scholarly journals COPII-coated membranes function as transport carriers of intracellular procollagen I

2017 ◽  
Vol 216 (6) ◽  
pp. 1745-1759 ◽  
Author(s):  
Amita Gorur ◽  
Lin Yuan ◽  
Samuel J. Kenny ◽  
Satoshi Baba ◽  
Ke Xu ◽  
...  
Keyword(s):  
Vesicle Budding ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Copii Vesicle ◽  
Coated Membranes ◽  
Er Exit Sites ◽  
A Cell ◽  
Bona Fide ◽  
Copii Vesicles ◽  
Optical Reconstruction

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.

scholarly journals COPII-coated membranes function as transport carriers of intracellular procollagen-1

2017 ◽  
Author(s):  
Amita Gorur ◽  
Lin Yuan ◽  
Samuel J Kenny ◽  
Satoshi Baba ◽  
Ke Xu ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Imaging Techniques ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Coated Membranes ◽  
Copii Vesicles ◽  
Optical Reconstruction

AbstractThe coat protein complex II (COPII) is essential for the secretion of large cargo, such as the 300 nm precursor fibrils of procollagen I (PC1). Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles to over 300 nm in diameter and accelerates the secretion of PC1; however, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM), and live cell imaging we report the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We have also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude from in vivo and in vitro evidence that large COPII vesicles are bona fide carriers of PC1.SummaryCOPII may play a direct or indirect role in the traffic of large protein complexes such as procollagen. Using high resolution imaging techniques in intact cells and in vitro reconstituted vesicles, Gorur et al. show that COPII coated vesicles carry procollagen1.

scholarly journals Endosome–mitochondria interactions are modulated by iron release from transferrin

2016 ◽  
Vol 214 (7) ◽  
pp. 831-845 ◽  
Author(s):  
Anupam Das ◽  
Sagarika Nag ◽  
Anne B. Mason ◽  
Margarida M. Barroso
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Time Lapse ◽  
Iron Release ◽  
Iron Transfer ◽  
Distance Transformation ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Kinetics Of

Transient “kiss and run” interactions between endosomes containing iron-bound transferrin (Tf) and mitochondria have been shown to facilitate direct iron transfer in erythroid cells. In this study, we used superresolution three-dimensional (3D) direct stochastic optical reconstruction microscopy to show that Tf-containing endosomes directly interact with mitochondria in epithelial cells. We used live-cell time-lapse fluorescence microscopy, followed by 3D rendering, object tracking, and a distance transformation algorithm, to track Tf-endosomes and characterize the dynamics of their interactions with mitochondria. Quenching of iron sensor RDA-labeled mitochondria confirmed functional iron transfer by an interacting Tf-endosome. The motility of Tf-endosomes is significantly reduced upon interaction with mitochondria. To further assess the functional role of iron in the ability of Tf-endosomes to interact with mitochondria, we blocked endosomal iron release by using a Tf K206E/K534A mutant. Blocking intraendosomal iron release led to significantly increased motility of Tf-endosomes and increased duration of endosome–mitochondria interactions. Thus, intraendosomal iron regulates the kinetics of the interactions between Tf-containing endosomes and mitochondria in epithelial cells.

scholarly journals Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

2015 ◽  
Vol 112 (25) ◽  
pp. E3199-E3206 ◽  
Author(s):  
Kanika Bajaj Pahuja ◽  
Jinzhi Wang ◽  
Anastasia Blagoveshchenskaya ◽  
Lillian Lim ◽  
M. S. Madhusudhan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Direct Interaction ◽  
Phosphatidyl Inositol ◽  
Adaptor Proteins ◽  
Secretory Proteins ◽  
Vesicle Budding ◽  
Cargo Proteins ◽  
A Cell ◽  
Copii Vesicles ◽  
Serum Dependent

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.

scholarly journals Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels

2020 ◽  
Vol 13 (629) ◽  
pp. eaaw7963 ◽  
Author(s):  
Shihab Shah ◽  
Chase M. Carver ◽  
Pierce Mullen ◽  
Stephen Milne ◽  
Viktor Lukacs ◽  
...  
Keyword(s):  
Close Association ◽  
Drg Neurons ◽  
Noxious Heat ◽  
Proximity Ligation ◽  
Trpv1 Channels ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Somatosensory Neurons ◽  
A Cell ◽  
Intricate Mechanism ◽  
Optical Reconstruction

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca2+-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca2+ dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca2+ stores reduced capsaicin-induced Ca2+ increases and CaCC activation, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane–localized TRPV1 channels was demonstrated with an ER-localized Ca2+ sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1, and the IP3 receptor IP3R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multichannel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca2+ release, which may be necessary to enhance ANO1 activation.

scholarly journals Poliovirus Infection Transiently Increases COPII Vesicle Budding

2012 ◽  
Vol 86 (18) ◽  
pp. 9675-9682 ◽  
Author(s):  
Meg Trahey ◽  
Hyung Suk Oh ◽  
Craig E. Cameron ◽  
Jesse C. Hay
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Vesicle Formation ◽  
Vesicle Budding ◽  
Precursor Pool ◽  
Copii Vesicle ◽  
Early Increase ◽  
Vesicle Biogenesis ◽  
Intermediate Compartment ◽  
Copii Vesicles

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication “organelles” of PV (N. Y. Hsu et al., Cell 141:799–811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

Nanoscale ◽  
2015 ◽  
Vol 7 (6) ◽  
pp. 2511-2519 ◽  
Author(s):  
Jing Gao ◽  
Ye Wang ◽  
Mingjun Cai ◽  
Yangang Pan ◽  
Haijiao Xu ◽  
...  
Keyword(s):  
Distribution Pattern ◽  
Lipid Rafts ◽  
Essential Role ◽  
Super Resolution ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Polarized Cells ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Cluster Maintenance

We investigate the distribution of membrane EGFR by direct stochastic optical reconstruction microscopy (dSTORM). Our results illustrate the clustering distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.

scholarly journals Superresolution imaging reveals spatiotemporal propagation of human replication foci mediated by CTCF-organized chromatin structures

2020 ◽  
Vol 117 (26) ◽  
pp. 15036-15046 ◽  
Author(s):  
Qian Peter Su ◽  
Ziqing Winston Zhao ◽  
Luming Meng ◽  
Miao Ding ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Nuclear Dynamics ◽  
Superresolution Imaging ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Replication Foci ◽  
Optical Reconstruction ◽  
Epigenetic Signatures

Mammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically or preferentially near certain chromatin features. To understand how DNA replication in single human cells is regulated at the sub-RD level, we directly visualized and quantitatively characterized the spatiotemporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase at superresolution using stochastic optical reconstruction microscopy. Importantly, we revealed a hierarchical radial pattern of RFi propagation dynamics that reverses directionality from early to late S-phase and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a “CTCF-organized REplication Propagation” (CoREP) model, which suggests a nonrandom selection mechanism for replication activation at the sub-RD level during early S-phase, mediated by CTCF-organized chromatin structures. Collectively, these findings offer critical insights into the key involvement of local epigenetic environment in coordinating DNA replication across the genome and have broad implications for our conceptualization of the role of multiscale chromatin architecture in regulating diverse cell nuclear dynamics in space and time.

scholarly journals Sec16p potentiates the action of COPII proteins to bud transport vesicles

2002 ◽  
Vol 158 (6) ◽  
pp. 1029-1038 ◽  
Author(s):  
Frantisek Supek ◽  
David T. Madden ◽  
Susan Hamamoto ◽  
Lelio Orci ◽  
Randy Schekman
Keyword(s):  
Vesicle Budding ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Thin Section Electron Microscopy ◽  
Transport Vesicle ◽  
Section Electron ◽  
A Cell ◽  
Acidic Phospholipids ◽  
Coat Protein Assembly ◽  
Stimulation Of

SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

scholarly journals Not just a cargo receptor for large cargoes; an emerging role of TANGO1 as an organizer of ER exit sites

2019 ◽  
Vol 166 (2) ◽  
pp. 115-119 ◽  
Author(s):  
Kota Saito ◽  
Miharu Maeda
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Vesicle Formation ◽  
Wide Range ◽  
Copii Vesicle ◽  
Cargo Receptor ◽  
Er Exit Sites ◽  
Golgi Organization

Abstract Proteins synthesized within the endoplasmic reticulum (ER) are exported from ER exit sites via coat protein complex II (COPII)-coated vesicles. Although the mechanisms of COPII-vesicle formation at the ER exit sites are highly conserved among species, vertebrate cells secrete a wide range of materials, including collagens and chylomicrons, which form bulky structures within the ER that are too large to fit into conventional carriers. Transport ANd Golgi Organization 1 (TANGO1) was initially identified as a cargo receptor for collagens but has been recently rediscovered as an organizer of ER exit sites. We would like to review recent advances in the mechanism of large cargo secretion and organization of ER exit sites through the function of TANGO1.

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