Susann Kummer

Fluorescence imaging of influenza H1N1 mRNA in living infected cells using single-chromophore FIT-PNA.

In light of the increasing importance assigned to RNA, significant efforts have been devoted to the development of fluorescent oligonucleotide probes that allow the imaging of RNA expression in living cells. Molecular beacons (MBs, Scheme 1a) probably are amongst the most widely used probes for RNA imaging. These hairpin-shaped probes rely on the interaction between two terminally appended chromophores which are separated upon formation of probe– target complexes. Unintended protein binding and/or nuclease-mediated probe degradation can also affect the distance between the chromophores. We have introduced so-called FIT-PNA probes (Scheme 1 b), which contain a single thiazole orange (TO) intercalator serving as artificial fluorescent nucleobase. These probes respond to changes of the local structure in the vicinity of the dye rather than to the more global changes of conformation that confer fluorescence signaling by the dual-labeled molecular beacons. High fluorescence enhancements require intercalation of the TO dye. This is expected to help in avoiding strong fluorescence signals upon inevitable binding to proteins. Amongst the many TOcontaining probes reported FIT probes are unique because a single fluorophore provides for both high enhancements of fluorescence upon matched hybridization and high target specificity at nonstringent hybridization conditions where both matched and mismatched probe–target complexes coexist. Further assets are the high affinity of the PNA probes for complementary RNA and the enhanced biostability provided by the peptide nucleic acid (PNA) backbone. Herein we demonstrate the advantageous properties of FITPNA probes in the imaging of mRNA from an influenza virus strain belonging to the same subtype as the recently emerged swine virus (A/Mexico/1/2009, H1N1). In a research program aiming at the characterization of the spatio-temporal pattern of virus assembly, we required a method that enables imaging of the mRNA coding for neuraminidase of influenza virus A/PR/8. FIT probes such as 1a and 1b were designed to target a sequence in the NA mRNA (nt 599–615, referred to the accession number NC_002018) which is essentially sequence identical to the NA mRNA from A/Mexico/1/2009/swine (H1N1, nt 625–640) (Figure 1). The accessibility of the target segment, which can be hindered by RNA folding and binding of proteins, has been previously demonstrated by Zhang and co-workers. A rapid screen, which required the synthesis of eight different PNA oligomers, suggested FIT probe 1 a as a suitable probe (see the Supporting Information). This sensor provided an 11-fold increase of the TO emission upon hybridization with complementary RNA target 3a at 37 8C (Figure 2a). Furthermore, we tested the subtype specificity. The RNA 4 from a different influenza strain (NA mRNA from A/X-31, H3N2, nt 16–32) includes seven continuous matched base pairs around the thiazole orange “base”. Nevertheless, the fluorescence of 1a remained virtually unchanged when RNA 4 was added. The TMR/Dabcyl-labeled molecular beacon 2 (Figure 1; TAMRA = tetramethyl-6-carboxyrhodamine; Dabcyl = 4-(4dimethylaminophenyl)diazenylbenzoic acid) was used in a Scheme 1. Nucleic acid detection with a) molecular beacon probes (MBs) and b) FIT-PNA probes. MBs change conformation upon binding of a complementary target. In FIT-PNA probes, an intercalator dye such as thiazole orange (TO) responds to changes of the local environment. Stacking interactions hinder twisting around the TO methine bridge and thus confer enhancements of fluorescence.

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology.

ABSTRACT The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

Clathrin-adaptor ratio and membrane tension regulate the flat-to-curved transition of the clathrin coat during endocytosis

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.The sequence of structural and molecular events during clathrin-mediated endocytosis is unclear. Here the authors combine correlative microscopy and simple mathematical growth laws to demonstrate that the flat patch starts to curve when around 70% of the final clathrin content is reached.

Combination of microdissection and single cell quantitative real-time PCR revealed intercellular mitochondrial DNA heterogeneities in fibroblasts of Kearns-Sayre syndrome patients

Kearns-Sayre syndrome (KSS) is a multisystemic disorder marked by aerobic cell metabolism dysfunction. Fibroblasts derived from KSS patient skin biopsy exhibit heterogeneous occurrence of mitochondrial genomes as those circular DNA molecules partially carry the common deletion. In our approach, we aim to evaluate the intercellular alterations in respect to mitochondrial DNA integrity by laser capture microdissection and multiplex quantitative real-time PCR in single cells. The obtained results give new insights into the understanding of mitochondrial genetics, e.g. postulated sorting of damaged mitochondria, and heterogeneity of cells. Further, we discuss the relevance of intercellular heterogeneities for human mitochondrial disorders in general.

Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

Sequence-specific imaging of influenza A mRNA in living infected cells using fluorescent FIT-PNA.

Significant efforts have been devoted to the development of techniques allowing the investigation of viral mRNA progression during the replication cycle. We herein describe the use of sequence-specific FIT-PNA (Forced Intercalation Peptide Nucleic Acids) probes which contain a single intercalator serving as an artificial fluorescent nucleobase. FIT-PNA probes are not degraded by enzymes, neither by nucleases nor by proteases, and provide for both high sensitivity and high target specificity under physiological conditions inside the infected living host cell.