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First published online 30 May 2006
doi: 10.1242/jcs.02977

Journal of Cell Science 119, 2532-2541 (2006)
Published by The Company of Biologists 2006
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ER storage diseases: a role for ERGIC-53 in controlling the formation and shape of Russell bodies

L. Mattioli1, T. Anelli2,3, C. Fagioli2, C. Tacchetti1, R. Sitia2,3,*,{ddagger} and C. Valetti1,{ddagger}

1 MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genova, Italy
2 DiBiT-San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy
3 Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy


Figure 1
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  		Fig. 1. RB-like structures form in the absence of L chains when µ{Delta}CH1 synthesis is increased. (A) Induction of RB in myeloma transfectants. N[µ{Delta}CH1], N[µ1] and J[µ{Delta}CH1] cells were treated with or without 1 mM sodium butyrate (NaBut) for 40 hours, fixed and stained with fluorescent anti-µ antibodies. Note that RB-like structures are induced by NaBut treatment in N[µ{Delta}CH1] but not in N[µ1]. As previously reported (Valetti et al., 1991Go), RB are present also in untreated, {lambda} producing J[µ{Delta}CH1] cells; bar, 5 µm. (B) The presence of RB correlates with accumulation of detergent-insoluble µ{Delta}CH1 chains. Cells were lysed in TX100 and equal amounts of soluble (s) and insoluble (i) fractions resolved under reducing conditions, blotted and decorated with anti-µ. (C,D) Different RB morphology in myeloma cells is dependent on L chain synthesis. J[µ{Delta}CH1] and N[µ{Delta}CH1] cells, the latter treated with 1 mM NaBut for 40 hours, were processed for both Epon embedding (C) and cryo-sectioning (D). Ultrathin cryo-sections were immuno-gold labeled using anti-µ antibodies and protein A-gold (15 nm). Arrowheads point to membrane-associated ribosomes. Bar, 100 nm. (E) The table summarizes the RB diameters (expressed in µm0 in N[µ{Delta}CH1] cells treated with 1 mM NaBut for 40 hours, and in untreated J[µ{Delta}CH1] cells. Measurements were carried out on electron microscope images. 60 and 30 RB were measured in N[µ{Delta}CH1] and J[µ{Delta}CH1] cells respectively, each from two independent preparations.

 

Figure 2
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  		Fig. 2. Pharmacological inhibition of proteasomal degradation favors sRB formation. N[µ{Delta}CH1] cells were cultured for 24 hours with or without 1 mM sodium butyrate (NaBut). Aliquots of cells treated with NaBut were exposed to MG132 for the last 2 hours in culture. The histogram depicts the percentage of cells containing µ{Delta}CH1 sRB structures and represents the average of two independent experiments.

 

Figure 3
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  		Fig. 3. Induction of sRB in non-lymphoid cells. (A) Time-dependent formation of sRB in H[µ{Delta}CH1] induced to synthesize µ{Delta}CH1 chains. After culture in the absence of tetracycline for the indicated periods, cells were harvested, fixed and stained with fluorescent anti-µ; bar 5 µm. The inset in the center panel (day 4) shows a magnification of a cell containing a small µ-positive dot. (B) sRB formation correlates with accumulation of detergent-insoluble µ{Delta}CH1 chains in H[µ{Delta}CH1]. Aliquots of detergent soluble (s) and insoluble (i) material from cells treated as in A were analyzed by western blotting as described in legend to Fig. 1. (C) sRB localize preferentially in the vicinity of the MTOC. H[µ{Delta}CH1] cells cultured for 5 days without tetracycline were simultaneously stained with anti-{gamma}-tubulin and anti-µ antibodies; bar 5 µm. (D) Dose-dependent formation of sRBs imply a threshold concentration for µ{Delta}CH1 condensation. H[µ{Delta}CH1] cells were cultured for 5 days in medium supplemented with decreasing doses of tetracycline as indicated. µ{Delta}CH1 chains were quantified in extracts containing total (TOT, lysis in SDS) or TX100 soluble (SOL) and insoluble (INS) proteins and expressed as arbitrary units. At the tetracycline concentration of 0.25 ng/ml or more, most µ{Delta}CH1 proteins are found in the soluble fraction. At 0.12 ng/ml detergent-insoluble chains become preponderant and continue to increase at lower concentrations, when more µ{Delta}CH1 chains are synthesized.

 

Figure 4
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  		Fig. 4. The presence or absence of L chains dictates the site of RB accumulation. HeLa Tet-Off cells were transiently transfected with µ{Delta}CH1 expression vectors alone (a,d,g) or in combination with wild-type {lambda} (L; b,e,h) or with a mutant {lambda} lacking the constant domain (VL; c,f). µ{Delta}CH1 appears red, whereas Ac38 and ERGIC-53 are green; bar 5 µm. g and h are imagines of resin-embedded EM sections. In cells expressing L chains, numerous ribosomes are uniformly distributed on the RB membrane. Electron density of RB content demonstrates high protein density. Bars, 200 nm.

 

Figure 5
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  		Fig. 5. In the absence of L chains, µ{Delta}CH1 condense primarily in tubular vesicles and saccules enriched in ERGIC-53. (a-f) H[µ{Delta}CH1] cells were cultured with (a,b) or without tetracycline for 3 (c,d) or 7 days (e,f) and stained with antibodies against ERGIC-53 (a,c,e in green), GM130 (b,d,f; green) or µ (c-f; red). Note that ERGIC-53 abandons its canonical distribution (a) to co-localize with µ{Delta}CH1-containing sRB upon tetracycline removal (panels c,e yellow staining). µ{Delta}CH1 accumulates close to GM130, but does not overlap with it (d,f); bar, 5 µm. (g,h) After 7 days of culture without tetracycline, cells were processed for cryo-EM with anti-µ (g) or anti-ERGIC-53 (h, G1/93) antibodies and revealed with protein-A coupled to 15 nm gold particles. µ{Delta}CH1 chains accumulate in saccules enriched in ERGIC-53. Bars, 100 nm. (i,j) H[µ{Delta}CH1] cells were kept for 7 days without tetracycline and processed for resin embedding and EM. Continuity between rough ER and µ{Delta}CH1-containing sRB are evident in j (see arrow; the arrowheads point to regions rich in ribosomes). Bars, 100 nm.

 

Figure 6
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  		Fig. 6. µ{Delta}CH1 binds to ERGIC-53 in a Ca2+-dependent manner. (A) Ca2+-dependent co-localization of ERGIC-53 and µ{Delta}CH1. After 5 days of culture without tetracycline, H[µ{Delta}CH1] cells were treated for 2 hours with or without 50 µM CPA (top right and left panels, respectively), a drug that reversibly depletes intracellular Ca2+ stores. CPA was then washed out and cells were cultured in Ca2+-containing medium for 15 and 45 minutes (bottom left and right panels, respectively) before fixation and co-staining with anti-µ (red) and anti-ERGIC-53 (green); bar 5 µm. (B) ERGIC-53 is recruited to the insoluble fraction in cells expressing µ{Delta}CH1. Detergent-soluble (s) and insoluble (i) fractions were obtained from cells treated with or without tetracycline as indicated. In order to preserve Ca2+-dependent interactions, EDTA was omitted from lysis buffer, and 1 mM CaCl2 added (lanes 3-6). An aliquot of cells was lysed in the presence of EDTA (lanes 1-2). Aliquots were resolved under reducing conditions, and blotted. The filter was sequentially decorated with anti-ERGIC-53 and anti-µ. (C) Ca2+-dependent co-immunoprecipitation of ERGIC-53 and µ{Delta}CH1. H[µ{Delta}CH1] cells were cultured without tetracycline for 2 days and then transfected with ERGIC-53 wt, the KKAA or the lectin-deficient mutant N156A. After a further 40 hours in culture without tetracycline, cells were treated with 50 µM CPA for 2 hours to deplete intracellular Ca2+ stores (lanes 3-4), and cross-linked with DSP. Equal aliquots of the lysates were immunoprecipitated with anti-µ or anti-ERGIC-53, as indicated. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-Myc, in order to detect co-precipitated exogenous ERGIC-53. (D) The amount of ERGIC-53 co-immunoprecipitated with µ{Delta}CH1 was quantified and expressed as the percentage relative to total immunoprecipitable material (IP with anti-ERGIC-53). Average of two independent experiments.

 

Figure 7
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  		Fig. 7. Mannose trimming is required for µ{Delta}CH1 condensation and efficient sRB formation. (A,B) Kifunensine causes accumulation of µ{Delta}CH1 but no sRB formation in myeloma transfectants. N[µ{Delta}CH1] were treated for 40 hours with sodium butyrate (NaBut, 1 mM) or with kifunensine (Kif, 2 µg/ml), to increase the expression or prevent the degradation of µ{Delta}CH1, respectively. Cells were then fixed and processed for immunofluorescence microscopy with anti-µ (A, bar 5 µm) or lysed in TX100. Equal amounts of soluble (s) and insoluble (i) lysate fractions were analyzed by western blotting (B). (C,D) Kifunensine causes accumulation of detergent-soluble µ{Delta}CH1 in the ER of HeLa cells. Cells were cultured for 5 days without tetracycline, with or without 2 µg/ml kifunensine, and processed for immunofluorescence microscopy (C, bar 5 µm) or western blotting (D). Kifunensine was re-added every 24 hours (Tokunaga et al., 2000Go). In both lymphoid and non-lymphoid cells, kifunensine caused accumulation of abundant µ{Delta}CH1, which was mainly in the soluble fraction when mannose trimming was prevented. As expected, µ{Delta}CH1 chains present in kifunensine-treated cells display slower electrophoretic mobility. (E,F) The association between ERGIC-53 and µ{Delta}CH1 is inhibited by kifunensine. H[µ{Delta}CH1] cells were cultured in the absence of tetracycline for 2 days, with or without kifunensine (Kif, 2 µg/ml, lanes 5-6) and then transfected with ERGIC-53 wt. After further 40 hours of culture, cells were treated with 50 µM CPA for 2 hours (lanes 3-4) before co-immunoprecipitation and quantification, as described in the legend to Fig. 6C. Kifunensine was present for the entire duration of the experiment.

 

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© The Company of Biologists Ltd 2006