scholarly journals The architecture of the endoplasmic reticulum is regulated by the reversible lipid modification of the shaping protein CLIMP-63

2018 ◽  
Author(s):  
Patrick A. Sandoz ◽  
Robin A. Denhardt-Eriksson ◽  
Laurence Abrami ◽  
Luciano Abriata ◽  
Gard Spreemann ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cysteine Residue ◽  
Relative Distribution ◽  
Post Translational Modification ◽  
Combining Data

AbstractThe endoplasmic reticulum (ER) has a complex morphology generated and maintained by membrane-shaping proteins and membrane energy minimization, though not much is known about how it is regulated. The architecture of this intracellular organelle is balanced between large, thin sheets that are densely packed in the perinuclear region and a connected network of branched, elongated tubules that extend throughout the cytoplasm. Sheet formation is known to involve the cytoskeleton-linking membrane protein 63 (CLIMP-63), though its regulation and the depth of its involvement remain unknown. Here we show that the post-translational modification of CLIMP-63 by the palmitoyltransferase ZDHHC6 controls the relative distribution of CLIMP-63 between the ER and the plasma membrane. By combining data-driven mathematical modeling, predictions, and experimental validation, we found that the attachment of a medium chain fatty acid, so-called S-palmitoylation, to the unique CLIMP-63 cytoplasmic cysteine residue drastically reduces its turnover rate, and thereby controls its abundance. Light microscopy and focused ion beam electron microcopy further revealed that enhanced CLIMP-63 palmitoylation leads to strong ER-sheet proliferation. Altogether, we show that ZDHHC6-mediated S-palmitoylation regulates the cellular localization of CLIMP-63, the morphology of the ER, and the interconversion of ER structural elements in mammalian cells through its action on the CLIMP-63 protein.Significance StatementEukaryotic cells subcompartmentalize their various functions into organelles, the shape of each being specific and necessary for its proper role. However, how these shapes are generated and controlled is poorly understood. The endoplasmic reticulum is the largest membrane-bound intracellular compartment, accounting for more than 50% of all cellular membranes. We found that the shape and quantity of its sheet-like structures are controlled by a specific protein, cytoskeleton-linking membrane protein 63, through the acquisition of a lipid chain attached by an enzyme called ZDHHC6. Thus, by modifying the ZDHHC6 amounts, a cell can control the shape of its ER. The modeling and prediction technique used herein also provides a method for studying the interconnected function of other post-translational modifications in organelles.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis

2021 ◽  
Author(s):  
Toshiyuki Goto ◽  
Shuhei Torii ◽  
Aoi Kondo ◽  
Junji Kawakami ◽  
Haruka Yagi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Translational Regulation ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Early Embryogenesis ◽  
Axis Formation ◽  
Second Phase ◽  
Dynamic Changes ◽  
Maternal Mrna ◽  
Maternal Mrnas

AbstractAxis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded “dense ER” using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.

scholarly journals Insight into the Characteristics of Novel Desmin-Immunopositive Perivascular Cells of the Anterior Pituitary Gland Using Transmission and Focused Ion Beam Scanning Electron Microscopy

2021 ◽  
Vol 22 (16) ◽  
pp. 8630
Author(s):  
Depicha Jindatip ◽  
Rebecca Wan-Yan Poh ◽  
Ken Fujiwara
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Type ◽  
Insight Into ◽  
Scanning Electron

Recently, another new cell type was found in the perivascular space called a novel desmin-immunopositive perivascular (DIP) cell. However, the differences between this novel cell type and other nonhormone-producing cells have not been clarified. Therefore, we introduced several microscopic techniques to gain insight into the morphological characteristics of this novel DIP cell. We succeeded in identifying novel DIP cells under light microscopy using desmin immunocryosection, combining resin embedding blocks and immunoelectron microscopy. In conventional transmission electron microscopy, folliculostellate cells, capsular fibroblasts, macrophages, and pericytes presented a flat cisternae of rough endoplasmic reticulum, whereas those of novel DIP cells had a dilated pattern. The number of novel DIP cells was greatest in the intact rats, though nearly disappeared under prolactinoma conditions. Additionally, focused ion beam scanning electron microscopy showed that these novel DIP cells had multidirectional processes and some processes reached the capillary, but these processes did not tightly wrap the vessel, as is the case with pericytes. Interestingly, we found that the rough endoplasmic reticulum was globular and dispersed throughout the cytoplasmic processes after three-dimensional reconstruction. This study clearly confirms that novel DIP cells are a new cell type in the rat anterior pituitary gland, with unique characteristics.

scholarly journals MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel

2020 ◽  
Vol 117 (51) ◽  
pp. 32380-32385
Author(s):  
Michael W. Martynowycz ◽  
Farha Khan ◽  
Johan Hattne ◽  
Jeff Abramson ◽  
Tamir Gonen
Keyword(s):  
Membrane Protein ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Anion Channel ◽  
Voltage Dependent Anion Channel ◽  
X Ray ◽  
X Ray Crystallography ◽  
Voltage Dependent ◽  
Viscous Media ◽  
Scanning Electron

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Investigation of Cell-Sensor Hybrid Structures by Focused Ion Beam (FIB) Technology

2006 ◽  
Vol 983 ◽  
Author(s):  
Andreas Heilmann ◽  
Frank Altmann ◽  
Andreas Cismak ◽  
Werner Baumann ◽  
Mirko Lehmann
Keyword(s):  
Cross Section ◽  
Mammalian Cells ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Nerve Cells ◽  
Cross Sectional ◽  
Dimensional Reconstruction ◽  
The Cross ◽  
Silicon Interface

AbstractFor the investigation of the adhesion of mammalian cells on a semiconductor biosensor structure, nerve cells on silicon neurochips were prepared for scanning electron microscopy investigations (SEM) and cross-sectional preparation by focused ion beam technology (FIB). The cross-sectional pattern demonstrates the focal adhesion points of the nerve cells on the chip. Finally, SEM micrographs were taken parallel to the FIB ablation to investigate the cross section of the cells slice by slice in order to demonstrate the spatial distribution of focal contact positions for a possible three-dimensional reconstruction of the cell-silicon interface.

scholarly journals Regulation of NRF1, a master transcription factor of proteasome genes: implications for cancer and neurodegeneration

2020 ◽  
Vol 31 (20) ◽  
pp. 2158-2163 ◽  
Author(s):  
Amy Northrop ◽  
Holly A. Byers ◽  
Senthil K. Radhakrishnan
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Membrane Protein ◽  
Neurodegenerative Diseases ◽  
Mammalian Cells ◽  
Therapeutic Strategy ◽  
De Novo ◽  
Degradation Pathway ◽  
Special Focus ◽  
Homeostatic Mechanism

The ability to sense proteasome insufficiency and respond by directing the transcriptional synthesis of de novo proteasomes is a trait that is conserved in evolution and is found in organisms ranging from yeast to humans. This homeostatic mechanism in mammalian cells is driven by the transcription factor NRF1. Interestingly, NRF1 is synthesized as an endoplasmic reticulum (ER) membrane protein and when cellular proteasome activity is sufficient, it is retrotranslocated into the cytosol and targeted for destruction by the ER-­associated degradation pathway (ERAD). However, when proteasome capacity is diminished, retrotranslocated NRF1 escapes ERAD and is activated into a mature transcription factor that traverses to the nucleus to induce proteasome genes. In this Perspective, we track the journey of NRF1 from the ER to the nucleus, with a special focus on the various molecular regulators it encounters along its way. Also, using human pathologies such as cancer and neurodegenerative diseases as examples, we explore the notion that modulating the NRF1-proteasome axis could provide the basis for a viable therapeutic strategy in these cases.

scholarly journals Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

2013 ◽  
Vol 24 (17) ◽  
pp. 2703-2713 ◽  
Author(s):  
Philip D. Fox ◽  
Christopher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Jenny L. Higgins ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Surface Proteins ◽  
Hek 293 ◽  
Cortical Endoplasmic Reticulum

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

scholarly journals Contacts between the endoplasmic reticulum and other membranes in neurons

2017 ◽  
Vol 114 (24) ◽  
pp. E4859-E4867 ◽  
Author(s):  
Yumei Wu ◽  
Christina Whiteus ◽  
C. Shan Xu ◽  
Kenneth J. Hayworth ◽  
Richard J. Weinberg ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Physiology ◽  
Organelle Biogenesis ◽  
Axon Terminals ◽  
Narrow Lumen ◽  
Dynamics And Control ◽  
Membrane Contacts ◽  
And Control

Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca2+ dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER–plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER–PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies.

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