Expression, purification and characterization of fungal and viral recombinant proteins

This work reports the production of recombinant yeast and viral proteins in a number of diverse in vivo model systems for enzymatic and structural studies. In the first part Hsp150Δ peptide, a derivative of the yeast (Saccharomyces cerevisiae) secretory heat-shock protein Hsp150, was investigated for its ability to act as a carrier in transporting the ectodomain of rat nerve growth factor (NGFRe) out from the yeast cell. The Hsp150Δ-NGFRe fusion protein was efficiently secreted into the growth medium, where it constituted the majority of total secreted proteins. Inhibition experiments with purified Hsp150Δ-NGFRe showed that Hsp150Δ did not prevent NGFRe from folding into a ligand-binding conformation. Circular dichroism (CD) analysis revealed that the Hsp150Δ-carrier did not have any specific secondary structure, which was also suggested by NMR analysis of a synthetic polypeptide corresponding to the repetitive consensus sequence of subunit II of Hsp150. These findings suggest that Hsp150Δ can successfully act as a carrier for foreign proteins, such as NGFRe, made and secreted by S. cerevisiae. The second part of this study involved the expression and purification of an RNA animal virus, Semliki Forest virus (SFV), nonstructural proteins (Nsp1-4) using a number of in vivo protein expression systems. To ensure quantities large enough for structural and enzymatic studies of the Nsps, each of them was expressed either in bacteria (Escherichia coli) or in insect cells (Sf9). All the proteins were expressed in high quantities (10-100 mg/l), and purified by affinity and size exclusion chromatography under nondenaturing or denaturing conditions. Independent of the expression system used, all the partially purified Nsps aggregated and precipitated either upon concentration, dialysis, storing or thawing. No detergents were found that could alleviate the aggregation problem or assist in the purification process. Despite the unsuccessful purification of Nsps for structural studies, the expression and partial purification of Nsp1 and Nsp3 permitted biochemical characterization of their enzyme activities and posttranslational modifications. Point mutational analysis of the Nsp1 methyltransferase domain revealed that residue His38 was essential for the guanylyltransferase activity of Nsp1. Furthermore, residues Asp64 and Asp90 were found to be important for the methyltransferase activity of Nsp1. Phosphorylation sites in Nsp3 were determinated by point mutational analysis, electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) as well as by phosphopeptide mapping and Edman sequencing. A phosphorylated domain (aa 320-368) was located in the C-terminal, non-conserved region of Nsp3, where 12 serines and 4 threonines could be modified by phosphates. The phosphorylation of Nsp3 seemed not to affect the membrane association or the localization of Nsp3 in either transfected or infected cells. Furthermore, Nsp3 phosphorylation deficient mutant viruses were capable of replication in infected mammalian cells a similar manner to the wild type SFV, but their neuropathogenicity in adult mice was greatly reduced.reviewe

Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes

Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

AbstractHere we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp150Δ) carrier. The hsp150Δ-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp150Δ-NGFRe fusion protein were highly selectively O-glycosylated

Microscopic characterization reveals the diversity of EVs secreted by GFP-HAS3 expressing MCF7 cells

We have shown the connection of hyaluronan synthesis activity with the enhanced shedding of extracellular vesicles, but detailed morphological analysis of those hyaluronan-induced EVs is still missing. In this study we utilized a comprehensive set of high-resolution imaging techniques to characterize in high detail the size and morphology of EVs originating from stable MCF7 breast cancer cell line and transiently transfected cells expressing GFP-HAS3. To avoid possible artefacts or loss of EVs resulting from the isolation process, special attention was paid to analysis of EVs in situ in monolayer and in 3D cultures. The results of this study show that GFP-HAS3 expressing MCF7 cells produce morphologically diverse EVs but also demonstrates the variation in results obtained with different experimental setup, which emphasizes the importance of comparison between different methods when interpreting the observations.Peer reviewe

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores

A Na,K-ATPase–Fodrin–Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores

A Na,K-ATPase–Fodrin–Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes

Microscopy Image Browser : A Platform for Segmentation and Analysis of Multidimensional Datasets

Understanding the structure-function relationship of cells and organelles in their natural context requires multidimensional imaging. As techniques for multimodal 3-D imaging have become more accessible, effective processing, visualization, and analysis of large datasets are posing a bottleneck for the workflow. Here, we present a new software package for high-performance segmentation and image processing of multidimensional datasets that improves and facilitates the full utilization and quantitative analysis of acquired data, which is freely available from a dedicated website. The open-source environment enables modification and insertion of new plug-ins to customize the program for specific needs. We provide practical examples of program features used for processing, segmentation and analysis of light and electron microscopy datasets, and detailed tutorials to enable users to rapidly and thoroughly learn how to use the program.Peer reviewe