From the Cover: STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum

Abstract

Store-operated calcium entry relies on the formation of a specialized compartment derived from the endoplasmic reticulum (ER) and closely apposed to the plasma membrane. In this study, detailed ultrastructural analysis revealed the existence of three distinct structures derived from conventional ER: precortical ER, cortical ER, and thin cortical ER. Precortical subdomains of the ER enriched in STIM1 can form without contacting the plasma membrane. Upon ER calcium depletion, these subdomains are translocated to the plasma membrane to form cortical ER, which is still connected to the conventional ER. Thin cortical ER, depleted of BiP and deprived of attached ribosomes, may represent a specialized region dedicated to calcium regulation and not engaged in protein translocation and folding. These observations form the basis for future structure-function analysis of cortical ER.

Fig. 1.

Calcium depletion stimulates the formation of cortical ER. HeLa cells were treated for 10 min with Tg (1 μM) to deplete ER calcium stores. They were then fixed, embedded in Epon resin, sectioned and observed. (A–C) In cells transfected with YFP-STIM1, calcium depletion induced the formation of large sheets of cER (asterisks) apposed to the PM (closed circle), which were often aligned along microtubules (arrowheads). Note the connection between cER and ER cisternae (ER) in A, as well as a region of very close contact between the PM and the cER (arrow). Ribosomes were seen only on thick regions of cER, but absent in thinner regions. The bottom of the culture dish appears in (B) as a straight, dense line on top of the picture. (D and E) In nontransfected HeLa cells, local apposition of the ER to the PM also became apparent upon Tg treatment. Both thick (D) and thin (E) cER was observed. The corresponding quantitative analysis is presented in Table 1.

Fig. 2.

Thin cER is enriched in STIM1 and depleted of BiP. HeLa cells expressing YFP-STIM1 were fixed and processed for cryo-immuno electron microscopy. (A and B) YFP-STIM1, revealed with an anti-GFP antibody and 15-nm diameter gold particles was highly enriched in thin cER (asterisks) apposed to the PM (closed circle) relative to conventional ER cisternae (ER). (C and D) Colabeling with anti-GFP (15-nm diameter gold) and anti-BiP (10-nm diameter gold). BiP is excluded from cER. The corresponding quantitative analysis is presented in Table 2. In an area distant from the PM, a thin ER region enriched in STIM1 and depleted of BiP is visible (arrowhead in D).

Fig. 3.

Formation of double-layered cortical ER. HeLa cells transfected with YFP-STIM1 and depleted of calcium occasionally exhibited two layers of cortical ER (asterisks) apposed to the PM (closed circle). The bottom of the culture dish appears as a straight, dense line below the cell. Analysis of serial sections revealed that the cisterna facing the cytosol did not establish a contact with the PM (Fig. S3).

Fig. 4.

A constitutively active mutant of STIM1 induces multilayered cortical ER elements. HeLa cells were transfected with a constitutively active STIM1 mutant. (A) These cells exhibited occasionally highly developed stacks of thin ER cisternae apposed to the PM. Of the entire stack, only one cisterna established a direct contact with the PM. (N) nucleus. (B) Labeling of cryosections with an anti-GFP antibody revealed the accumulation of mutant YFP-STIM1 in these stacks.

Fig. 5.

Precortical ER can form in the cytosol. HeLa cells transfected with YFP-STIM1 and treated with Tg were fixed and processed for electron microscopy. (A and B) In Epon sections, thinner regions of the ER deprived of ribosomes (arrows) were seen aligned along microtubules (arrowheads). These elements were still connected to ER cisternae (ER) or to the nuclear envelope (star). Their morphology was similar to that of cER (asterisks) apposed to the PM (closed circles). Ly, lysosome; N, nucleus; np, nuclear pore. (C) In cryosections, labeling with an anti-GFP antibody (15-nm diameter gold) revealed a high concentration of YFP-STIM1 in cytosolic precortical elements. (D) In sections colabeled with an anti-BiP antibody (10-nm diameter gold) exclusion of BiP from thin YFP-STIM1-enriched domains was visible.