correlative-super-resolution-electron-microscopy-to-resolve-protein

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

University of Zurich

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

Although the biochemistry of early trimming reactions by glucosidases and ER mannosidases occurring on asparagine-linked oligosaccharides has been known for a long time, their involvement in quality control of protein folding has become apparent only more recently. Here we review the evidence for the involvement of specific oligosaccharide trimming intermediates such as Glc(1)Man(9)GlcNAc(2) and Man(8)GlcNAc(2) B isomer in this fundamental cellular process and the subcellular distribution of components of the protein quality control machinery which indicates the involvement of both the ER and pre-Golgi intermediates in this process. In addition, recent studies on the subcellular distribution of endomannosidase in conjunction with previously obtained biochemical data will be reviewed which demonstrate that an alternative deglucosylation pathway exists in pre-Golgi intermediates and the Golgi apparatus.

The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control.

A missense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds results in intracellular accumulation of misfolded proinsulin. We analyzed the secretory pathway of pancreatic beta cells by electron microscopy and morphometry and identified sites of proinsulin accumulation by quantitative immunogold electron microscopy in this protein-folding disease. In Akita mice beta cells, the volume density of dilated endoplasmic reticulum subdomains was increased by 2.9-fold, resulting in a 1.7-fold increased volume density of the entire rough endoplasmic reticulum. The volume density of pre-Golgi intermediates was increased by 4.9-fold, and that of the Golgi apparatus was increase by 3.4-fold. The relative labeling intensity for proinsulin was 2.1-fold higher in dilated endoplasmic reticulum subdomains and 2.9-fold higher in pre-Golgi intermediates as compared with narrow endoplasmic reticulum, resulting in a significantly different distribution pattern between Akita and control mice beta cells (Chi2= 29.97, P<0.001). The numerical density of insulin secretory granules was equal in Akita and control mice beta cells. However, their volume density and average volume were reduced to 20% and their average diameter to 58% in Akita mice. Together, these data demonstrate that misfolded proinsulin accumulates mainly in pre-Golgi intermediates and to a lesser extent in dilated endoplasmic reticulum subdomains, providing evidence for the importance of pre-Golgi intermediates in a protein folding disease.

Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice.

This review provides an update on the use of postembedding immunogold labeling, preferentially of the protein A-gold technique, for electron microscopic research in diseased states. In the first part, some helpful antigen recovery techniques for use of immunogold labeling of ultrathin sections prepared from routinely aldehyde (and osmium tetroxide) fixed and conventionally epoxy resin-embedded tissue are cited. In the second part, selected applications for studies of tumor cell surface conjugates, such as polysialic acid in Wilms tumor, proinsulin-insulin conversion in human insulinoma, and the importance of pre-Golgi intermediates for retention of a misfolded polypeptide hormone in a protein misfolding disease, are discussed.

Applications of immunogold labeling in ultrastructural pathology.

Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing alpha-mannosidase-like protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to approximately 150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and approximately 11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of alpha-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.

EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites.

In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.

Protein quality control: the who's who, the where's and therapeutic escapes.

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or alpha1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to 18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies.

Glucose residues from N-linked oligosaccharides are removed by glucosidases I and II in the endoplasmic reticulum (ER) or by the alternate endomannosidase pathway in the Golgi apparatus. Our morphological analysis demonstrates that recombinant rat endomannosidase exhibited a cis- and medial-Golgi localization alike the endogenous enzyme and its ER to Golgi transport is COP II mediated. Recombinant endomannosidase undergoes a posttranslational modification, which is not related to N-or O-glycosylation. A shift in molecular mass of recombinant endomannosidase was observed upon phosphatase digestion but not for ER-retained CHO cell endomannosidase. Furthermore, immunoprecipitation of (35)S- and (33)P-labeled endomannosidase expressed in CHO-K1 cells suggests that recombinant endomannosidase undergoes phosphorylation. Substitution of the single cytoplasmic threonine residue of rat endomannosidase by either an alanine or valine residue resulted in the same posttranslational modification alike the wild-type enzyme. The subcellular localization and the in vivo activity of the mutant endomannosidase were not affected. Thus, endomannosidase phosphorylation is occurring in luminal sequences. Modification was prevented when endomannosidase was synthesized using reticulocyte lysates in the presence of canine microsomes. Treatment of cells with brefeldin A blocked the posttranslational modification of endomannosidase, suggesting that phosphorylation is occurring in the Golgi apparatus, the residence of endomannosidase.

Endomannosidase undergoes phosphorylation in the Golgi apparatus.

Mycoplasma suis belongs to the haemotrophic mycoplasmas which colonise red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. Due to the lack of a cultivation system, identification of adhesion structures has been difficult. So far, only one adhesion protein, i.e. MSG1 was identified. In order to determine further adhesion molecules of M. suis, we screened genomic M. suis libraries and performed Southern blot hybridisation analyses of genomic M. suis DNA. The α-enolase of M. suis was identified and analysed genetically and functionally. The encoding gene has 1623 bp in size. The deduced amino acid sequence showed an overall identity of 59.6-65.1% to α-enolases of other pathogenic mycoplasmas. The 540 aa M. suis α-enolase displays a size extension of about 90 aa in comparison to α-enolases of other mycoplasmas. Recombinant α-enolase expressed in Escherichia coli demonstrated immunogenicity in experimentally infected pigs. Immunoblot, confocal laser scanning microscopy and immune electron microscopy analysis using antibodies against recombinant α-enolase, indicate the membrane and surface localisation of native α-enolase in M. suis, though no typical signal sequences exist. Furthermore, we showed that recombinant α-enolase binds to porcine erythrocyte lysate in a dose-dependent manner. E. coli transformants which express α-enolase on their surface acquire the ability to adhere to porcine red blood cells. In conclusion, our observations indicate that α-enolase could be involved in the adhesion of M. suis to porcine red blood cells.

The surface-localised α-enolase of Mycoplasma suis is an adhesion protein.

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy.

Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients.

Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing.

Epstein Barr virus (EBV) persists as a latent herpes virus infection in the majority of the adult human population. The virus can reactivate from this latent infection into lytic replication for virus particle production. Here, we report that autophagic membranes, which engulf cytoplasmic constituents during macroautophagy and transport them to lysosomal degradation, are stabilized by lytic EBV replication in infected epithelial and B cells. Inhibition of autophagic membrane formation compromises infectious particle production and leads to the accumulation of viral DNA in the cytosol. Vice versa, pharmacological stimulation of autophagic membrane formation enhances infectious virus production. Atg8/LC3, an essential macroautophagy protein and substrate anchor on autophagic membranes, was found in virus preparations, suggesting that EBV recruits Atg8/LC3 coupled membranes to its envelope in the cytosol. Our data indicate that EBV subverts macroautophagy and uses autophagic membranes for efficient envelope acquisition during lytic infection.

Macroautophagy Proteins Assist Epstein Barr Virus Production and Get Incorporated Into the Virus Particles.

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.

Article	Year
Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control. Molecular biology of the cell\| PubMed ID: 11102520	2000
Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 11535823	2001
The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control. Histochemistry and cell biology\| PubMed ID: 11935292	2002
Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 15033933	2004
Applications of immunogold labeling in ultrastructural pathology. Ultrastructural pathology\| PubMed ID: 16036886	2005
EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17360537	2007
Protein quality control: the who's who, the where's and therapeutic escapes. Histochemistry and cell biology\| PubMed ID: 18075753	2008
A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies. Histochemistry and cell biology\| PubMed ID: 19381673	2009
Endomannosidase undergoes phosphorylation in the Golgi apparatus. Glycobiology\| PubMed ID: 19759276	2010
The surface-localised α-enolase of Mycoplasma suis is an adhesion protein. Veterinary microbiology\| PubMed ID: 22047714	2012
Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy. Cellular and molecular life sciences : CMLS\| PubMed ID: 23338832	2013
Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing. The Journal of cell biology\| PubMed ID: 24322427	2013
Macroautophagy Proteins Assist Epstein Barr Virus Production and Get Incorporated Into the Virus Particles. EBioMedicine\| PubMed ID: 26137519	2014
Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy. Scientific reports\| PubMed ID: 27666401	2016

Bruno Guhl

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