scholarly journals Unveiling the cryo-EM structure of retromer

2020 ◽  
Vol 48 (5) ◽  
pp. 2261-2272 ◽  
Author(s):  
Mintu Chandra ◽  
Amy K. Kendall ◽  
Lauren P. Jackson
Keyword(s):  
Electron Tomography ◽  
Transmembrane Protein ◽  
Brain Diseases ◽  
Actin Remodeling ◽  
Sorting Nexin ◽  
Multiple Partners ◽  
Cryo Electron Microscopy ◽  
Golgi Network ◽  
Neuronal Maintenance ◽  
In Cells

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans-Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3P)-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments

2018 ◽  
Vol 217 (8) ◽  
pp. 2691-2708 ◽  
Author(s):  
J. Richard McIntosh ◽  
Eileen O’Toole ◽  
Garry Morgan ◽  
Jotham Austin ◽  
Evgeniy Ulyanov ◽  
...  
Keyword(s):  
High Pressure ◽  
Brownian Dynamics ◽  
Electron Tomography ◽  
Tubulin Polymerization ◽  
Guanosine Triphosphate ◽  
Simple Mechanism ◽  
In Cells

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate–tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

scholarly journals Yeast synaptobrevin, Snc1, engages distinct routes of postendocytic recycling mediated by a sorting nexin, Rcy1-COPI, and retromer

2020 ◽  
Vol 31 (9) ◽  
pp. 944-962
Author(s):  
Jordan T. Best ◽  
Peng Xu ◽  
Jack G. McGuire ◽  
Shannon N. Leahy ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Multiple Modes ◽  
Sorting Nexin ◽  
Trans Golgi Network ◽  
Golgi Network

The yeast synaptobrevin, Snc1, uses multiple modes of postendocytic recycling to facilitate its return to either the trans-Golgi network or the plasma membrane. Snc1 primarily recycles via pathways dependent on either Rcy1/COPI or Snx4, while a smaller portion of the SNARE appears to be capable of being retrieved by retromer.

scholarly journals A molecular pore spans the double membrane of the coronavirus replication organelle

Science ◽  
2020 ◽  
Vol 369 (6509) ◽  
pp. 1395-1398 ◽  
Author(s):  
Georg Wolff ◽  
Ronald W. A. L. Limpens ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Ulrike Laugks ◽  
Shawn Zheng ◽  
...  
Keyword(s):  
Drug Target ◽  
Rna Synthesis ◽  
Transmembrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Genome Replication ◽  
Messenger Rnas ◽  
Double Membrane ◽  
The Core ◽  
Cryo Electron Microscopy

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking

2015 ◽  
Vol 89 (20) ◽  
pp. 10145-10155 ◽  
Author(s):  
David Pim ◽  
Justyna Broniarczyk ◽  
Martina Bergant ◽  
Martin P. Playford ◽  
Lawrence Banks
Keyword(s):  
Pdz Domain ◽  
Endosomal Trafficking ◽  
Sorting Nexins ◽  
Human Papillomavirus 16 ◽  
Sorting Nexin ◽  
Papillomavirus Infection ◽  
Infection Dynamics ◽  
And Function ◽  
Golgi Network ◽  
Interfering Rna

ABSTRACTPrevious studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.IMPORTANCEThe trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.

scholarly journals Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

Structure ◽  
2016 ◽  
Vol 24 (7) ◽  
pp. 1031-1043 ◽  
Author(s):  
Marion Jasnin ◽  
Mary Ecke ◽  
Wolfgang Baumeister ◽  
Günther Gerisch
Keyword(s):  
Electron Tomography ◽  
Live Imaging ◽  
Actin Organization ◽  
Cryo Electron Tomography ◽  
In Cells

scholarly journals A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

1990 ◽  
Vol 10 (2) ◽  
pp. 830-836 ◽  
Author(s):  
M Hamaguchi ◽  
M Matsuda ◽  
H Hanafusa
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Transmembrane Protein ◽  
Temperature Sensitive ◽  
Morphological Transformation ◽  
Fibronectin Receptor ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Potential Substrate ◽  
In Cells

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.

scholarly journals Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes

2019 ◽  
Author(s):  
Amy K. Kendall ◽  
Boyang Xie ◽  
Peng Xu ◽  
Jue Wang ◽  
Rodger Burcham ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Core Complex ◽  
Sorting Nexin ◽  
Flat Chains ◽  
Biophysical Studies ◽  
Cargo Proteins ◽  
Golgi Network ◽  
Multi Body ◽  
Cargo Sorting

AbstractIn metazoans, retromer (VPS26/VPS35/VPS29) associates with sorting nexin (SNX) proteins to form coats on endosomal tubules and sort cargo proteins to the trans-Golgi network (TGN) or plasma membrane. This core complex is highly conserved from yeast to humans, but molecular mechanisms of metazoan retromer assembly remain undefined. Here we combine single particle cryo-electron microscopy with biophysical methods to uncover multiple oligomer structures formed by mammalian retromer. Two-dimensional class averages in ice reveal the retromer heterotrimer; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical studies in solution. We provide cryo-EM reconstructions of all species, including pseudo-atomic resolution detail for key sub-structures. Multi-body refinement demonstrates how retromer heterotrimers and dimers adopt a range of conformations. Our structures identify a flexible yet highly conserved electrostatic interface in dimers formed by interactions between VPS35 subunits. We generate a structure-based mutant to disrupt this key interface in vitro and introduce equivalent mutations into S. cerevisiae to demonstrate the mutant exhibits a cargo sorting defect. Together, structures and complementary functional data in budding yeast imply a conserved assembly interface across eukaryotes. These data further suggest mammalian retromer acts as an adaptable and plastic scaffold that accommodates interactions with different SNXs to sort multiple cargoes from endosomes their final destinations.

scholarly journals Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

2022 ◽  
Author(s):  
Martin Obr ◽  
Wim JH Hagen ◽  
Robert A Dick ◽  
Lingbo Yu ◽  
Abhay Kotecha ◽  
...  
Keyword(s):  
Field Emission ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Energy Filtering ◽  
Cryo Electron Microscopy ◽  
Electron Detection ◽  
Thermo Fisher Scientific ◽  
Subtomogram Averaging ◽  
New Generation ◽  
Fisher Scientific

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

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