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The endoplasmic reticulum adopts two distinct tubule forms.

ABSTRACT: The endoplasmic reticulum (ER) is a versatile organelle with diverse functions. Through superresolution microscopy, we show that the peripheral ER in the mammalian cell adopts two distinct forms of tubules. Whereas an ultrathin form, R1, is consistently covered by ER-membrane curvature-promoting proteins, for example, Rtn4 in the native cell, in the second form, R2, Rtn4 and analogs are arranged into two parallel lines at a conserved separation of ∼105 nm over long ranges. The two tubule forms together account for ∼90% of the total tubule length in the cell, with either one being dominant in different cell types. The R1–R2 dichotomy and the final tubule geometry are both coregulated by Rtn4 (and analogs) and the ER sheet–maintaining protein Climp63, which, respectively, define the edge curvature and lumen height of the R2 tubules to generate a ribbon-like structure of well-defined width. Accordingly, the R2 tubule width correlates positively with the Climp63 intraluminal size. The R1 and R2 tubules undergo active remodeling at the second/subsecond timescales as they differently accommodate proteins, with the former effectively excluding ER-luminal proteins and ER-membrane proteins with large intraluminal domains. We thus uncover a dynamic structural dichotomy for ER tubules with intriguing functional implications.

SUBMITTER: Wang B

PROVIDER: S-EPMC9170160 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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The endoplasmic reticulum adopts two distinct tubule forms.

Wang Bowen B Zhao Zhiheng Z Xiong Michael M Yan Rui R Xu Ke K

Proceedings of the National Academy of Sciences of the United States of America 20220426 18

The endoplasmic reticulum (ER) is a versatile organelle with diverse functions. Through superresolution microscopy, we show that the peripheral ER in the mammalian cell adopts two distinct forms of tubules. Whereas an ultrathin form, R1, is consistently covered by ER-membrane curvature-promoting proteins, for example, Rtn4 in the native cell, in the second form, R2, Rtn4 and analogs are arranged into two parallel lines at a conserved separation of ∼105 nm over long ranges. The two tubule forms t ...[more]

PMID: 35471903

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Project description:UnlabelledAlthough many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming.ImportanceIntracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

Project description:The water-soluble Nrf2 (nuclear factor, erythroid 2-like 2, also called Nfe2l2) is accepted as a master regulator of antioxidant responses to cellular stress, and it was also identified as a direct target of the endoplasmic reticulum (ER)-anchored PERK (protein kinase RNA-like endoplasmic reticulum kinase). However, the membrane-bound Nrf1 (nuclear factor, erythroid 2-like 1, also called Nfe2l1) response to ER stress remains elusive. Herein, we report a unity of opposites between these two antioxidant transcription factors, Nrf1 and Nrf2, in coordinating distinct cellular responses to the ER stressor tunicamycin (TU). The TU-inducible transcription of Nrf1 and Nrf2, as well as GCLM (glutamate cysteine ligase modifier subunit) and HO-1 (heme oxygenase 1), was accompanied by activation of ER stress signaling networks. Notably, the unfolded protein response (UPR) mediated by ATF6 (activating transcription factor 6), IRE1 (inositol requiring enzyme 1) and PERK was significantly suppressed by Nrf1-specific knockout, but hyper-expression of Nrf2 and its target genes GCLM and HO-1 has retained in Nrf1a-/- cells. By contrast, Nrf2-/-ΔTA cells with genomic deletion of its transactivation (TA) domain resulted in significant decreases of GCLM, HO-1 and Nrf1; this was accompanied by partial decreases of IRE1 and ATF6, rather than PERK, but with an increase of ATF4 (activating transcription factor 4). Interestingly, Nrf1 glycosylation and its trans-activity to mediate the transcriptional expression of the 26S proteasomal subunits, were repressed by TU. This inhibitory effect was enhanced by Nrf1a-/- and Nrf2-/-ΔTA, but not by a constitutive activator caNrf2ΔN (that increased abundances of the non-glycosylated and processed Nrf1). Furthermore, caNrf2ΔN also enhanced induction of PERK and IRE1 by TU, but reduced expression of ATF4 and HO-1. Thus, it is inferred that such distinct roles of Nrf1 and Nrf2 are unified to maintain cell homeostasis by a series of coordinated ER-to-nuclear signaling responses to TU. Nrf1 (i.e., a full-length form) acts in a cell-autonomous manner to determine the transcription of most of UPR-target genes, albeit Nrf2 is also partially involved in this process. Consistently, transactivation of ARE (antioxidant response element)-driven BIP (binding immunoglobulin protein)-, PERK- and XBP1 (X-box binding protein 1)-Luc reporter genes was mediated directly by Nrf1 and/or Nrf2. Interestingly, Nrf1 is more potent than Nrf2 at mediating the cytoprotective responses against the cytotoxicity of TU alone plus tBHQ (tert-butylhydroquinone). This is also further supported by the evidence that the intracellular reactive oxygen species (ROS) levels are increased in Nrf1a-/- cells, but rather are, to our surprise, decreased in Nrf2-/-ΔTA cells.

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The endoplasmic reticulum adopts two distinct tubule forms.

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The endoplasmic reticulum adopts two distinct tubule forms.

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