovery of the IRE1a subdomain suggests intriguing possibilities for how these specialized ER structures could serve regulatory functions in the UPR. IRE1a recruitment into long-lived, topologically distinct structures may scaffold proposed downstream effectors (9) and/or affect the switch in IRE1a RNase output between XBP1 mRNA splicing and RIDD activities (17). Such regulation may profoundly affect the UPR's life/death decision and hence be of crucial importance in designing UPRcentered therapies in disorders characterized by a breakdown of proteostasis.