SUMMARY
One of the major cellular mechanisms to ensure cellular protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is triggered by accumulation of misfolded proteins in the ER lumen. The ER stress response is also activated in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we explore the mechanism of activation of the ER stress response in HGPS. We find that aggregation of the diseases-causing progerin protein at the nuclear envelope triggers ER stress. Induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nucleoplasmic protein aggregates can be sensed, and signaled to the ER lumen, via clustering of SUN2. These results identify a mechanism of communication between the nucleus and the ER and provide insight into the molecular disease mechanisms of HGPS.
Graphical Abstract
In brief
Vidak et al. find that aggregation of the disease-causing progerin protein at the nuclear periphery promotes sequestration of ER chaperones and induces ER stress. The inner nuclear membrane protein SUN2 senses progerin aggregation and transmits this information to the ER lumen, establishing an alternative mechanism to activate the ER stress response.
INTRODUCTION
The cellular levels of proteins and their homeostasis in response to environmental cues are determined by a finely tuned balance between protein synthesis and degradation. In addition to regulated synthesis of proteins, several major cellular pathways are involved in the maintenance of the proteome, including the proteasomal system and autophagy, which are responsible for removal of damaged, misfolded, and aggregated proteins from cells.1 Furthermore, to ensure proper folding of nascent proteins and to prevent the accumulation of disease-causing aggregates, cells employ a repertoire of chaperones that resolubilize protein aggregates and assist in refolding of misfolded or unfolded proteins.2,3
A prominent cellular mechanism of protein quality control involves the endoplasmic reticulum (ER), the site of protein synthesis and folding of secreted and membrane-bound proteins.4 Stringent quality control systems in the lumen of the ER ensure that only correctly folded and functional proteins are released into the secretory pathway and that misfolded proteins are degraded.5 Proper functioning of the ER and its protein quality control mechanisms are essential for cellular activities and cell survival. Stimuli that interfere with ER function, such as hypoxia, aberrant calcium homeostasis, viral infections, or overproduction of secreted proteins, disrupt ER homeostasis and cause accumulation of unfolded or misfolded proteins in the ER lumen, resulting in ER stress.6-8 ER transmembrane proteins, including inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor (ATF)-6, detect the onset of ER stress and initiate the unfolded protein response (UPR) signaling pathway to restore normal ER functions.9-11 Under physiological conditions, the activation of these sensors is inhibited by the binding of their luminal domains by the main ER-resident chaperone GRP78/BiP (78-kDa glucose-regulated protein). Due to its higher affinity to unfolded proteins compared with ER stress sensors, accumulated unfolded proteins cause the dissociation of GRP78/BiP from ER stress sensors and subsequent activation of the UPR pathway.12 Prolonged ER stress caused by conditions that lead to increased secretory load or the presence of mutated proteins that cannot properly fold in the ER often result in cellular dysfunction and disease.13 The UPR and ER stress have also been linked to physiological and premature aging.1,14
Naturally occurring premature aging disorders are powerful model systems to study aging and aging-related pathologies.15,16One of the most prominent premature aging diseases is Hutchinson-Gilford progeria syndrome (HGPS), an extremely rare, fatal genetic condition caused by a de novo heterozygous mutation in the LMNA gene encoding for the nuclear architectural proteins lamin A and C.16-18 The HGPS mutation activates a cryptic splice donor site in LMNA, resulting in the expression of progerin, a lamin A isoform that lacks 50 amino acids and permanently retains a C-terminal farnesyl group, leading to its stable association with the inner nuclear membrane (INM) and predominant localization at the nuclear periphery.16,19 Progerin acts in a dominant-negative fashion and causes a variety of cellular defects including compromised nuclear architecture, loss of heterochromatin, DNA repair deficiencies, and altered cell proliferation and differentiation, as well as post-transcriptional reduction of select cellular proteins.20-24 Furthermore, progerin has been shown to form static protein aggregates at the periphery of nuclei of patient-derived primary and immortalized fibroblasts.25-28 Progerin aggregates are thought to be deleterious due to the entrapment of normal cellular proteins and compromising their function, including the anti-oxidative master regulator NRF2.27 Activation of autophagy through rapamycin-mediated mTOR inhibition has been shown to promote the solubilization and clearance of progerin and to alleviate cellular aging defects,29 indicating progerin aggregation as one of the drivers of HGPS etiology.
Here, we have investigated the cellular chaperone response to the presence of progerin aggregates in HGPS. We find that progerin aggregates at the nuclear periphery promote sequestration of chaperones in the ER lumen and induce chronic ER stress. The progerin interactor and INM transmembrane protein SUN2 is necessary and sufficient to trigger ER stress and contributes to the ER stress phenotype via clustering of its luminal domain, leading to induction of ER stress. These observations identify an unanticipated mechanism of communication between the nucleus and the ER, and they provide insight into the molecular disease mechanisms of HGPS.
RESULTS
Progerin alters expression and localization of select molecular chaperones
The premature aging disorder HGPS is caused by the progerin protein, a mutant isoform of the nuclear intermediate filament protein lamin A.16-18 Progerin is incorporated into the nuclear lamina and forms insoluble protein aggregates with low protein turnover dynamics both at the nuclear periphery and in the nuclear interior.28 HGPS cells are characterized by ER stress,14 and we hypothesized that the presence of progerin aggregates leads to activation of a cellular chaperone response.
To assess the effect of progerin on the expression and localization of major molecular chaperones, we performed high-throughput imaging analysis to quantitatively determine protein levels and the cellular distribution of several chaperones in a well-characterized cellular HGPS model system of hTERT-immortalized skin fibroblasts expressing doxycycline-inducible GFP-progerin30 and in HGPS patient cells (Figure S1A). Upon induction of GFP-progerin for 4 days (Figure S1A), which is sufficient to induce most major cellular defects associated with HGPS,22 the localization of several major cytosolic chaperones (Hsp40, Hsc70, Hsp90, and Hsp110) was unaffected by the presence of progerin (Figure 1A). Single-cell immunofluorescence analysis using high-throughput spinning-disc confocal microscopy revealed only minor changes in the average mean levels of Hsp90 and Hsp40 upon GFP-progerin expression (Figure 1C; p = 0.006 and 0.033, respectively). No significant change in the levels of Hsp90 and Hsp110 was observed (Figure 1C). Similar observations were made in immortalized HGPS patient skin fibroblasts (Figures S1E and S1F).
Figure 1. Altered expression of various chaperones in progerin-expressing cells.
(A) Immunofluorescence (IF) staining of indicated cytosolic chaperones in paraformaldehyde (PFA)-fixed GFP-progerin inducible fibroblasts. Scale bar: 10 mm.
(B) IF staining of indicated ER chaperones in PFA-fixed GFP-progerin inducible fibroblasts. Scale bar: 10 μm.
(C and D) High-throughput IF quantification of chaperone levels in GFP-progerin inducible fibroblasts represented as the average mean fluorescence values ± SD from 3 experiments (number of cells in each experiment = 1,300–1,500). Statistical differences were analyzed by t test for the fold change (FC) indicated in the figure.
Interestingly, despite unchanged cytosolic chaperone levels, single-cell high-throughput imaging demonstrated a significant increase in the mean protein levels of several ER chaperones including PDI, calnexin, GRP78, and GRP94 in the presence of progerin (Figures 1B, 1D, and S1B), independent of cell density (Figures S1C and S1D). The increase was more moderate and more variable in immortalized patient fibroblasts (Figures S1G and S1H), in line with the well-established heterogeneity of cellular phenotypes in HGPS cells.31,32 Single-cell analysis revealed high heterogeneity of expression within the cell population, as well as between biological replicates in HGPS patient cells, with 10%–40% of cells showing an increase in expression relative to the wild-type control (Figure S2A). The same pattern was observed in several primary HGPS patient-derived fibroblasts, with 10%–40% of cells showing an increase in GRP78 and PDI and up to 80% of cells an increase in calnexin and GRP94 expression levels relative to control (wild-type [WT] 1) (Figure S2B). Taken together, our data point to changes in the expression levels of several ER chaperones in the presence of progerin.
Progerin expression induces chronic ER stress in vitro and in vivo
Disturbances in ER homeostasis result in the accumulation of misfolded proteins within the ER lumen, leading to ER stress.33 These luminal aggregates activate the UPR, which alters the expression of numerous genes involved in ER quality control.9 In addition, ER stress can result in widespread cytoplasmic protein aggregation and generate proteotoxic stress responses across cellular compartments.34,35 Given the observed increase in the levels of ER chaperones in progerin-expressing cells, we sought to determine whether progerin expression causes ER stress by measuring UPR activation in vitro and in vivo. Quantitative RT-PCR analysis showed transcriptional activation of several UPR genes, including DDIT3, HSPA5, XBP1, and ERN1/IRE1, upon induction of GFP-progerin (Figure 2A). The same increase was observed in multiple immortalized and, to a lesser extent, in primary HGPS patient fibroblasts (Figures 2B and S2C).14 Importantly, induction of these genes was a specific response to the presence of progerin and did not occur upon expression of WT lamin A (Figure 2A). These changes represented physiological responses, as they were also present in the heart and aorta of homo- and heterozygous progerin transgenic HGPS mice36 and a homozygous knockin HGPS mouse37 (Figure 2C), in agreement with earlier observations of UPR upregulation in an atherosclerosis-prone HGPS mouse model.14 Furthermore, immunoblot analysis demonstrated increased phosphorylation of PERK and IRE1α, two major mediators of the UPR pathway, in the presence of progerin (Figure 2D). We conclude that progerin triggers ER stress pathways.
Figure 2. Progerin expression induces ER stress in vitro and in vivo.
(A and B) Quantification of mRNA levels of UPR genes in GFP-progerin- and GFP-lamin A-expressing inducible fibroblasts relative to uninduced control (A) and in two patient-derived immortalized human HGPS fibroblasts relative to healthy control (B) by qRT-PCR. GAPDH and TBP1 were used for normalization. Values represent the mean of 3 biological replicates ± SD. Statistical differences were analyzed by t test for GFP-progerin/GFP-lamin A and HGPS/WT. *p < 0.05, **p < 0.01. Dashed lines represent control value of WT cells normalized to 1.
(C) mRNA expression levels of several UPR genes in three different progeria mouse models (G608G+/−, G608+/+, and G609G+/+) relative to C57BL6 control determined by qRT-PCR. GAPDH and TBP1 were used for normalization. Values represent the mean of 4–15 biological replicates ± SD. Dashed lines represent control normalized to 1. Statistical differences were analyzed by t test between biological replicates. *p < 0.05, **p < 0.01.
(D) Immunoblot analysis of phospho-IRE1 α, total IRE1 α, phospho-PERK, and total PERK levels in total cell lysates of GFP-progerin inducible and patient-derived fibroblasts. GAPDH and γ-tubulin served as a loading control. Vertical line in IRE1 α blot marks a lane that was removed between the WT and HGPS samples.
(E) qRT-PCR analysis of mRNA levels of several UPR genes in GFP-progerin inducible fibroblasts treated with Thapsigargin or DMSO (vehicle control). Values for GFP-progerin OFF/Thapsigargin and GFP-progerin ON/DMSO were calculated relative to GFP-progerin OFF/DMSO control, whereas GFP-progerin ON/ Thapsigargin is shown relative to GFP-progerin ON/DMSO. GAPDH and TBP1 were used for normalization. Values represent means of 3 biological replicates ± SEM. Statistical differences were analyzed by t test. *p < 0.05, **p < 0.01.
(F) Number of cells in GFP-progerin inducible fibroblasts treated with 25 mM TUDCA or H2O (vehicle control) for 48 h. Values represent means of 3 biological replicates ± SD (n = 350–1,550). Statistical differences were analyzed by t test. *p < 0.05, **p < 0.01.
(G) Average mean GFP-progerin levels in inducible fibroblasts treated with 25 μM TUDCA or H2O (vehicle control) for 48 h. Values represent means of 3 biological replicates ± SD (n = 350–1,550). Statistical differences were analyzed by t test.
(H) Average mean GRP78 levels in inducible fibroblasts treated with 25 μM TUDCA or H2O (vehicle control) for 48 h. Values represent means of 3 biological replicates ± SD (n = 350–1,550). Statistical differences were analyzed by t test.
Elevated levels of ER chaperones and transcriptional activation of the UPR genes are typically associated with a heightened ER stress response.38 To assess whether the progerin-mediated increase in key ER stress response factors affects the ability of cells to respond to stress, we challenged progerin-expressing cells with the ER stressors Thapsigargin or Tunicamycin. Remarkably, while control cells were able to significantly upregulate UPR genes, progerin-expressing cells failed to respond to additional ER stress above their elevated baseline level of UPR activity (Figures 2E and S2D). These results suggest that progerin chronically activates ER stress response pathways to a level that prevents further activation upon additional stressors.
Previous studies have shown that tauroursodeoxycholic acid (TUDCA), an inhibitor of ER stress, can significantly relieve ER stress and reduce ER stress-mediated cell death.39-42 Treatment of GFP-progerin inducible cells with 25 μM TUDCA for 48 h significantly increased cell number compared with vehicle-treated control (Figure 2F) but had no effect on progerin levels (Figure 2G) nor total GRP78 levels (Figure 2H), consistent with a role of ER stress in progerin-mediated cell death, one of the major HGPS cellular phenotypes.25,43 Together with recent findings demonstrating beneficial effects of TUDCA on vascular phenotype and lifespan of atherosclerosis-prone progeria mice,14 our data suggest a contribution of ER stress to the development of the HGPS phenotypes.
Progerin aggregates sequester ER chaperones at the nuclear periphery
To investigate the mechanism by which the nuclear protein progerin induces ER stress, we probed the distribution of several ER chaperones in progerin-expressing cells using super-resolution structured illumination microscopy (SIM) (Figure 3). We find accumulation of GRP78/BiP and PDI in ER regions immediately juxtaposed to the nuclear periphery, mirroring the distribution of progerin (Figures 3A and 3B). Quantification of single-cell high-throughput imaging of GRP78/BiP distribution shows significant recruitment to the nuclear periphery in both GFP-progerin-expressing and patient-derived fibroblasts, as assessed by measuring the mean fluorescence intensity of a band of 5%–35% distance from the nucleus, where 0% is adjacent to DAPI and 100% represents the cell border (Figures 3C and 3D; see STAR Methods). Expression of WT lamin A did not induce any changes in GRP78/BiP localization or UPR activity (Figures S2E-S2G). Co-immunoprecipitation of GRP78/BiP with progerin, but not with WT lamin A (Figures 3E, S2H, and S2I), confirmed the physical association, most likely indirectly (see below), of progerin with the resident ER chaperone GRP78/BiP.
Figure 3. ER chaperones accumulate around the nuclear periphery of progerin-expressing cells.
(A) Representative structured illumination microscopy (SIM) images of GRP78 and PDI staining in GFP-progerin inducible fibroblasts fixed with PFA. Scale bar: 10 μm.
(B) Enlarged view of the nuclear periphery in GFP-progerin inducible fibroblasts from (A). Scale bar: 5μm.
(C and D) High-throughput IF quantification of GRP78 recruitment to the nuclear periphery in GFP-progerin inducible fibroblasts (C) and in patient-derived HGPS fibroblasts (D) assessed by measuring the mean fluorescence intensity of a band of 5%–35% distance from the DAPI-based nuclear region of interest, where 0% is adjacent to DAPI and 100% represents the cell boundary (number of cells for each experiment >500). Data are represented as the average mean fluorescence values ± SD from 3 experiments (see STAR Methods). Statistical differences were analyzed by t test for the FC indicated in the figure.
(E) Western blot analysis of interaction between immunoprecipitated GRP78 with GFP-lamin A or GFP-progerin in inducible fibroblasts.
(F) GRP78 IF staining in PFA-fixed, non-farnesylated GFP-progerin C661S inducible fibroblasts (see STAR Methods). Scale bar: 10 μm.
(G) qRT-PCR analysis of mRNA levels of several UPR genes in non-farnesylated GFP-progerin inducible fibroblasts relative to uninduced control. GAPDH and TBP1 were used for normalization. Dashed lines represent control normalized to 1. Values represent means of 3 biological replicates ± SD. Statistical differences were analyzed by t test. *p < 0.05, **p < 0.01.
(H) High-throughput IF quantification of ER chaperone expression levels in non-farnesylated GFP-progerin inducible fibroblasts (number of cells for each experiment >1,500) represented as the average mean fluorescence values ± SD from 3 experiments. Statistical differences were analyzed by t test for the FC indicated in the figure.
(I) High-throughput IF quantification of GRP78 recruitment to the nuclear periphery in non-farnesylated GFP-progerin inducible fibroblasts (number of cells for each experiment >1,500) shown as the average mean fluorescence values ± SD from 3 experiments. Statistical differences were analyzed by t test for the FC indicated in the figure.
(J) Western blot analysis of interaction between immunoprecipitated GRP78 with GFP-progerin C661S in inducible fibroblasts.
To determine whether the accumulation of GRP78/BiP at the nuclear periphery was a direct consequence of the presence of progerin aggregates at the nuclear envelope or an indirect, non-specific stress response, we expressed the non-farnesy-lated C661S mutant of progerin, which accumulates in the nucleoplasm but not at the nuclear periphery (Figure 3F).30 Progerin- C661S, in contrast to progerin, does not lead to the induction of UPR signaling or increased levels of ER chaperones (Figures 3G and 3H) nor to significant GRP78/BiP sequestration (Figure 3I) to the nuclear periphery. In addition, co-immunoprecipitation of GRP78/BiP with progerin-C661S showed no physical association between non-farnesylated progerin and GRP78/BiP (Figures 3J and S2H). These results demonstrate that the presence of progerin aggregates at the nuclear periphery triggers GRP78/BiP sequestration to the ER regions juxtaposed to the nuclear envelope and to the induction of ER stress.
The INM protein SUN2 mediates the ER stress phenotype triggered by nuclear aggregation
Progerin is a nucleoplasmic protein tightly associated with the nuclear envelope (NE),28 yet it appears to trigger the UPR in the ER lumen and can be biochemically pulled down with the ER-luminal chaperone GRP78/BiP (see Figure 3). However, direct interaction between progerin and ER chaperones is unlikely since progerin does not extend into the perinuclear space, pointing to the existence of adaptor proteins that sense and transmit the presence of nucleoplasmic progerin aggregates to the ER lumen. Because the ER lumen is contiguous with the perinuclear space between the INM and the outer nuclear membrane (ONM), we hypothesized that the prominent INM proteins SUN1 and SUN2 may mediate the nuclear-triggered ER stress response.44,45 SUN1 and SUN2 are transmembrane proteins whose N terminus interacts with the nuclear lamin proteins, including progerin, on the nucleoplasmic face of the nuclear envelope, whereas their highly conserved luminal C terminus extends into the perinuclear space, where it interacts with nesprin transmembrane proteins located in the ONM.46 As part of the linker of nucleoskeleton and cytoskeleton complex (LINC), which spans the lumen of the NE, SUN proteins act as signal transmitters between the nucleus and the cytoplasm, in particular of mechanical forces.47 Interestingly, SUN1/2 have been linked to progeric and dystrophic laminopathies.48
In line with a role for SUN proteins in sensing progerin aggregates and transmitting their presence to the ER lumen, singlecell high-throughput imaging revealed that both SUN1 and SUN2 levels were increased upon GFP-progerin expression (Figure S3A), as well as in various immortalized and primary patient-derived fibroblast cell lines (Figures S3B-S3D). Furthermore, SIM revealed accumulation of progerin, SUN1, and SUN2 and co-localization with GRP78/BiP or PDI at the nuclear periphery in GFP-progerin-expressing cells (Figures 4A and S3E), as well as local enrichment of SUN2, GRP78, and PDI in the presence of progerin aggregates (Figure 4B), whereas no significant enrichment of GRP78 with SUN1 was observed (Figure S3F). Similar accumulation was observed in highly lobulated nuclei of patient-derived fibroblasts as assessed by confocal microscopy and measuring local enrichment of SUN2, GRP78, and PDI at the nuclear periphery (Figures S3G and S3H). The observed accumulations of SUN2, GRP78, and PDI were not merely a consequence of altered nuclear membrane morphology, such as membrane folds or formation of ER sheets, since no enrichment was observed for WT lamin A (Figures S2E-S2G). Co-immunoprecipitation of GRP78/BiP with SUN2 confirmed the association of SUN2 in progerin-expressing cells but not in WT lamin A-expressing cells (Figure S3I). Activation of the ER stress response was not dependent on the interaction of SUN2 with KASH proteins in the ONM since expression of a dominant negative KASH domain of Nesprin-1 (DN-KASH1)49 or a KASH domain of Nesprin-2 (mScarlet-KASH2),50,51 which have been shown to disrupt SUN2 interactions, in contrast to the negative control (mScarlet-KASH2ΔL),50,51 did not affect ER stress induction (Figures S4A and S4B), suggesting that SUN2 acts via a distinct mechanism from its canonical interaction with KASH proteins.
Figure 4. SUN2 contributes to the ER stress phenotype in progerin-expressing cells.
(A) Representative SIM images of GRP78, PDI, and SUN2 staining in GFP-progerin inducible fibroblasts fixed with PFA. Scale bar: 10 μm.
(B) Scatterplot of fluorescence intensities of the peripheral SUN2, GRP78, and PDI localization (n = 4–15). Line represents the median. Statistical differences were analyzed by t test. **p < 0.005, ***p < 0.0005.
(C) Confocal images of GRP78 IF staining in siSUN1- and siSUN2-treated GFP-progerin inducible fibroblasts and their respective controls. Scale bar: 10 μm.
(D and E) High-throughput IF quantification of total GRP78 levels (D) and GRP78 recruitment to the nuclear periphery (E) in siSUN1- and siSUN2-treated GFP-progerin inducible fibroblasts and their respective controls (number of cells for each experiment >200) represented as the average mean fluorescence values ± SD from 3 experiments. Statistical differences were analyzed by t test for the FC indicated in the figure.
(F) qRT-PCR analysis of mRNA levels of several UPR genes in siSUN2- and control-treated GFP-progerin inducible fibroblasts relative to uninduced control. GAPDH and TBP1 were used for normalization. Dashed lines represent control normalized to 1. Values represent means of 3 biological replicates ± SD. Statistical differences were analyzed by t test. *p < 0.05, **p < 0.01.
To determine whether SUN1 and/or SUN2 indeed contribute to the progerin-mediated accumulation of ER proteins at the nuclear periphery, we performed small interfering RNA (siRNA) knockdown of each SUN protein and measured the recruitment of GRP78/BiP to the nuclear periphery upon induction of progerin (Figures 4C, S4C, and S4D). While loss of SUN1 had no effect on GRP78/BiP accumulation at the nuclear periphery, knockdown of SUN2 prevented GRP78/BiP accumulation and restored normal GRP78/BiP localization even when progerin was induced (Figures 4C and 4E). In addition, SUN2 but not SUN1 downregulation decreased total GRP78/BiP levels (Figure 4D) and prevented the progerin-mediated transcriptional upregulation of UPR genes (Figure 4F). siRNA knockdown of each SUN protein had no effect on the total GRP78/BiP levels or GRP78/BiP accumulation at the nuclear periphery in uninduced control cells (Figure S4E). No obvious sequence features to account for the differential role of SUN1 and SUN2 proteins in ER stress activation were noted (Figure S4F), suggesting that the differential behavior of SUN1 and SUN2 is a result of more efficient GRP78 sequestration by SUN2 aggregates at the nuclear periphery (Figures 4A and 4B) compared with SUN1 (Figures S3E and S3F). We conclude that the progerin interactor SUN2 contributes to the ER stress phenotype observed when progerin aggregates are present in the nucleoplasm.
SUN2 clustering at the nuclear periphery recruits GRP78/BiP
To uncover how protein aggregates at the nucleoplasmic face of the nuclear envelope can cause a change in the localization of ER chaperones and activate ER stress pathways via SUN2, we analyzed the expression and the localization of SUN2 in more detail. Stimulated emission depleted (STED) super-resolution microscopy revealed that SUN2 proteins are not uniformly localized at the nuclear periphery, but accumulate in cluster-like structures in the INM in the presence of progerin but not of WT lamin A (Figure 5A, arrows). Interestingly, SUN2 clusters coincide with the highest levels of GRP78/BiP accumulation (Figure 5A, arrows). The accumulation of SUN2 was not merely a reflection of higher local levels of progerin or altered nuclear envelope morphology, since the ratio of SUN2 relative to progerin increased compared with WT lamin A, pointing to local enrichment of SUN2 in the presence of progerin aggregates (Figure 5B). Similarly, GRP78/BiP was enriched at sites of SUN2 clusters induced by progerin aggregation (Figure S4G), indicating recruitment of GRP78/BiP in the NE lumen to the newly formed SUN2 clusters.
Figure 5. SUN2 clustering causes changes in the localization of ER chaperones.
(A) Representative stimulated emission depletion microscopy (STED) images of GRP78 and SUN2 staining in GFP-lamin A and GFP-progerin inducible fibroblasts. Arrows indicate SUN2 and GRP78 clusters. Scale bar: 2 μm.
(B) SUN2 mean fluorescence intensity normalized to the GFP signal in GFP-lamin A(n = 18) and GFP-progerin (n = 20) inducible fibroblasts. The measurements were done on maximum projection STED images by measuring the whole region of interest (ROI). Statistical differences were analyzed by t test. *p < 0.05.
(C) Schematic representation of SUN2 and its deletion constructs. The conserved SUN domain is depicted in red, the transmembrane segment in blue, additional hydrophobic stretches in green, and C-terminal SUN1 fragment in purple.
(D) Representative confocal images of MP029-CRY2olig-mCherry SUN2 variants driven by the ubiquitin promoter for low-level expression of SUN2 and stained for GRP78 in HEK293FT cells fixed with PFA after cluster induction. Scale bar: 10 μm. Arrows indicate SUN2 and GRP78 clusters. Drawings on the right depict localization of SUN2 variants in the INM and their level of GRP78 recruitment.
(E) Mean fluorescence intensities of the mCherry and GRP78 in non-clustered and clustered cells (+488 nm) transfected with various MP029-CRY2mCherry constructs (n = 20). Statistical differences were analyzed by t test relative to no plasmid control. **p < 0.005.
(F) Representative confocal images of the MP029-CRY2olig-mCherry SUN2-N-2 variant fused to SUN1 C-terminal fragment (aa 245–511) expressed in HEK293FT cells after cluster induction. Cells were fixed with PFA and stained with GRP78 antibody. Scale bar: 10 μm. Arrows indicate SUN2-N-SUN1-C and GRP78 clusters. Drawings on the right depict localization of SUN2-N-SUN1-C variant in the INM and its level of GRP78 recruitment.
(G) Mean fluorescence intensities of the mCherry and GRP78 in non-clustered and clustered cells (+488 nm) transfected with MP029-CRY2olig-mCherry SUN2- N-SUN1-C construct (n = 20). Statistical differences were analyzed by t test relative to non-clustered control. *p < 0.05, **p < 0.005.
(H) qRT-PCR analysis of mRNA levels of several UPR genes in HEK293FT cells transfected with MP029-CRY2olig-mCherry SUN2-N variants. Data are shown as fold increase of cluster-induced over non-induced samples. Constructs were under control of a ubiquitin promoter for low-level expression. GAPDH and TBP1 were used for normalization. Dashed lines represent non-transfected control used to normalize the data (set to 1). Values represent means of 3 biological replicates ± SD. Statistical differences were analyzed by t test. *p < 0.05, **p < 0.01.
The requirement for SUN2 in activation of the luminal ER stress response combined with the observed local clustering of SUN2 and the accumulation of GRP78/BiP at SUN2 clusters points to a model in which SUN2 senses the density of lamin A/progerin on the nucleoplasmic side of the INM. One possible mechanism to transmit the presence of nucleoplasmic progerin aggregates to the ER lumen is via clustering of SUN2 proteins in the INM (Figures 5A and S4H). In this model, SUN2 would detect the presence of progerin aggregates via its known interaction of its nucleoplasmic N terminus with progerin, while SUN2 clustering would be sensed in the ER lumen by interaction of GRP78/BiP with the clustered SUN2 C-terminal domain (Figure S4H). This model predicts that forced clustering of SUN2 in the absence of progerin should be sufficient to recruit GRP78/BiP to SUN2 clusters in the ER and that this accumulation should be dependent on the C-terminal domain of SUN2 (Figure S4H).
To test these predictions, we developed an optimized optogenetic clustering approach based on the Arabidopsis thaliana CRY2-derived optogenetic module CRY2olig, which induces rapid and robust protein oligomerization upon exposure to blue light.52 Several SUN2 variants (Figure 5C) were fused to CRY2olig-mCherry and expressed under the control of a ubiquitin promoter for moderate to low expression in the absence of progerin. As expected based on prior observations of SUN2 localization upon exogenous expression,53 all of the variants localized to both the ER and the NE and showed no visible cluster formation (Figure S5A). In addition, overexpression of any of the SUN2 variants did not affect cell proliferation and did not induce any cellular progeric or senescence phenotypes (Figure S5B) nor significant changes in the localization of endogenous Nesprin-1 and -2 (Figure S5C). SUN2 clustering was induced in a controlled fashion upon low-level expression of CRY2olig-SUN2 driven by a ubiquitin promoter and using ten 200-ms light pulses over 90 min (see STAR Methods). As previously described, live cell imaging revealed rapid clustering of CRY2olig-mCherry (Figure S5D, panel 1).52 Consistent with slow lateral diffusion dynamics of SUN proteins in the INM,53,54 the full-length SUN2 started to reorganize over 30 min and reached full clustering at 90 min (Figure S5D, panel 2), whereas SUN2-N-1 and SUN2-N-2 variants, which lack part or all of the luminal domain, respectively (Figure 5C), clustered more readily than full-length SUN2 (Figure S5D, panels 3 and 4), most likely due to significantly smaller sizes of these proteins (260 and 226 aa, respectively, compared with 717 aa for the full-length SUN2). Fluorescence recovery after photobleaching (FRAP) analysis showed very little recovery of induced clusters with a recovery rate of 0.086 ± 0.0075 s−1, demonstrating cluster formation (Figure S5E).
Using this optogenetic system, we find that GRP78/BiP accumulated strongly at light-induced SUN2 clusters at the nuclear periphery in the absence of progerin (Figures 5D, top panel, and 5E), recapitulating the phenotype observed in progerin-expressing cells. Similarly, clusters of SUN2-N-1, which contains a short portion of the luminal domain, recruited GRP78/BiP to the nuclear periphery (Figures 5D, middle panel, arrows, and 5E), whereas induced clusters of SUN2-N-2, which completely lacks the luminal domain, failed to recruit GRP78/BiP (Figures 5D, bottom panel, and 5E). These data demonstrate that the luminal portion of SUN2 is necessary and that parts of the luminal domain are sufficient for the recruitment of GRP78/ BiP to the nuclear periphery. Interestingly, clustering of a construct consisting of the nucleoplasmic and transmembrane domains of SUN2 fused to the luminal domain of SUN1 (Figure S5D, panel 5) still efficiently recruits GRP78 to the induced clusters (Figures 5F and 5G), suggesting that GRP78/BiP recognition of clusters is sequence independent.
To finally test whether the peripheral recruitment of the GRP78/BiP can lead to the activation of the UPR response, we enriched mCherry-positive cells by fluorescence-activated cell sorting (FACS) and performed quantitative RT-PCR (qRT-PCR) analysis of UPR genes after optogenetic clustering of SUN2 variants. qRT-PCR analysis showed transcriptional activation of several UPR genes, including DDIT3, ERN1/IRE1, and EIF2AK3, upon induction of SUN2-N-1 clusters (1.5- to 1.9-fold increase), whereas clustering of the SUN2-N-2 variant completely lacking the C-terminal luminal domain did not further increase expression of select UPR genes (Figures 5H and S5F). Expression of the C-terminal luminal domain of SUN2 (SUN2-C; Figure S5G) also led to transcriptional activation of several UPR genes, including DDIT3, ERN1/IRE1, EIF2AK, HSPA5, HSP90B1, and spliced XBP1 (sXBP1) upon induction of SUN2-C clusters (Figure S5H). These data demonstrate that clusters of SUN2 protein in the INM lead to the activation of the UPR response through the luminal domain of SUN2.
DISCUSSION
Sustained ER stress and a faulty UPR response have been linked to physiological aging, as well as to many aging-associated diseases.55 The naturally occurring premature aging disorder HGPS mimics several aspects of normal aging, including increased DNA damage, epigenetic changes, and the emergence of vascular defects.15 Our work now extends these parallels by demonstrating activation of the ER stress response in progerin-expressing cells and in HGPS patient cells as well as in several progeria mouse models. These findings complement the recent observation of transcriptional misregulation of ER stress and UPR components in two mouse models of HGPS with ubiquitous or vascular smooth muscle cell-specific progerin expression,14 pointing to a significant role of ER stress pathways in HGPS. Interestingly, chronic activation of UPR as well as the inability to activate it upon challenge as found in HGPS cells has previously been associated with normal aging.56 In addition, previous studies have shown that nuclear irregularities in HGPS fibroblasts correlate with SUN1 overexpression and that knocking down of SUN1 alleviates some of the HGPS nuclear defects.48 We show here that SUN2 overexpression in WT cells does not recapitulate HGPS cellular phenotypes but rather contributes to the disease development by chronic activation of the ER stress response, ultimately leading to cell death. These findings point to distinct functions for SUN1 and SUN2 in HGPS, which is in line with prior findings demonstrating both redundant and separate functions for SUN isoforms depending on their context.57,58
Our finding of UPR activation in HGPS may also have therapeutic implications. Treatment with the chemical chaperone TUDCA has previously been reported to efficiently alleviate ER stress in a number of HGPS-relevant physiological systems including models of aortic valve calcification, hypertrophy, and diabetes.59-61 Furthermore, treatment with TUDCA ameliorates vascular pathology and prolongs lifespan in atherosclerosis-prone HGPS mouse models.14 These findings identify ER stress and the UPR as drivers of cardiovascular and cellular phenotypes in HGPS, and they suggest that alleviation of ER stress may offer a therapeutic strategy for HGPS.
ER stress response pathways, including the UPR, are triggered by the accumulation of misfolded and unfolded proteins in the ER lumen.62 The UPR is activated when the major ER-resident chaperone GRP78/BiP dissociates from the major UPR transducers IRE1α, PERK, and ATF6 and binds to unfolded proteins in the ER lumen,63 leading to an increases in the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression.64 This is reflected in the observed increase in the mean protein levels of several ER chaperones including PDI, calnexin, GRP78, and GRP94 in the presence of progerin. Since calnexin is an ER transmembrane protein,65,66 its membrane localization was more pronounced than that of GRP78, GRP94, and PDI, which are located in the ER lumen and get recruited to the periphery upon progerin expression. The UPR can be triggered by numerous stimuli including redox disruption, hypoxia, aberrant Ca2+ regulation, energy balance disruption, viral infections, overproduction of secreted/transmembrane proteins, and secretion of folding-incompetent mutated proteins, as well as a number of xenobiotics.7 Our findings expand these mechanisms of UPR activation by demonstrating that ER stress pathways can also be triggered by nucleoplasmic protein aggregates at the periphery of the nucleus. Our observations suggest that the transmembrane INM protein SUN2 senses progerin aggregates juxtaposed to the nuclear envelope via interactions of its N terminus with progerin, leading to SUN2 clustering in the INM. It is possible that, analogous to typical UPR activation, clustering of SUN2 may then be sensed by GRP78/BiP in the perinuclear space, which is contiguous with the ER lumen, via recognition of the C-terminal luminal regions of SUN2, thus triggering the UPR pathway. This signaling axis would represent an uncommon mechanism of ER stress activation induced by a nuclear trigger. Whether other nuclear proteins can trigger similar ER stress by the same or related mechanisms remains to be determined.
Several lines of evidence support the conclusion that clustering of the luminal portion of SUN2 induces recruitment of the GRP78/BiP to SUN2 clusters. First, artificial clustering by optogenetic methods of the full-length SUN2 or a SUN2 N-terminal variant that still contains a portion of the C terminus is sufficient to promote GRP78/BiP sequestration, similar to the high-density aggregates observed in progerin-expressing cells. Second, optogenetic clustering of a SUN2 variant that completely lacks the C-terminal luminal domain does not recruit GRP78/BiP and does not induce transcriptional activation of UPR genes, in contrast to full-length SUN2- or SUN2-N-1-induced clusters. The observation that the SUN2-N-1 variant containing only a small portion of the luminal domain (34 aa) still efficiently recruits GRP78/BiP demonstrates that GRP78/BiP recognition of clusters is a sequence-independent effect, as confirmed by clustering the construct consisting of nucleoplasmic and transmembrane domains of SUN2 fused to the luminal domain of SUN1.
Our results document a communication pathway between the nuclear interior and the ER lumen and, as such, provide an unexplored mechanism of intercompartment communication. We identify the INM protein SUN2 as a mediator of this communication. SUN2, together with SUN1, is a member of the SUN (Sad1p, Unc-84) domain family of proteins67 which share a conserved SUN domain at their luminal C termini,68 at least one transmembrane domain, and a less-characterized N terminus that interacts with the nuclear lamin A protein on the nucleoplasmic face of the INM.46 SUN proteins interact in the lumen of the nuclear envelope with the ONM KASH (Klarsicht, Anc-1, Syne-1 homology) domain proteins to form the core of the LINC complex involved in connecting the nuclear lamina and the cytoskeleton.69 As such, the LINC complex plays an important role in mechano-transduction, nuclear migration, cell polarity, cytoskeletal organization, meiotic chromosome movement, and protein recruitment to the ONM.69 Our findings expand the mode of action of SUN proteins by demonstrating that SUN2 does not only function via interaction with the KASH domains in nesprins. Whether the interaction of SUN2 with GRP78/BiP in the presence of progerin reflects an amplification of more sporadically occurring binding events in a non-disease state or whether the interaction is specifically triggered under pathological conditions by the presence of progerin remains to be determined.
The ability of SUN2 to sense the aggregation of progerin is based on its intrinsic propensity to interact with nuclear lamins as part of the LINC complex.46 In HGPS, the disease-causing progerin protein becomes integrated into the nuclear lamina and accumulates at the nuclear periphery, leading to thickening of the lamina and formation of static protein aggregates.28 The presence of these nucleoplasmic, membrane-proximal aggregates results in abnormal clustering of SUN2 and activation of ER stress responses. In addition to the interaction of the SUN2 nuclear domain with lamins, clustering is likely also facilitated by the intrinsically low mobility of SUN proteins in the INM.54 Our finding that SUN2 acts as a sensor of nuclear protein aggregates, transmits the signal to the ER, and activates stress response pathways to restore homeostasis describes a LINC-independent function for SUN2. Taken together, our observations suggest that protein aggregation at the nuclear periphery may be responsible for prominent defects associated with premature, and possibly physiological, aging.
Limitations of the study
Our study is limited to the analysis of the clustering behavior of SUN2 in cultured cells, and the proposed mechanism will need to be validated in vivo. Furthermore, it remains unclear why GRP78 specifically interacts with SUN2 but not SUN1, especially since sequence alignment of SUN1 and SUN2 proteins show high sequence similarity and no significant difference in their predicted disordered domain formation in the C-terminal region. In addition, we cannot completely rule out that the anti-nesprin-1 and -2 antibodies cross-react with cytoskeletal filaments in the cytoplasm, making it harder to discern the nuclear envelope localization of these proteins. Finally, it remains to be determined whether other INM proteins are able to activate ER stress responses by clustering.
STAR★METHODS
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tom Misteli ([email protected]).
Materials availability
Cell lines generated in this study have been deposited at NCI, Frederick Repository.
Plasmids generated in this study are available by request.
Data and code availability
All original code has been deposited at Github and is publicly available as of the date of publication at https://doi.org/10.5281/zenodo.7829552.
Raw data used for high-throughput image analysis have been deposited at Figshare, https://doi.org/10.6084/m9.figshare. 22253287.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell culture and treatments
hTERT-immortalized GFP-lamin A, GFP-progerin, and GFP-progerin C661S doxycycline inducible dermal fibroblast cell lines were maintained and induced (96 hr) as described.30 Immortalized patient-derived HGPS (72T=HGPS 1; 97T=HGPS 2) and age-matched wild-type control (CRL-1474) cell lines were cultured as described.70-72 Experiments in immortalized cells were performed within the first 15-20 passages after transformation. Cells were grown in MEM containing 15% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U ml− 1 penicillin, 100 μgml− 1 streptomycin, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids at 37°C in 5% CO2. Primary human dermal fibroblast cell lines were obtained from the Progeria Research Foundation (PRF) and National Institute of Aging (NIA) Cell Repository distributed by the Coriell Institute. Four of the cell lines were from healthy donors and five were from patients that have the classic mutation in LMNA Exon 11, heterozygous c.1824C > T (p.Gly608Gly). The list of the cell lines used together with the source and the age at donation are listed in Table S1 and in key resources table. For all experiments, cells were passage matched between P10-20. Cells were grown in high glucose DMEM containing 15% fetal bovine serum (FBS), 1% GlutaMAX (ThermoFisher #35050-061) and 1% Penicillin-Streptomycin (ThermoFisher #15140-122) at 37°C in 5% CO2. HEK293FT cells (Thermo Fisher Scientific, R70007) were grown in DMEM containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U ml− 1 penicillin and 100 mgml− 1 streptomycin, at 37°C in 5% CO2. All cell lines were mycoplasma negative as shown by routine testing (EZ-PCR™ Mycoplasma Detection Kit, Biological Industries).
KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal anti-Hsp110 | StressMarq Biosciences | Cat#SPC-195; RRID:AB_2119373 |
| Rat monoclonal anti-Hsp90 | Enzo Life Sciences | Cat#ADI-SPA-835; RRID:AB_10621845 |
| Rat monoclonal anti-Hsc70 | Abcam | Cat#ab19136; RRID:AB_444764 |
| Rabbit polyclonal anti-Hsp40 | StressMarq Biosciences | Cat#SPC-100; RRID:AB_2261861 |
| Rabbit polyclonal anti-GRP94 | Abcam | Cat#ab3674; RRID:AB_303992 |
| Rabbit polyclonal anti-GRP78/BiP | Abcam | Cat# ab21685; RRID:AB_2119834 |
| Mouse monoclonal anti-GRP78 | StressMarq Biosciences | Cat# SMC-195; RRID:AB_10964368 |
| Mouse monoclonal anti-PDI, Clone RL90 | Abcam | Cat# ab2792; RRID:AB_303304 |
| Rabbit polyclonal anti-Calnexin | Cell Signaling Technology | Cat# 2433; RRID:AB_2243887 |
| Rabbit polyclonal anti-SUN1 | Sigma-Aldrich | Cat# HPA008346; RRID:AB_1080462 |
| Rabbit polyclonal anti-SUN2 | Sigma-Aldrich | Cat# HPA001209; RRID:AB_1080465 |
| Rabbit monoclonal anti-Nesprin 1/SYNE1 [EPR14196] | Abcam | Cat# ab192234; RRID:AB_2917992 |
| Rabbit monoclonal anti-SYNE2 (Nesprin 2) | Produced by Gregg Gundersen50 | N/A |
| Rabbit polyclonal anti-LAP2 alpha antibody | Abcam | Cat# ab5162; RRID:AB_304757 |
| Rabbit polyclonal anti-Lamin B1 antibody - Nuclear Envelope Marker | Abcam | Cat# ab16048; RRID:AB_443298 |
| Rabbit monoclonal anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade | Abcam | Cat# ab192985, RRID:AB_2650559 |
| Anti-acetyl-Histone H4 (Ac-Lys16) antibody produced in rabbit | Sigma - Aldrich | Cat# H9164; RRID:AB_1079064 |
| Mouse Anti-Ki-67 Monoclonal Antibody, Unconjugated, Clone 35 | BD Biosciences | Cat# 610968; RRID:AB_398281 |
| Mouse Anti-PCNA Monoclonal Antibody, Unconjugated, Clone PC10 | Abcam | Cat# ab29; RRID:AB_303394 |
| Rabbit monoclonal anti-IRE1 (Phospho S724) [EPR5253] | Abcam | Cat# ab124945; RRID:AB_11001365 |
| Rabbit polyclonal anti-IRE1 | Abcam | Cat# ab37073; RRID:AB_775780 |
| Rabbit monoclonal anti-PERK (PhosphoThr982) | Abcam | Cat# ab192591; RRID:AB_2728666 |
| Rabbit monoclonal anti-PERK (D11A8) | Cell Signaling Technology | Cat# 5683; RRID:AB_10841299 |
| Rabbit polyclonal anti-GFP | Abcam | Cat# ab290; RRID:AB_303395 |
| Mouse monoclonal anti-GAPDH [6C5] | Abcam | Cat# ab8245; RRID:AB_2107448 |
| Mouse monoclonal γ-tubulin [GTU-88] | Sigma - Aldrich | Cat#T6557; RRID:AB_477584 |
| Alexa Flour 568-conjugated goat anti-mouse | Thermo Fisher Scientific | Cat# A-11004; RRID:AB_2534072 |
| Alexa Flour 647-conjugated donkey anti-mouse | Thermo Fisher Scientific | Cat# A-31571; RRID:AB_162542 |
| Alexa Flour 568-conjugated donkey anti-rabbit | Thermo Fisher Scientific | Cat# A10042; RRID:AB_2534017 |
| Alexa Flour 647-conjugated donkey anti-rabbit | Thermo Fisher Scientific | Cat# A-31573; RRID:AB_2536183 |
| Alexa Flour 568-conjugated goat anti-rat | Thermo Fisher Scientific | Cat# A-11077; RRID:AB_2534121 |
| Alexa Flour 647-conjugated chicken anti-rat | Thermo Fisher Scientific | Cat# A-21472; RRID:AB_2535875 |
| Abberior Goat-anti-Rabbit STAR RED | Abberior | Cat# STRED-1002-500UG; RRID:AB_2833015 |
| Alexa Fluor 594-AffiniPure Fab Fragment Goat Anti-Mouse | Jackson ImmunoResearch Labs | Cat# 115-587-003; RRID:AB_2338900 |
| Mouse-anti-Rabbit IgG-HRP | Santa Cruz Biotechnology | Cat# sc-2357; RRID:AB_628497 |
| Goat-anti-Mouse IgG-HRP | Santa Cruz Biotechnology | Cat# sc-2005; RRID:AB_631736 |
| Chemicals, peptides, and recombinant proteins | ||
| Thapsigargin | Sigma-Aldrich | Cat#T9033 |
| Tunicamycin | Sigma-Aldrich | Cat# T7765 |
| TUDCA | Milipore | Cat##580549 |
| Critical commercial assays | ||
| NucleoSpin RNA Kit | Takara Bio | Cat# 740984 |
| RNeasy Mini Kit | Quiagen | Cat# 74004 |
| iScript™ cDNA Synthesis Kit | Bio-Rad | Cat# 1708890 |
| iQ SYBR Green Supermix | Bio-Rad | Cat#1708887 |
| Lipofectamine LTX with Plus Reagent | Thermo Fisher Scientific | Cat# 15338100 |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | Cat#13778150 |
| Deposited data | ||
| Raw data | This paper | https://doi.org/10.6084/m9.figshare.22253287 |
| Analyzed data | This paper | https://doi.org/10.5281/zenodo.7829552 |
| Experimental models: Cell lines | ||
| hTERT-immortalizedGFP-lamin A | Produced by Nard Kubben30 | N/A |
| hTERT-immortalizedGFP-progerin | Produced by Nard Kubben30 | N/A |
| hTERT-immortalizedGFP-progerin C661S | Produced by Nard Kubben30 | N/A |
| hTERT-immortalizedCRL-1474 | Produced by Paola Scaffidi70-72 | N/A |
| hTERT-immortalized 72T | Produced by Paola Scaffidi70-72 | N/A |
| hTERT-immortalized 97T | Produced by Paola Scaffidi70-72 | N/A |
| GM00038 | Coriell | Cat# GM00038; RRID: CVCL_7271 |
| AG08470 | Coriell | Cat# AG08470; RRID: CVCL_L270 |
| HGMDFN090 | Progeria Research Foundation | RRID: CVCL_1Y94 |
| HGFDFN168 | Progeria Research Foundation | RRID: CVCL_1Y93 |
| AG08466 | Coriell | Cat# AG08466; RRID: CVCL_L272 |
| HGADFN127 | Progeria Research Foundation | RRID: CVCL_1Y88 |
| GM01972 | Coriell | Cat#GM01972 |
| HGADFN167 | Progeria Research Foundation | RRID: CVCL_1Y92 |
| HGADFN178 | Progeria Research Foundation | RRID: CVCL_1Y96 |
| 293FT | Thermo Fisher Scientific | Cat# R70007 |
| Experimental models: Organisms/strains | ||
| C57BL/6J | N/A | |
| HGPS LMNA G608G+/+ transgenic mice expressing human progerin | Gift from Francis Collins36 | N/A |
| HGPS LMNA G608G+/− transgenic mice expressing human progerin | Gift from Francis Collins36 | N/A |
| HGPS LMNA G609G+/+ knock-in mouse strain | Mouse tissues were gift from Carlos Lopez-Otin37 | N/A |
| Oligonucleotides | ||
| Silencer Select negative control | Ambion | Cat#4390843 |
| Silencer Select UNC84A siRNA | Ambion | Cat#4392420 |
| Custom UNC84B siRNA 5’-CGUACCAGGUGGUGGAGCUGCGGAU-3’ | Dharmacon | N/A |
| Primers for quantitative real-time PCR, see Table S2 | This paper | N/A |
| Recombinant DNA | ||
| MP029 | Produced by Murali Palangat73 | N/A |
| pcDNA3.1+/C-(K)DY-SUN1 | GenScript | OHu26731, Cat# NM_001130965.3 |
| pcDNA3.1+/C-(K)DY-SUN2 | GenScript | OHu01874, Cat# NM_001199579.1 |
| MP029-CRY2olig-mCherry | Derived from MP029 vector by inserting CRY2olig-mCherry in the 5’ of MCS | N/A |
| MP029-CRY2-mCherry-SUN2 | Derived from MP029-CRY2olig-mCherry by inserting full length human SUN2 in the MCS after CRY2olig-mCherry construct | N/A |
| MP029-CRY2-mCherry-SUN2-N-1 | Derived from MP029-CRY2olig-mCherry by inserting N-terminal part of SUN2 (AA 1-260) in the MCS after CRY2olig-mCherry construct | N/A |
| MP029-CRY2-mCherry-SUN2-N-2 | Derived from MP029-CRY2olig-mCherry by inserting N-terminal part of SUN2 (AA 1-226) in the MCS after CRY2olig-mCherry construct | N/A |
| MP029-CRY2-mCherry-SUN2-N-SUN1-C | Derived from MP029-CRY2olig-mCherry-SUN2-N-2 by inserting C-terminal part of SUN1 (AA 245-511) in the MCS after CRY2olig-mCherry-SUN2-N-2 construct | N/A |
| MP029-CRY2-mCherry-SUN2-C | Derived from MP029-CRY2olig-mCherry by inserting C-terminal part of SUN2 (AA 440-717) in the MCS after CRY2olig-mCherry construct | N/A |
| DN-KASH | Addgene | Cat#125553; RRID:Addgene_125553 |
| pmScarlet-I-C4 hNesp2-KASH | Produced by Gregg Gundersen50 | N/A |
| pmScarlet-I-C4 hNesp2-KASH delta lumen | Produced by Gregg Gundersen50 | N/A |
| pmCherry-N1 | Clontech | Cat#632523 |
| Software and algorithms | ||
| Image Lab Software | Bio-Rad | https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z; RRID:SCR_014210 |
| Bio-Rad CFX Maestro | Bio-Rad | https://www.bio-rad.com/en-us/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15; RRID:SCR_018057 |
| GraphPad Prism | Graphpad Software | https://www.graphpad.com/scientific-software/prism/; RRID:SCR_002798 |
| Fiji | ImageJ Software | https://fiji.sc/; RRID:SCR_002285 |
| Columbus 2.8.1/2.9.1 | Perkin Elmer | https://www.perkinelmer.com/product/image-data-storage-and-analysis-system-columbus |
| ZEN Digital Imaging for Light Microscopy | Zeiss | https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html; RRID:SCR_013672 |
| Huygens Professional software version 14.10.1 | Scientific Volume Imaging | https://svi.nl/Huygens-Professional |
| Adobe Photoshop | Adobe | https://www.adobe.com/products/photoshop.html; RRID:SCR_014199 |
| Adobe Illustrator | Adobe | https://www.adobe.com/products/illustrator.html; RRID:SCR_010279 |
| BioRender | Biorender | http://biorender.com; RRID:SCR_018361 |
| BD FACSDiva Software | BD Biosciences | https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software; RRID:SCR_001456 |
| R Project for Statistical Computing | R project | https://www.r-project.org/; RRID:SCR_001905 |
| Other | ||
| Bio-Rad CFX384 Real-Time Detection System | Bio-Rad | https://www.bio-rad.com/en-us/product/cfx384-touch-real-time-pcr-detection-system?ID=LJB22YE8Z; RRID:SCR_018057 |
| Bio Rad ChemiDoc MP Imaging System | Bio-Rad | https://www.bio-rad.com/en-in/product/chemidoc-mp-imaging-system?ID=NINJ8ZE8Z; RRID:SCR_019037 |
| Cell Voyager CV7000S | Yokogawa | https://www.yokogawa.com/solutions/discontinued/cv7000s/ |
| Cell Voyager CV8000 | Yokogawa | https://www.yokogawa.com/us/solutions/products-and-services/life-science/high-content-analysis/cv8000/ |
| Zeiss LSM 780 Confocal Laser Scanning Microscope | Zeiss | https://www.zmbh.uni-heidelberg.de/Central_Services/Imaging_Facility/info/780ZEN2010.pdf; RRID:SCR_020922 |
| Zeiss Elyra PS.1 Super Resolution Microscope | Zeiss | https://www.zeiss.com/microscopy/us/local/zen-knowledge-base-home/zen-knowledgebase-elyra.html |
| Leica SP8 STED 3X System | Leica Microsystems | https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/ |
| BD FACSAria III Cell Sorting System | BD Biosciences | https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/research-cell-sorters/bd-facsaria-iii?gclid=Cj0KCQiAofieBhDXARIsAHTTldr2PcQKxSFDwsx94dE8px09lCB9JY0ualwd4u8_13Rd_TococCLFuYaApDSEALw_wcB |
| UV LED light source | ThorLabs | Cat#M405L4 |
| LED Driver | ThorLabs | Cat#DC2200 |
Cell treatments included 1μM Thapsigargin (Sigma, T9033), 1μM Tunicamycin (Sigma, T7765), 25μM TUDCA (Milipore, #580549) and their respective vehicle controls. Thapsigargin was dissolved in DMSO, Tunicamycin in EtOH and TUDCA in sterile cell-culture grade H2O. Cells were treated with Thapsigargin and Tunicamycin for 2h and with TUDCA for 48h at 37°C.
Mice
Animal experimental procedures were carried out according to guidelines in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The mice were fed a chow diet and housed in a virus-free barrier facility with a 12h light and dark cycle. Experimental mice used in this study were HGPS (LmnaG608/+) transgenic mice expressing a copy of human progerin,36 C57BL/6J mice were used as a control. Mice were sacrificed at 3.5 and 5.5 months of age, and liver, heart, and aorta tissues were collected for analysis. Number of mice used for analysis was as follows: C57BL/6J (liver, n=4; heart, n=4; aorta, n=4, samples were pooled into 1), LmnaG608/+ (liver, n=12; heart, n=10; aorta, n=15, samples were pooled into 6), LmnaG608/− (liver, n=8; heart, n=8; aorta, n=8, samples were pooled into 3). Tissues from a LmnaG609/− knock-in mouse strain (liver, n=4-5; heart, n=4-5; aorta, n=4-5, samples were pooled into 2) carrying the HGPS mutation (c.1827C>T;p.Gly609Gly mutation) equivalent to the HGPS c.1824C>T;p.Gly608Gly mutation in the human LMNA gene37 were kindly provided by Carlos Lopez-Otin (University of Oviedo, Spain). Equal number of male and female mice was used in studies.
METHOD DETAILS
Plasmid construction and expression
MP029-CRY2olig-mCherry plasmid was created by amplifying CRY2oligo-mCherry from Addgene Plasmid #60032 by PCR using 5′- CTGTGCGGCCGCGCCATGAAGATGGACAA-3′ and 5′- CTGTGCCGCTGTACAGCTCGTCCAT-3′ primers, and subsequent cloning into the MP029 vector using the NotI/KpnI restriction sites. MP029 is a lentiviral vector with a ubiquitin promoter allowing for moderate to low expression levels, a kind gift from Dr. Murali Palangat (National Cancer Institute, NIH).73 MP029-CRY2-mCherry-SUN2 variants were created by amplifying human full-length SUN2 or SUN2-N-1 (AA 1-260) and SUN2-N-2 (AA 1-226) fragments by PCR from pcDNA3.1+/C-(K)DY-SUN2 vector (OHu01874,GenScript # NM_001199579.1) and subsequent cloning into MP029-CRY2-mCherry lentiviral vector using the NheI/XbaI restriction sites. Forward PCR primer for full length SUN2, and SUN-N-1 and -N-2 variants was 5- CTGTGCTAGCGCCATGTCCCGAAGAAGCCA-3′ and reverse primers were 5′ CTGTTCTAGAGCCCTAGTGGGCGGGCTCC-3′ for full length SUN2, 5′- CTGTTCTAGAGCTCAGCCCTCATCCGGCCTC-3′ for SUN2-N-1 variant and 5′- CTGTTCTAGACTACAGGCACGTCAGCAAGAG-3′ for SUN2-N-2 variant. The construct consisting of nucleoplasmic and transmembrane domains of SUN2 and the luminal domain of SUN1 was created by amplifying SUN1 C-terminal fragment (AA 245-511) by PCR from pcDNA3.1+/C-(K)DY-SUN1 vector (OHu26731,GenScript # NM_001130965.3) using
5′-CTGTTCTAGAATGTTGGCTGGCCGTGG-3′ forward and
5′-CTGTCTCGAGGCCCTGACTTGCACGTCCA-3′ reverse primers, and subsequent cloning into MP029-CRY2-mCherry-SUN2-N-2 vector using the XbaI/XhoI restriction sites. MP029-CRY2-mCherry-SUN2-C variant was created by amplifying C-terminal part of human SUN2 (AA 440-717) by PCR from pcDNA3.1+/C-(K)DY-SUN2 vector (OHu01874,GenScript # NM_001199579.1) using 5′-CTGTGCTAGCATGTCCCGGGTACACTCTC-3′ forward and
5′-CTGTTCTAGACTAGTGGGCGGGCTCCCCATGC-3′ reverse primers, and subsequent cloning into MP029-CRY2-mCherry lentiviral vector using the NheI/XbaI restriction sites.
mCherry-DN-KASH1 plasmid was purchased from Addgene (#125553) and pmCherry-N1 plasmid was purchased from Clontech (#632523). mScarlet-KASH2 and mScarlet-KASH2ΔL plasmids were gift from Dr. Gregg Gundersen (Columbia University).50
Constructs were transfected into HEK293T and hTERT-immortalized inducible fibroblasts using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions.
Immunofluorescence staining
Cells were grown either on glass coverslips (for confocal, SIM and STED microscopy) or on 384-well (Perkin Elmer, 6057300) and 96-well (Matrical, MGB096-1-2-LG-L) microplates for high-throughput imaging, washed once with PBS and fixed for 15 min with 4% paraformaldehyde (PFA). Fixed cells were then washed one time with PBS, permeabilized for 10 min (PBS/0.5% Triton X-100) and washed again. Next, cells were incubated for 1.5 hour with primary antibodies diluted in blocking buffer (PBS, 0.05% Tween20, 5% bovine serum albumin). After two consecutive washes with PBS, cells were incubated for 1 hour with secondary antibodies diluted in blocking buffer and washed once with PBS. SIM samples were mounted in Vectashield antifade mounting media with DAPI (Vector Laboratories), while STED samples were mounted in ProLong glass antifade mountant (Thermo Fisher Scientific). Cells grown on microplates were counterstained with DAPI (Biotium, #40043) or DAPI and HCS CellMask Deep Red Stain (Thermo Fisher Scientific, H32721) for 30 min and washed once with PBS. After washing, cells could be stored for extended periods of time in PBS at 4°C. All steps for IF staining were performed at ambient temperature. Primary antibodies used for immunofluorescence were: α-Hsp110 rabbit polyclonal (1:100, StressMarq, SPC-195), α-Hsp90 rat monoclonal (1:200, Enzo, ADI-SPA-835), α-Hsc70 rat monoclonal (1:500, Abcam, ab19136), α-Hsp40 rabbit polyclonal (1:500, StressMarq, SPC-100), α-GRP94 rabbit polyclonal (1:200, Abcam, ab3674), α-GRP78/BiP rabbit polyclonal (1:500, Abcam, ab21685), α-GRP78 mouse monoclonal (1:50, StressMarq, SMC-195), α-PDI P4HB mouse monoclonal (1:500, Abcam, ab2792), α-Calnexin rabbit polyclonal (1:100, Cell Signaling, 2433S), α-SUN1 rabbit polyclonal (1:100, Sigma, HPA008346), α-SUN2 rabbit polyclonal (1:500, Sigma, HPA001209), α -Nesprin 1 rabbit monoclonal (1:100, Abcam, ab192234),α-Nesprin 2 rabbit monoclonal (1:250, described in50), α-LAP2alpha rabbit polyclonal (LAP2a, 1:500, Abcam, ab5162), α-Lamin B1 rabbit polyclonal (1:200, Abcam, ab16048), α-Histone H3 (tri methyl K27) rabbit monoclonal (1:400, Abcam, ab192985), α-acetyl-Histone H4 (Ac-Lys16) rabbit polyclonal (1:400, Sigma-Aldrich, # H9164), α-Ki67 mouse monoclonal (1:200, DB Biosciences, #610968), and α-PCNA mouse monoclonal (1:200, Abcam, ab29). Secondary antibodies used for immunofluorescence detection in confocal and SIM microscopy were Alexa Fluor Goat-anti-Mouse 568 (1:500, Invitrogen, A11004) and Donkey-anti-Mouse 647 (1:500, Invitrogen, A31571), Alexa Fluor Donkey-anti-Rabbit 568 and 647 (1:500, Invitrogen, A10042 and A31573), Alexa Fluor Goat-anti-Rat 568 (1:500, Invitrogen, A11077) and Chicken-anti-Rat 647 (1:500, Invitrogen, A21472). Secondary antibodies used for STED detection were Abberior Goat-anti-Rabbit STAR RED (1:400, Abberior, STRED-1002-500UG) and AffiniPure Fab Fragment Goat Anti-Mouse Alexa Fluor 594 (1:200, Jackson Immunoresearch, 115-587-003).
Immunoprecipitation
hTERT-immortalized doxycycline inducible dermal fibroblasts were lysed in IP buffer (20mM Tris-HCl pH7.5, 150mM NaCl, 2mM EGTA, 2mM MgCl2, 0.5% NP-40, 1mM DTT, 1U/ml Benzonase, 1x Protease inhibitor cocktail set from Milipore) for 1h at 4°C and cleared of insoluble material by centrifugation. 1mg of extract was then incubated with 2μg of α-GRP78/BiP rabbit polyclonal antibody (Abcam, ab21685) or 2μg normal rabbit IgG antibody (Cell Signaling, 2729S) overnight, followed by incubation with Pierce protein A/G magnetic beads (Thermo Fisher Scientific) at 4°C for 4h. Beads were thoroughly washed 3x in IP buffer (w/o Benzonase), and precipitated proteins were eluted using 1x Laemmli sample buffer (Bio-Rad), followed by Western blot analysis. Antibody used for Western blot detection was α-GFP rabbit polyclonal (Abcam, ab290) and α-GRP78/BiP rabbit polyclonal (Abcam, ab21685).
Western blotting
Total cell lysates were prepared by dissolving ~750, 000 cells in 150μί 1x Laemmli sample buffer (Bio-Rad) and subsequently denatured for 10 min at 95°C. Equal amounts of protein extract were loaded onto 4-15% Mini-PROTEAN TGX precast gels (Bio-Rad), separated by SDS-PAGE, and transferred onto PVDF membrane using a Trans-Blot turbo transfer system according to manufacturer instructions (Bio-Rad). Membranes were blocked for 30 min in 5% BSA/TBS-T block buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20) and incubated with primary antibody diluted in 2% BSA/TBS-T overnight at 4°C. Membranes were washed 3x 15 min in TBS-T and incubated with appropriate HRP-conjugated secondary antibodies for 1h at room temperature. Protein detection was performed with the ECL western blotting detection system (Amersham) and imaged using a Bio-Rad ChemiDoc imaging system and ImageLab 6.0.1 software. Primary antibodies used for western blotting were: α-IRE1 (phospho S724) rabbit monoclonal (1:2000, Abcam, ab124945), α-IRE1 rabbit polyclonal (1:2000, Abcam, ab37073), α-Phospho-PERK (Thr982) rabbit monoclonal (1:1000, Abcam, ab192591), α-SUN1 rabbit polyclonal (1:2000, Sigma, HPA008346), α-SUN2 rabbit polyclonal (1:5000, Sigma, HPA001209), α-GFP rabbit polyclonal (1:5000, Abcam, ab290), γ-tubulin (1:5000, Sigma T6557) and α-GAPDH mouse monoclonal (1:10000, Abcam, ab8245). Secondary antibodies used were Mouse-anti-rabbit IgG-HRP (1:15000, Santa Cruz, sc-2357) and Goat-anti-Mouse IgG-HRP (1:15000, Santa Cruz, sc-2005).
RNA isolation and quantitative analysis of gene expression
Total RNA was extracted from cells using the NucleoSpin RNA Kit (Takara Bio) according to manufacturer instructions. Total RNA from flash frozen liver, heart and aorta tissues was isolated from 25-50 mg ground tissue. Briefly, tissue was homogenized in 600 μl lysis buffer using a Bullet Blender, 0.9-2.0 mm stainless steel beads for 3 mins, setting 12 at 4°C, total RNA was isolated using RNeasy Mini Kit (Qiagen) and the integrity of the RNA was assessed by Bioanalyzer using RNA 6000 Nano Chip (Agilent, Inc.). mRNA levels of UPR genes in control and progerin-expressing cells and mice were measured by reverse transcribing 1 μg of RNA using iScript cDNA synthesis kit containing a blend of oligo(dT), random hexamer primers and iScript™ Reverse Transcriptase according to manufacturer recommendation (Bio-Rad). Equal volumes of cDNA were used as template in a real-time quantitative PCR reaction using iQ SYBR Green Supermix (Bio-Rad) on a CFX384 Touch Real-Time PCR System (Bio-Rad). Reaction conditions were: 3 min at 95°C, 1 cycle; 20 s at 95°C, 30 s at 58°C, 40 cycles. Melting curves of the amplified product were generated to verify that a single amplicon was generated. All the values were normalized to the internal controls glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-box-binding protein 1 (TBP1) genes. Primer combinations are indicated in Table S2. Expression values are shown as means ± s.d. of biological triplicates and statistical significance between biological replicates was determined by paired two-tailed Student t-test in Graphpad Prism and Excel.
siRNA transfection
For siRNA treatment hTERT immortalized GFP-progerin doxycycline inducible dermal fibroblasts were plated on a 10 cm2 culture well (6-well) at 70-80% confluence in high glucose DMEM containing 15% FCS and 0.2mM L-glutamine 16 h prior to transfection. Cells were transfected on two consecutive days with each siRNA (working concentration of 10nM) and 5μl Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to manufacturer instructions. After 48h, cells were collected and plated onto 384-well plates for high-throughput imaging analysis. Silencing efficiency of each protein was monitored by western blot analysis and immunofluorescence. All experiments were performed in triplicates. siRNAs used were Silencer Select negative control (Ambion, #4390843), Silencer Select UNC84A siRNA (Ambion, #4392420) and custom UNC84B siRNA 5′-CGUACCAGGUGGUGGAGCUGCGGAU-3′ (Dharmacon).
High-throughput image acquisition
Cells were imaged in four channels (405, 488, 561, and 640 nm excitation lasers) in an automated fashion using a dual spinning disk high-throughput confocal microscope (Yokogawa CV7000S) with a 405/488/561/640 nm excitation dichroic mirror, a 40 air objective lens (NA 0.95) or a 60X water immersion lens (NA = 1.2), a 568 nm excitation dichroic mirror, and two 16-bit sCMOS cameras (Andor, 2560 X 2160 pixels) with binning set to 2 (Pixel size: 0.325 nm for 40X and 0.216 nm for 60X nm). Emission bandpass filters were used for each channel: 445/45, 525/50, 600/37, and 690/29 nm. Nine randomly selected fields of view were imaged per well in a single optimal focal plane. Images were corrected on the fly using a geometric correction for camera background, illumination field (vignetting), camera alignment, and chromatic aberrations. Corrected images were saved and stored as 16-bit TIFF files.
Light microscopy
Laser scanning confocal microscopy was performed using a Carl Zeiss LSM880 inverted microscope controlled by ZEN software with definite focus. Cells were imaged using Plan Apochromat 63×/1.46 NA oil objective and images were acquired in three channels (405, 488 and 561nm excitation lasers). Raw images were reconstructed using the appropriate tool in the Zeiss Zen software and ImageJ. In vivo FRAP experiments were performed using a Carl Zeiss LSM780 inverted microscope controlled by ZEN software and with definite focus. Live cells were imaged using 63×/1.46 NA oil objective and a CO2/heating control stage insert. Induced MP029-CRY2olig-mCherry-SUN2-N clusters were bleached using 488 and 561 nm light with maximum laser power and recovery was monitored over a period of 2–5 min.
Super resolution microscopy and image quantification
Structured Illumination Microscopy was performed on fixed cells using a Zeiss Elyra PS.1 on an AxioObserver Z1 inverted microscope controlled by Zen software (Carl Zeiss). Images were acquired in four channels (405, 488, 561, and 640 nm excitation lasers) using Plan Apochromat 63x/1.4 NA oil immersion objective (Carl Zeiss) and Pco.edge sCMOS camera in a 30°C environmental chamber. Raw images were reconstructed using the appropriate tool in the Zeiss Zen software (Black version with Structured Illumination module). Stimulated Emission Depleted (STED) microscopy was performed on fixed cells using a commercial Leica SP8 STED 3X system (Leica Microsystems), equipped with a white light laser (range, 470–670 nm) and 592-nm, 660-nm and pulsed 775-nm STED depletion lasers. Special STED white objective 100×/1.4-NA oil-immersion objective lens (HCX PL APO STED white, Leica Microsystems) was used for imaging and sequential confocal and STED images were taken in three channels via three sequential excitations. GFP labelled probes were excited with the 488 nm white light laser and depleted with a 592 nm STED laser at 25% power, Alexa Fluor 594 probes were excited with the 570 nm wavelength white light laser and depleted with a 775 nm STED laser at 20% power and Abberior STAR RED probes were excited with the 647 nm white light laser and depleted with a 775 nm STED laser at 20% power. All images were acquired in 2D STED mode with a Z-stack size of 0.12μm, a scan speed of 600 lines per second, a pixel size of 50–80 nm (512 × 512 pixels) and 8 lines averages. Deconvolution of STED data was carried out using the STED module in Huygens Professional software version 14.10.1 (Scientific Volume Imaging) using the classical maximum likelihood estimation algorithm and a STED saturation factor of 9.
Live cell imaging and protein clustering induction
96-well plates (Matrical, MGB096-1-2-LG-L) were coated for 1h with 0.1% gelatin solution (ATCC, PCS-999-027) and washed twice with PBS (pH 7.4, Thermo Fisher Scientific). HEK293FT cells were plated on the gelatin coated dish and grown overnight in normal growth medium to reach ~ 70% confluency. Constructs were transfected on the next day using Lipofectamine LTX (Thermo Fisher Scientific) according to manufacturer instructions and live cell imaging was performed 24h after the transfection. Just prior to imaging, the medium was replaced with 150μl of imaging medium (Phenol Red free DMEM medium containing 15% fetal bovine serum). Livecell high-throughput imaging experiments were performed using a Yokogawa CV8000 high-throughput dual spinning disk confocal microscope with 60x water immersion lens (NA = 1.2). Cells were incubated in an environmental chamber at 37°C, 5% CO2 and 80% humidity. For protein clustering induction, cells were imaged by use of two laser wavelengths (488 nm for CRY2 activation /560 nm for mCherry imaging). To execute CRY2 activation, the repetitive ON/OFF cycle was applied every 10 min for 90 min (488nm activation duration was fixed to 200ms with 60% laser power in all measurements). Image z-stacks of 5 planes at a 1 μm interval were acquired using a heated Olympus PlanApoChromat 60X water lens objective (NA 1.2), and 2 sCMOS cameras (Hamamatsu, QE 82%, 2048X 2048 pixels) using camera binning of 2X2. A total of 16 fields of view were acquired. Images were corrected on the fly using a geometric correction for camera alignment and optical aberrations and saved and stored as 16-bit TIFF files. Data was reconstructed with Fiji software and images were montaged with Adobe Photoshop CC 2019 and Adobe Illustrator 2023 software.
Cell sorting and protein clustering induction on a 24-well plate
For fluorescence-activated cell sorting (FACS), cells were transfected with MP029-CRY2olig-mCh, MP029-CRY2olig-mCh-SUN2- N-1, MP029-CRY2olig-mCh-SUN2-N-2 or MP029-CRY2olig-mCh-SUN2-C using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) according to manufacturer instructions. After 24h cells were trypsinized, collected in PBS and examined for mCherry expression using a BD FACSAria Cell Sorting System with BD FACSDiva Software (BD Biosciences). Non-fluorescent cells were negatively selected, and the remaining gated cells were sorted based on the presence of mCherry fluorescent protein. Sorted cells were plated on a 24-well plate at a density of 5-6x105 cells/well for RNA isolation or on a 8-well microscopy chamber at a density of 1x105 cells/well for immunofluorescence and left to recover overnight. The next day CRY2 activation was executed using a UV LED light source (M405L4, ThorLabs) operated by LED Driver (DC2200, ThorLabs). Repetitive ON/OFF cycle was applied every 10 sec for 60 min (405nm activation duration was fixed to 200ms with 5% power in all measurements). Cells were then either fixed with 4% PFA for immunofluorescence analysis or collected for RNA isolation and subsequent RT-qPCR analysis as described above.
QUANTIFICATION AND STATISTICAL ANALYSIS
Captured images from high-throughput imaging experiments were analyzed using Columbus 2.8.1 or 2.9.1 (Perkin Elmer). Briefly, nuclei regions of interest (ROI) were segmented using the DAPI (405 nm) channel and cytoplasmic based ROIs were segmented using the CellMask HCS channel (640 nm). Cytoplasm and nucleus ROIs adjacent to the image edges were excluded from subsequent image analysis steps. The mean fluorescence intensity for the nucleus and cytoplasmic ROI was measured in the 561 nm channel. Analysis of the recruitment of GRP78/BiP to the nuclear periphery was done by creating a ring ROI covering 5-35% of the distance from the DAPI-based nuclear ROI (where 0% is adjacent to DAPI and 100% represents the cell border) and the mean fluorescence intensity per well for ring ROI was measured in 561nm channel. Columbus results were exported as comma-separated text files and analyzed using R. 300-1500 cells were analyzed per condition. Data are plotted as means of 3 biological replicates +/− standard deviation (SD).
High-throughput imaging experiments in inducible cell lines were performed on three technical replicates (wells) and averaged to obtain the measurement for that experiment. The experiments were repeated independently three times (n = 3), and the values for each experiment were averaged. Data calculations were performed using R software and paired two-tailed Student’s t-test for the fold change (FC) indicated in the figures was used for statistical analyses. High-throughput imaging experiments in immortalized and primary patient-derived fibroblasts were performed on three technical replicates (wells) and the experiments were repeated three times (n = 3) for subsequent passage numbers. Single cell data analysis was performed using R software and the data are shown as the average mean values of 3 different experiments and/or the percentage of cells showing increase in the phenotype relative to the wild-type control. The threshold for quantification of increases was set as mean of the wild-type +/− 1 SD. Statistical analysis for the average mean values of single cell data was performed by an ANOVA one-way test, followed by a post-hoc Dunnett’s test using the cell line as the predictor variable and fixing WT as the negative control. Statistical analysis to determine any difference between cell lines for the percentage of cells showing increase or decrease was performed by Chi-square test. Corresponding p-value is shown in every figure.
For FRAP experiments, all FRAP measurements were pooled together and averaged (n=20). Fitted FRAP curve was obtained by fitting the recovery portion to the equation R = C + P[1 – exp(-kt)].
Images obtained from light and super-resolution microscopy were reconstructed with Fiji software and montaged with Adobe Photoshop CC 2019 and Adobe Illustrator 2023 software. Local enrichment of SUN2, GRP78 and PDI was measured by drawing ROI at the nuclear periphery using the corresponding tools in Fiji or Zeiss Zen software for the number of cells indicated in each figure. Paired two-tailed Student’s t-test was used for statistical analyses and corresponding p-value is shown in every figure.
All other calculations were performed using Graphpad Prism software and Excel and paired two-tailed Student’s t-test was used for statistical analyses. Error bars represent standard deviation (s.d.) or standard error of the mean (s.e.m.) as indicated. Statistical significance was classified as follows: *p<0,05, **p<0,005, ***p<0,0005. Biological replicates represent 3 different experiments performed at different times and with different batches of cells. Graphical abstract was created with BioRender (BioRender.com).
Supplementary Material
Highlights.
Aggregation of the progerin protein in the nucleus triggers ER stress
Induction of ER stress in progeria depends on the nuclear membrane protein SUN2
SUN2 forms progerin-induced protein clusters at the nuclear periphery
SUN2 clusters sequester ER chaperones and trigger an ER stress response
ACKNOWLEDGMENTS
We thank the Misteli lab members for sharing feedback, data, and reagents. We are grateful to Susan Shackleton (University of Leicester, UK), Kyle Roux (Sanford School of Medicine, University of South Dakota), Chandra Tucker (University of Colorado School of Medicine), Gregg Gundersen (Columbia University, NYC), and Murali Palangat (National Cancer Institute, NIH) for providing plasmids; Gregg Gundersen (Columbia University, NYC) for providing the nesprin-2 antibody; Francis Collins (National Human Genome Research Institute, NIH) for providing HGPS G608G mice; Carlos Lopez-Otin (University of Oviedo, Spain) for providing tissue of HGPS G609G mice; Tatiana Karpova and David Ball for help with SIM, laser scanning confocal microscopy, and FRAP experiments as part of the NIH/NCI/CCR LRBGE Optical Imaging Core; the NIH/NCI/CCR High Throughput Imaging Facility (HiTIF) for help with high-throughput imaging and automated liquid handling; and Daniela Malide and Christian Combs at the NIH/NHLBI Light Microscopy Core for help with STED imaging. The T.M. lab is supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research, and S.V. was supported by FWF Austrian Science Fund (Vienna, Austria), grant number J 3849.
Footnotes
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2023.112534.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All original code has been deposited at Github and is publicly available as of the date of publication at https://doi.org/10.5281/zenodo.7829552.
Raw data used for high-throughput image analysis have been deposited at Figshare, https://doi.org/10.6084/m9.figshare. 22253287.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.