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. 2019 Dec 20;22:70-80.
doi: 10.1016/j.isci.2019.10.067. Epub 2019 Nov 2.

Calumenin-1 Interacts With Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets

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Free PMC article

Calumenin-1 Interacts With Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets

Birong Shen et al. iScience. .
Free PMC article

Abstract

The ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

Keywords: Cell Biology; Membrane Architecture; Molecular Biology; Molecular Interaction; Organizational Aspects of Cell Biology.

Conflict of interest statement

The authors declare no competing interests.

Figures

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Figure 1
Figure 1
Climp63 Determines the Luminal Width of ER Sheets (A) Structure prediction of full-length Climp63 by coiled-coil domain prediction software tool LOGICOIL. It reveals that four coiled coils are formed by the luminal segment of Climp63. The red line stands for the Marcoil confidence level. Diagram depicting Climp63 domains is shown at the bottom. Numbers indicate amino acids (a.a.) of Climp63. Cyto, cytoplasmic domain; TM, transmembrane domain; Lumi, luminal domain. (B) Cell lysates from wild-type (WT) and Climp63 knockout (KO) cells were immunoblotted with anti-Climp63 antibody. GAPDH serves as a loading control. (C) Representative electron microscopy images of ER in wild-type or Climp63-knockout U2OS cells transfected with the indicated Climp63 mutant plasmids. Boxed regions are magnified below. Scale bar: 100 nm. (D) Quantification of the ER profile sheet luminal widths for (C). Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, determined by unpaired two-tailed Student's t tests. (E) Quantification of the luminal widths of nuclear envelope profile for (C). Data represent mean ± SD. (F) Electron microscopy images of the ER in COS7 cells with overexpressed GFP or SP-GFP-Climp63 (132–602). SP, signal peptide (of Calu1). The boxed regions are magnified below. Scale bar: 100 nm. (G) Quantification of the ER profile sheet luminal widths for (F). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t test.
Figure 2
Figure 2
Calu1 Interacts with Climp63 and Regulates ER Sheet Luminal Width through Climp63 (A) Representative electron microscopy images of the ER sheets of COS7 cells with overexpressed GFP or GFP-Calu1. Boxed regions are magnified below. Scale bar: 100 nm. (B) Quantification of the ER profile sheet luminal widths for (A). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t test. (C) Immunoprecipitation (IP) assays of Climp63-Flag by overexpressed GFP or GFP-Calu1 in HEK293T cells. Cell extracts of Climp63-HA were mixed with cell extracts of GFP or GFP-Calu1 and then used for immunoprecipitation with anti-GFP antibody. The precipitates were immunoblotted with anti-GFP, anti-HA, and anti-GAPDH antibodies. GAPDH serves as a loading control. (D and E) Reciprocal endogenous immunoprecipitation assays with anti-Calu1 (D) or anti-Climp63 (E) antibodies in HEK293T cells. Normal IgG was used as a negative control. (F) Immunoprecipitation assays of Climp63-HA by GFP-Calu1 or GFP-Calu2 with anti-GFP antibody in HEK293T cells. (G) Representative electron microscopy images of the ER sheets of Climp63-knockout U2OS cells with overexpressed GFP or GFP-Calu1. Boxed regions are magnified below. Scale bar: 100 nm. (H) Quantification of the ER profile sheet luminal widths for (G). Data represent mean ± SD. n.s., not significant, determined by unpaired two-tailed Student's t test. See also Figures S1, S4, and S5.
Figure 3
Figure 3
Loss of Calu1 Causes ER Sheet Accumulation (A) Western blotting of wild-type (WT) and Calu1-knockout (KO) COS7 cells by anti-Calu1 antibody. GAPDH serves as a loading control. (B) Representative confocal microscope images of wild-type, Calu1-knockout, and Calu1-knockout COS7 cells transfected with GFP-Calu1 plasmid (rescue). All cells are expressing mCherry-KDEL as an ER marker. Scale bar: 10 μm. (C) Quantification of relative distribution area of ER sheets to total ER of cells as in (B). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t tests. (D) Quantification of the relative average intensities of the perinuclear ER to peripheral ER in cells labeled by mCherry-KDEL as in (B). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t tests. (E) Quantification of the number of cells with juxtanuclear accumulation of ER as in (B). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t test. (F) Representative immunofluorescence images of wild-type and Calu1-knockout COS7 cells labeled with anti-Calnexin antibody. Scale bar: 10 μm. (G) Quantification of relative average intensities of the perinuclear ER to peripheral ER in cells labeled by endogenous Calnexin as in (F). Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t test. (H) Western blotting of wild-type, Calu1-knockout COS7 cells, and Calu1-knockout COS7 cells restored with GFP-Calu1 (rescue) by anti-Climp63, anti-Rtn4 (Reticulon, Rtn4b), and anti-Calu1 antibodies. GAPDH serves as a loading control. Note that the band for Calu1 in the third lane is a degraded band. (I) Quantification of relative band intensities between Climp63 and Rtn4b as in (H). Data represent mean ± SD. *p < 0.05, determined by unpaired two-tailed Student's t tests. See also Figure S2.
Figure 4
Figure 4
Calu1 Regulates ER Sheet Distribution in a Climp63-Dependent Manner (A) Western blotting of Calu1-knockout (KO) COS7 cells transfected with Climp63 shRNA and detected by anti-Climp63 antibody. GAPDH serves as a loading control. (B) Representative confocal microscope images of Calu1-knockout COS7 cells transfected with control shRNA, Climp63 shRNA, and Climp63 shRNA together with Climp63-GFP plasmid (Climp63 shRNA-resistant). All cells were expressing mCherry-KDEL as an ER marker. Scale bar: 10 μm. (C) Quantification of the relative average intensities of the perinuclear ER to peripheral ER in cells labeled by mCherry-KDEL as in (B). Calu1-knockout COS7 cells were transfected with control shRNA, Climp63 shRNA, or Climp63 shRNA and shRNA-resistant Climp63-GFP plasmid. Data represent mean ± SD. ***p < 0.001, determined by unpaired two-tailed Student's t tests. (D and E) Western blotting analysis and quantification of the knockdown efficiency of Calu1 in wild-type (WT) and Climp63-knockout U2OS cells. Data represent mean ± SD. *p < 0.05, **p < 0.01, determined by unpaired two-tailed Student's t tests. (F) Representative confocal microscope images of wild-type or Climp63-knockout U2OS cells transfected with mCherry-KDEL and the indicated shRNAs. Scale bar, 10 μm. (G) Quantification of the relative average intensities of the perinuclear ER to peripheral ER in cells labeled by mCherry-KDEL as in (F). Data represent mean ± SD, ***p < 0.001, n.s., not significant, determined by unpaired two-tailed Student's t tests. See also Figure S3.
Figure 5
Figure 5
Calu1 Inhibits Climp63-Microtubule Binding Instead of Affecting Self-Association of Climp63 (A) Immunoprecipitation assays of Climp63-3×Flag by Climp63-HA using anti-HA antibody in HEK293T cells in the presence of GFP or GFP-Calu1. The number on top of the bands is the ratio between Climp63-3×Flag and Climp63-HA with GFP or GFP-Calu1 overexpression. (B) FRAP assay of COS7 cells with overexpressed Climp63-mApple and GFP or GFP-Calu1. The relative intensity of Climp63-mApple in the bleached area was recorded. Data shown are the average traces from 25 cells. (C) Microtubule co-sedimentation assay of HEK293T cells with overexpressed Climp63-HA and GFP or GFP-Calu1. At 37°C, less Climp63 exists in the pellet fraction (P) upon GFP-Calu1 overexpression (P% = 36%) compared with GFP overexpression (P% = 60%). Note that all Climp63 is in the supernatant fraction (S) at 4°C because microtubules are depolymerized at low temperature. P, pellet, microtubule fraction; S, supernatant, free tubulin fraction.

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