scholarly journals Cargo crowding contributes to sorting stringency in COPII vesicles

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Natalia Gomez-Navarro ◽  
Alejandro Melero ◽  
Xiao-Han Li ◽  
Jérôme Boulanger ◽  
Wanda Kukulski ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Secretory Pathway ◽  
Light And Electron Microscopy ◽  
Secretory Proteins ◽  
Vesicle Size ◽  
Cargo Protein ◽  
Er Retention ◽  
Biophysical Process ◽  
Membrane Bound ◽  
Copii Vesicles

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.

scholarly journals Cargo crowding drives sorting stringency in COPII vesicles

2020 ◽  
Author(s):  
Natalia Gomez-Navarro ◽  
Alejandro Melero ◽  
Jerome Boulanger ◽  
Wanda Kukulski ◽  
Elizabeth A. Miller
Keyword(s):  
Electron Microscopy ◽  
Secretory Pathway ◽  
Light And Electron Microscopy ◽  
Bulk Flow ◽  
Secretory Proteins ◽  
Er Quality Control ◽  
Vesicle Size ◽  
Cargo Protein ◽  
Biophysical Process ◽  
Copii Vesicles

AbstractAccurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy dictates ER retention: in the absence of abundant cargo, non-specific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein, or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates lumenal steric pressure that drives selectivity. Sorting stringency is thus an emergent property of the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.SummaryCombining correlative light and electron microscopy with yeast genetics and biochemistry, Gomez-Navarro, Melero and colleagues show that cargo recruitment into a constrained COPII vesicle restricts bulk flow, thereby contributing to sorting stringency and ER quality control.

scholarly journals SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Xiao-Wei Chen ◽  
He Wang ◽  
Kanika Bajaj ◽  
Pengcheng Zhang ◽  
Zhuo-Xian Meng ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Plasma Cholesterol ◽  
Negative Regulator ◽  
Secretory Proteins ◽  
Clearance Mechanism ◽  
Cargo Proteins ◽  
Survival And Development ◽  
Copii Vesicles ◽  
Partial Overlap

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes.

scholarly journals Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles

2016 ◽  
Vol 27 (12) ◽  
pp. 1938-1947 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transforming Growth Factor Α ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Copii Vesicles ◽  
Factor Α ◽  
Cytosolic Factor

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.

scholarly journals Distinct stages in the recognition, sorting and packaging of proTGFα into COPII coated transport vesicles

2016 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Factor Alpha ◽  
Copii Vesicles ◽  
Cytosolic Factor

AbstractIn addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former employs more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor alpha (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.AbbreviationsCNIHCornichon

Cytology of mucilage production in the seed coat of Candle canola (Brassica campestris)

1981 ◽  
Vol 59 (3) ◽  
pp. 292-300 ◽  
Author(s):  
L. Van Caeseele ◽  
J. T. Mills ◽  
M. Sumner ◽  
R. Gillespie
Keyword(s):  
Electron Microscopy ◽  
Seed Coat ◽  
Brassica Campestris ◽  
Light And Electron Microscopy ◽  
Epidermal Cells ◽  
Starch Grains ◽  
Golgi Bodies ◽  
Tangential Wall ◽  
Membrane Bound

The development of mucilage in the epidermal cells of canola seeds (Brassica campestris L. cv. Candle) was studied with light and electron microscopy from 5 days after pollination to maturity. During the first 17 days starch was deposited in amyloplasts. At or near the 17th day mucilage appeared between the plasmalemma and the outer tangential wall of the epidermal cells. As the volume of mucilage increased, starch grains disappeared and were totally absent by 25 days. Membrane-bound structures and Golgi bodies were visible within the cytoplasm adjacent to the site of mucilage deposition. At maturity the seed epidermal cells were totally devoid of cytoplasm and engorged with mucilage.

scholarly journals Photonic-chip assisted correlative light and electron microscopy

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jean-Claude Tinguely ◽  
Anna Maria Steyer ◽  
Cristina Ionica Øie ◽  
Øystein Ivar Helle ◽  
Firehun Tsige Dullo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Total Internal Reflection ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Large Field ◽  
Target Area ◽  
Liver Sinusoidal Endothelial Cells ◽  
Area Of Interest ◽  
Cargo Protein ◽  
Complex Organization

AbstractCorrelative light and electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The photonic chips are used as a substrate to hold, to illuminate and to provide landmarking of the sample through specially designed grid-like numbering systems. Using this approach, we demonstrate its applicability for tracking the area of interest, imaging the three-dimensional (3D) structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations (trans-cytoplasmic nanopores), and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis.

Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis

1991 ◽  
Vol 100 (1) ◽  
pp. 119-131 ◽  
Author(s):  
C.R. Morales ◽  
Y.K. Kwon ◽  
N.B. Hecht
Keyword(s):  
Electron Microscopy ◽  
Translational Regulation ◽  
Light And Electron Microscopy ◽  
Cytoplasmic Localization ◽  
Chromatoid Body ◽  
Translational Arrest ◽  
Membrane Bound ◽  
Transition Protein ◽  
Protamine 1

During spermatogenesis in mammals, the transcripts of transition protein 1 (TP 1) and protamine 1 (Prm 1) are under translational regulation. Following their transcription in round spermatids, the mRNAs for TP 1 and Prm 1 are stored in the cytoplasm from 3–7 days before being translated towards the end of spermatogenesis. To test the hypothesis that the inactivation or activation of transcripts during spermiogenesis could be mediated by mRNA compartmentalization in the cytoplasm of spermatids, light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of stored and translated mRNAs for these two testis-specific transcripts. During early spermiogenesis (before step 7) nuclear transcripts of both TP 1 and Prm 1 were seen. After step 7 the TP 1 and Prm 1 mRNAs were only detected in the cytoplasm. Throughout spermiogenesis the cytoplasmic mRNAs were not localized to any membrane-bound organelles such as the endoplasmic reticulum or mitochondria or to non-membrane-bound structures such as the chromatoid body. These studies demonstrate that the translational arrest of the TP 1 and Prm 1 mRNAs is not primarily controlled by compartmentalized storage in the cytoplasm of spermatids. Moreover, when translation of these mRNAs occurs in elongated spermatids, the mRNAs are present throughout the cytoplasm.

scholarly journals Inhibiting Endoplasmic Reticulum (ER)-associated Degradation of Misfolded Yor1p Does Not Permit ER Export Despite the Presence of a Diacidic Sorting Signal

2007 ◽  
Vol 18 (9) ◽  
pp. 3398-3413 ◽  
Author(s):  
Silvere Pagant ◽  
Leslie Kung ◽  
Mariana Dorrington ◽  
Marcus C.S. Lee ◽  
Elizabeth A. Miller
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Er Quality Control ◽  
Er Retention ◽  
Copii Vesicles ◽  
Er Export ◽  
Cystic Fibrosis Transmembrane

Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the “B-site” cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-ΔF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-ΔF by inhibiting its degradation does not permit access of Yor1p-ΔF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.

scholarly journals Differential screening identifies molecules specifically inhibiting CCR5 transport to the cell surface and HIV infection

2018 ◽  
Author(s):  
Gaelle Boncompain ◽  
Floriane Herit ◽  
Sarah Tessier ◽  
Aurianne Lescure ◽  
Elaine Del Nery ◽  
...  
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Secretory Proteins ◽  
Target Cells ◽  
Anterograde Transport ◽  
Chemical Libraries ◽  
Membrane Bound ◽  
Inhibitory Molecules ◽  
G Protein Coupled ◽  
Hiv 1

AbstractProteins destined to the cell surface are conveyed through membrane-bound compartments using the secretory pathway. Multiple secretory routes exist in cells, which paves the way to the development of inhibitory molecules able to specifically perturb the transport of a chosen cargo. We used differential high-content screening of chemical libraries to identify molecules reducing the secretion of CCR5, the major co-receptor for HIV-1 entry. Three molecules strongly affected the anterograde transport of CCR5, without inhibiting the transport of the related G protein-coupled receptors CCR1 and CXCR4. These three molecules perturb the transport of endogenous CCR5 and decrease the entry of HIV in human primary target cells. Two molecules were found to share the same mode of action, inhibiting palmitoylation of CCR5. Our results demonstrate that secretory routes can be specifically targeted which allows to envisage novel strategies to provoke the intracellular retention or rerouting of secretory proteins involved in disease development.

The Morphology of Vacuolated Cells in the Liver Following Administration of Vitamin A.

1973 ◽  
Vol 31 ◽  
pp. 408-409
Author(s):  
Odell T. Minick ◽  
Hidejiro Yokoo ◽  
Fawzia Batti
Keyword(s):  
Electron Microscopy ◽  
Body Weight ◽  
Vitamin A ◽  
Light And Electron Microscopy ◽  
Liver Cells ◽  
Prominent Feature ◽  
Vascular Space ◽  
Vacuolated Cell ◽  
Vacuolated Cells ◽  
Vacuolated Cytoplasm

Vacuolated cells in the liver of young rats were studied by light and electron microscopy following the administration of vitamin A (200 units per gram of body weight). Their characteristics were compared with similar cells found in untreated animals.In rats given vitamin A, cells with vacuolated cytoplasm were a prominent feature. These cells were found mostly in a perisinusoidal location, although some appeared to be in between liver cells (Fig. 1). Electron microscopy confirmed their location in Disse's space adjacent to the sinusoid and in recesses between liver cells. Some appeared to be bordering the lumen of the sinusoid, but careful observation usually revealed a tenuous endothelial process separating the vacuolated cell from the vascular space. In appropriate sections, fenestrations in the thin endothelial processes were noted (Fig. 2, arrow).

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