Inhibition of Rtn4a by antibody addition to the in vitro reaction did not block vesicle fusion but prevented the fused vesicles from adopting an elongated tubule-like morphology. The reticulon family member Rtn4a was identified based on its modification by sulfhydryl reagents that inhibit the assembly of ER tubules ( Voeltz et al., 2006). However, the development of systems for assembly of ER tubules from vesicles in vitro has led to some molecular insight. Relatively little is known about the factors that shape ER tubules and sheets, how the domains within the contiguous ER network maintain their distinct morphologies, or how transitions in the organization of the ER network during cell cycle progression are orchestrated. This has been particularly well characterized in Xenopus laevis oocytes, where electron microscopy revealed the formation of “mitotic ER clusters” between 1 and 5 μm in diameter composed of packed smooth ER tubules and cisternae ( Terasaki et al., 2001). In eggs from a variety of vertebrate and invertebrate species, there is a dramatic clustering of the peripheral ER network during mitosis ( Bobinnec et al., 2003 Poteryaev et al., 2005 Stricker, 2006). The peripheral ER also undergoes cell cycle–dependent changes. After the chromosomes separate in anaphase, nuclear envelopes reform around each of the separated chromatin masses ( Mattaj, 2004 Margalit et al., 2005 Prunuske and Ullman, 2006). One prominent example is in animal cells, where the nuclear envelope disassembles during mitotic entry to promote spindle assembly. The ER is also structurally reorganized during cell cycle progression. ER tubules in the periphery of mammalian cells continuously form and fuse, generating a meshwork characterized by the presence of “three-way” junctions between tubules that can move relative to one another ( Lee and Chen, 1988 Waterman-Storer and Salmon, 1998 Estrada de Martin et al., 2005). Visualization in living cells has revealed the dynamic nature of the ER network. Resident INM proteins pass from the ONM to the INM by diffusion or active transport through the nuclear pores and concentrate in the INM as a result of interactions with the underlying chromatin and the nuclear lamina ( Gerace and Burke, 1988 Soullam and Worman, 1995 Ellenberg et al., 1997 Holmer and Worman, 2001 Ohba et al., 2004 Gruenbaum et al., 2005 King et al., 2006). Nuclear pores, gated channels between the cytoplasm and the nuclear interior, pass through both membrane bilayers and are sites where the INMs and ONMs are fused to each other ( Salina et al., 2001 Hetzer et al., 2005 Tran and Wente, 2006). The membrane on one side of the sheet, the outer nuclear membrane (ONM), faces the cytoplasm, and on the opposite side of the lumen, the inner nuclear membrane (INM) faces the chromatin. The nuclear envelope, perhaps the most highly differentiated region of the ER, is a polarized sheet that regulates the movement of macromolecules between the nuclear space and the cytoplasm ( Hetzer et al., 2005 Prunuske and Ullman, 2006). In contrast, smooth ER, a site for lipid synthesis, contact with other organelles, and vesicle budding and fusion, lacks ribosomes and is often tubular ( Baumann and Walz, 2001). Rough ER, specialized for protein synthesis and folding, is often found in ribosome-studded sheets. The differing morphologies exhibited by ER domains likely contribute to their distinct functions. The thickness of ER sheets is similar to the diameter of ER tubules, typically 60–100 nm, suggesting that common structural elements underlie these morphologically distinct forms ( Shibata et al., 2006). Structurally, the ER network consists of membrane tubules, flattened sheets, and cisternae.
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