Ratiometric sensing of BiP-client versus BiP levels by the unfolded protein response determines its signaling amplitude

Abstract

Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µ_s), is well-tolerated in HeLa cells. Upon µ_s expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µ_s model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.

Keywords: cell biology; endoplasmic reticulum; human; proteostasis; unfolded protein response.

Figure 1.. Tunicamycin has pleiotropic cytotoxic effects.

(A) HeLa S3 cells were treated with Tm at various concentrations and for various durations, as indicated, before mRNA was isolated from cells for RT-PCR analysis with oligos specific for XBP1. PCR fragments corresponding to spliced (XBP^S) and unspliced (XBP^U) XBP1 were separated on gel. A hybrid product that is formed during the PCR reaction (Shang and Lehrman, 2004) is denoted by an asterisk. The ratio of (XBP^S)/(XBP^S +XBP^U) is indicative of the extent of IRE1α activation. (B) Cells as in (A) were seeded upon 1:5 serial dilution into 24-well plates and treated continuously or only for the first 4 hr upon seeding with various concentrations of Tm, as indicated. After 7 days of growth, cells were fixed and stained with crystal violet. (C) Quantitation of the crystal violet staining shown in (B) as a measure of cell growth, except for conditions that fully abrogated growth; n.a. = not assessed. Staining of untreated cells was set at 100%, and values are shown in a bar graph. Mean and s.e.m. are shown; n = 3. Statistical significance in a one-sample t-test of differences in crystal violet staining compared to untreated samples in (B) and replicate experiments was determined as a proxy for growth (**p≤0.01). (D) Cell growth assay as in (B) of HeLa S3 cells in which UPR transducers were deleted (IRE1α KO) or silenced (PERK KD and ATF6α KD) or not (WT), treated with Tm at various concentrations, as indicated.

Figure 1—figure supplement 1.. Efficiency of ATF6α and PERK silencing.

HeLa-µ_s cells were transfected with siRNAs (KD) targeting ATF6α or PERK or not (WT). Levels of ATF6α and PERK were assessed as in Figure 2A; 1 mM DTT treatment for 1 hr was used as a positive reference for ATF6α cleavage.

Figure 2.. A model for proteostatically induced ER stress.

(A–F,H–J) Expression of µ_s in HeLa-µ_s cells was induced with 0.5 nM Mif. (A,G,H) The UPR was pharmacologically induced as a reference for 4 hr with 5 µg/ml Tm, or—for analysis of ATF6α in (A) only—for 1.5 hr with 300 nM Tg (since Tm leads to deglycosylation of ATF6α). (A,D) Immunoblotting revealed levels of µ_s, CHOP, BiP and α-tubulin. A shift to a lower mobility phosphorylated form, P-PERK, and the appearance of CHOP revealed activation of PERK. The release of the p50 cleavage product from the p90 precursor revealed activation of ATF6α. Cross-reaction of the secondary antibody against anti-ATF6α with µ_s is denoted. (A,G) Splicing of XBP1 was assessed as in Figure 1A. (A) Three different clones of HeLa-µ_s cells with decreasing µ_s expression levels: high (Hi), medium (Me), or low (Lo) were induced for 16 hr. Non-treated (nt) or Tm-treated Hi HeLa-µ_s served as references. (B,C,H) Immunofluorescence revealed µ_s (red) and CRT (blue), which marks the ER (C) at the indicated times before or after induction (B) or after 8 hr induction (C,H). Nuclei were stained with DAPI (blue) (B). (C,H) The area that is boxed is shown by 3.5-fold magnification; scale bars represent 10 µm. (D) Samples were derived from equal numbers (7 × 10⁴) of HeLa-µ_s or I.29µ⁺ lymphomas induced with Mif or stimulated with 20 µg/ml lipopolysaccharide (LPS), respectively, for the indicated times. Ponceau staining of the blot serves as a loading control. (E) HeLa-µ_s cells were pulse labeled with ³⁵S labeled methionine and cysteine for 10 min at the indicated times after induction. Immunoprecipitated µ_s was resolved by gel electrophoresis (F). Levels of radio-labeled µ_s in (E) were quantified by phosphor imaging. The maximal signal for µ_s (at 64 hr) was set at 100. (G) HeLa-MifON cells were treated with a high dose of Mif (10 nM) for the indicated times. (H) In HeLa-µ_s cells in which IRE1α was replaced with doxycycline (Dox)-inducible IRE1α-GFP (green), IRE1α-GFP expression was tuned with 10 nM Dox to levels that allowed satisfactory detection of IRE1α-GFP by fluorescence microscopy. (I) Cell growth assay as in Figure 1B of HeLa-MifON and HeLa-µ_s induced continuously with Mif or not. (J) Quantitation of (I), performed as in Figure 1C. Mean and s.e.m. are shown, n = 2. There is no statistical significance in a one-sample t-test of differences in growth between conditions.

Figure 2—figure supplement 1.. Functional reconstitution of IRE1α-KO cells with IRE1α-GFP.

Wild-type (WT) or IRE1α knock out (KO) HeLa cells, whether reconstituted with IRE1α-GFP or not, were treated (+) or not (–) with 5 µg/ml Tm for 4 hr. XBP1 mRNA splicing was assessed as in Figure 1A. Functional reconstitution did not require Dox induction of IRE1α-GFP, as ‘leaky’ expression was sufficient.

Figure 3.. The accumulation of clients in the ER rather than the flux of secretory load drives the UPR.

(A–D) HeLa clones constructed to express λ inducibly, either alone (λ) or in conjunction with µ_s (µ_s + λ) at different stoichiometries, ranging from µ_s being in excess to λ being in excess, all under control of Mif, were induced with 0.5 nM in parallel to the HeLa-µ_s cells for 16 hr to express IgM subunits. Non-treated (nt) HeLa-µ_s cells served as a reference. Supernatants (secreted) and cell lysates (intracellular) were separated by reducing (A) or non-reducing (B) gel electrophoresis. Levels of µ_s, λ (A), CHOP, BiP (C) and α-tubulin (A,C), as well as activation of the UPR pathways (C) were assessed as in Figure 2A. (B) Immunobloting of µ_s (red) and λ (green) reveals disulfide linked assembly intermediates, as indicated: µλ, µ₂, µ₂λ₂, and high molecular weight (HMW) polymeric assemblies of µ_s and λ. Simultaneous immunodetection of both IgM subunits resulted in yellow signal. (D) Quantitation of BiP levels shown in (C) and replicate experiments. Mean and s.e.m. are shown, n = 3. Statistical significance in a one-sample t-test of differences in BiP levels is indicated (*p≤0.05). (E) Schematic of BiP associating with the C_H1 domain of µ_s until it is displaced by λ (if available).

Figure 4.. ER homeostatic readjustment entails a transition from acute to chronic UPR signaling.

(A–C) HeLa-µ_s cells were induced with 0.5 nM Mif for various times as indicated. Levels of µ_s, BiP, PDI (A) and α-tubulin (A,C), as well as activation of the UPR pathways (C), were assessed as in Figure 2A. (B) Levels of µ_s and BiP were quantified using known quantities of BiP and µ_s as standards. The X-axis displays time in hrs in a logarithmic scale to show better detail of the early phase of the time course. Experiments were normalized to the level of BiP at t = 64 hr, which was reproducibly ~10 fold that at t = 0 hr. Quantities of BiP and µ_s are expressed in equivalents of BiP levels at t = 0 hr; mean and s.e.m. are shown, n = 5. Fitting was performed with Prism software to obtain a sigmoidal dose-response curve. As quantities were not assessed at the same time points in each experiment, some values were inferred from fit curves to obtain the s.e.m. Statistical significance of differences in expression levels between BiP and µ_s were tested by two-tailed t-test, and are depicted in a color-based heat map ranging from dark blue for [BiP] > [µ_s] (***p≤0.001), via purple for when there is no statistical significance for differences between the levels of BiP and µ_s, that is [BiP] ≈ [µ_s], to red for [µ_s] > [BiP] (*p≤0.05). (D) Activation of the UPR branches in (C) and replicate experiments were quantified (n = 2–20, depending on the time point assessed): for IRE1α, the percentage of XBP1 mRNA splicing; for PERK, the levels of expression of its downstream effector CHOP; and for ATF6α, the levels of ATF6α-p50 (both the latter normalized on the levels of α-tubulin). Mean values of UPR signaling output at each time point were calculated as a percentage of the maximal level, and are depicted in a color-based heat map ranging from no color (min) to full color (max).

Figure 4—figure supplement 1.. Transitioning from acute, full-geared to chronic, submaximal IRE1α signaling upon µ_s expression.

Percentages of XBP1 mRNA splicing in Figure 4C and replicate experiments were quantified and expressed in percentages of the maximal XBP1 mRNA splicing that was reached. Mean and s.e.m. are shown (n = 3–22 depending on the time point assessed).

Figure 4—figure supplement 2.. Transitioning from acute, full-geared to chronic, submaximal PERK signaling upon µ_s expression.

Levels of CHOP in Figure 4C and replicate experiments were quantified, normalized to α-tubulin levels, and expressed in percentages of the maximal CHOP level that was reached. Mean and s.e.m. are shown (n = 1–10 depending on the time point assessed).

Figure 4—figure supplement 3.. Transitioning from acute, full-geared to chronic, submaximal ATF6α signaling upon µ_s expression.

Levels of ATF6α-p50 in Figure 4C and replicate experiments were quantified, normalized to α-tubulin levels, and expressed in percentages of the maximal ATF6α-p50 level that was reached. Mean and s.e.m. are shown (n = 1–3 depending on the time point assessed).

Figure 4—figure supplement 4.. UPR pathways are not exhausted upon chronic µ_s overexpression.

HeLa-µ_s cells were induced for 128 hr to express µ_s or not, prior to treatment with 5 mg/ml Tm for 4 hr or 300 nM Tg for 1.5 hr (for analysis of ATF6α). Levels of µ_s and activation of the UPR pathways were assessed as in Figure 2A.

Figure 5.. ER expansion in the course of homeostatic readjustment to µ_s overexpression.

HeLa-µ_s-derived cells, harboring Dox-inducible APEX-KDEL, were induced with 0.5 nM Mif to express µ_s for various days as indicated, and APEX-KDEL expression was induced with 100 nM Dox for 2 days. APEX-KDEL was exploited to obtain DAB precipitates (dark), revealing the extent of the ER in electron micrographs. Boxed areas are shown by 3-fold magnification; scale bars represent 1 µm. The percentage of the area within the cytoplasm corresponding to ER was determined and depicted in bar graphs; mean and s.e.m. are shown, n = 10. Statistical significance of differences in the extent of ER occupying cytosolic area in the electron micrographs was tested by ANOVA (*p≤0.05; ***p≤0.001).

Figure 5—figure supplement 1.. APEX-KDEL expression hardly interferes with µ_s-driven UPR.

HeLa-µ_s cells expressing APEX-KDEL induced at low levels with 100 nM Dox for 2 days (+) or not (–) were induced with 0.5 nM Mif to express µ_s for the indicated times (days), and XBP1 mRNA splicing was assessed as in Figure 1A.

Figure 6.. BiP becomes dominant in the ER when it expands upon µ_s overexpression.

HeLa-µ_s cells were induced with Mif to express µ_s for various numbers of days as indicated. Proteins were identified at each time point by mass spectrometry and expression levels were approximated by label-free proteomics. The ~600 most abundant proteins, accounting for over 90% of the total protein content, were categorized according to localization in the cell—cytosolic, nuclear, mitochondrial, ER resident, or residing in other organelles—by the use of Uniprot entries. From the approximated quantities of these proteins, the quantities of proteins per organelle were calculated as a percentage of the total proteome and depicted in pie charts (upper panels). For the ER, the resident proteins (gold) and µ_s (red) are shown separately. Quantities of ER resident proteins including µ_s were calculated as a percentage of the ER proteome and are depicted in pie charts (lower panels) that are size-proportioned commensurate with the difference in combined quantity of ER and µ_s at different days. For details on calculations, see Supplementary file 1. Color-coding of pie charts is as annotated in the legend embedded in the figure.

Figure 7.. The UPR is essential to sustain ER homeostatic readjustment upon µ_s expression.

(A,B,C) All UPR transducers were ablated, either by deletion (KO, IRE1α) or by silencing (KD, ATF6α and PERK) in HeLa-µ_s cells, and µ_s expression was induced with 0.5 nM Mif (+) or not (–), as indicated. (A,B) Cell growth was assessed as in Figure 1B and quantitated as in Figure 1C (B). Mean and s.e.m. are shown in a bar graph; n = 2. (C) Percentages of Annexin-V-positive cells before or upon induction of µ_s expression for 3 days were assessed by cytometric analysis. Means and s.e.m. are shown in a bar graph, n = 2. (B,C) Statistical significance of differences in growth (B) or Annexin V staining (C) were tested by ANOVA (**p≤0.01; ***p≤0.001).