A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA

Macrophage Identification In Situ

Understanding the processes of inflammation and tissue regeneration after injury is of great importance. For a long time, macrophages have been known to play a central role during different stages of inflammation and tissue regeneration. However, the molecular and cellular mechanisms by which they exert their effects are as yet mostly unknown. While in vitro macrophages have been characterized, recent progress in macrophage biology studies revealed that macrophages in vivo exhibited distinctive features. Actually, the precise characterization of the macrophages in vivo is essential to develop new healing treatments and can be approached via in situ analyses. Nowadays, the characterization of macrophages in situ has improved significantly using antigen surface markers and cytokine secretion identification resulting in specific patterns. This review aims for a comprehensive overview of different tools used for in situ macrophage identification, reporter genes, immunolabeling and in situ hybridization, discussing their advantages and limitations

Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ

Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ

Hybridization‐chain‐reaction is a relevant method for in situ detection of M2d‐like macrophages in a mini‐pig model

International audienceMacrophages are a heterogeneous population of cells with an important role in innate immunity and tissue regeneration. Based on in vitro experiments, macrophages have been subdivided into five distinct subtypes named M1, M2a, M2b, M2c, and M2d, depending on the means of their activation and the cell surface markers they display. Whether all subtypes can be detected in vivo is still unclear. The identification of macrophages in vivo in the regenerating muscle could be used as a new diagnostic tool to monitor therapeutic strategies for tissue repair. The use of classical immunolabeling techniques is unable to discriminate between different M2 macrophages and a functional characterization of these macrophages is lacking. Using in situ hybridization coupled with hybridization-chain-reaction detection (HCR), we achieved the identification of M2d-like macrophages within regenerating muscle and applied this technique to understand the role of M2 macrophages in the regeneration of irradiated pig-muscle after adipose tissue stem cell treatment. Our work highlights the limits of immunolabeling and the usefulness of HCR analysis to provide valuable information for macrophage characterization

New insights on the organization and regulation of the fatty acid biosynthetic network in the model higher plant Arabidopsis thaliana

In the plastids of plant cells, fatty acid (FA) production is a central biosynthetic process. It provides acyl chains for the formation of a variety of acyl lipids fulfilling different biological functions ranging from membrane synthesis to signaling or carbon and energy storage. The biochemical pathway leading to the synthesis of FA has been described for a long time. Over the last 15 years, and after the genome of the model higher plant Arabidopsis thaliana has been sequenced, the scientific community has deployed approaches of functional genomics to identify the actors comprising this pathway. One of the puzzling aspects of the emerging molecular biology of FA synthesis resided in the occurrence of multigene families encoding most enzymes of the pathway. Studies carried out to investigate these families led to the conclusion that most members have acquired non-redundant roles in planta. This is usually the consequence of divergent expression patterns of these isogenes and/or of different substrate specificities of the isoforms they encode. Nevertheless, much remains to be elucidated regarding the molecular bases underpinning these specificities. Protein biochemistry together with emerging quantitative proteomic technologies have then led to a better understanding of the structure of the network, which is composed of multiprotein complexes organized within the stromal compartment of plastids: whereas growing evidence suggests that the early steps of the pathway might be associated to the inner envelope membrane, several late enzymes might be localized next to the thylakoids. The question of the existence of a large integrated protein assembly channeling substrates through the whole pathway that would span the stroma remains uncertain. Finally, recent discoveries regarding the post-translational regulation of the pathway open new research horizons and may guide the development of relevant biotechnological strategies aimed at monitoring FA production in plant systems

In Situ Gene Expression in Native Cryofixed Bone Tissue

Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues

The N-terminal region of GBF1 is important for rescue of polio replication from BFA inhibition and for localization to sites of viral RNA synthesis.

<p>A. HeLa cells were transfected with vectors expressing YFP-GBF1 fusions with BFA-resistance mutation A795E with either the wild type N-terminus or with a deletion of 37 N-terminal amino-acids (Δ37). The next day the cells were transfected with polio <i>Renilla</i> replicon RNA and incubated in the presence or absence of 1 µg/ml BFA while monitoring viral RNA synthesis. B. HeLa cells were transfected with plasmids expressing Venus-GBF1 fusions with either wild type N-terminus (panels A–D) or with a deletion of 37 N-terminal amino-acids (Δ37) (panels E–H). The next day cells were infected with poliovirus at a multiplicity of 10 PFU/cells (panels A–C and E–G) or mock-infected (panels D and H), incubated for 4 h, fixed and processed for immunofluorescent staining with anti-polio 3A antibodies (red). Nuclear chromatin is stained with Hoechst 33342.</p

Polio-induced vesicles form in the presence of BFA, but fail to induce Arf1 relocalization.

<p>A. Polio non-structural proteins were expressed from non-replicating RNA. The cells were incubated in the presence or absence of 2 µg/ml of BFA. An equivalent amount of DMSO solvent was added to the incubation medium in the sample without BFA. After 4.5 h incubation, cells were fixed and processed for electron microscopy. B. Poliovirus non-structural proteins were expressed from non-replicating RNA in HeLa cells expressing Arf1-EGFP fusion. The cells were incubated in the presence of 2 µg/ml of BFA where indicated (panels E–H). An equivalent amount of DMSO solvent was added to the incubation medium in samples without BFA (panels A–D). To observe the localization of Arf1-EGFP fusion protein in cells without expression of polio proteins, an empty plasmid was substituted for the polio cDNA-bearing plasmid (panels D and H).</p

Synthesis of poliovirus protein 3A stimulates GBF1-dependent Arf activation in vitro.

<p>RNA coding for poliovirus 3A was translated in HeLa cell S10 extracts. The samples contained 80 µg/ml BFA where indicated (or the corresponding amount of solvent DMSO in other samples). The membranes were collected by centrifugation and assessed by western blot with anti-GBF1, anti-Arf and anti-COPI antibodies. Translation efficiency was monitored by labeling an aliquot of the translation reaction with ³⁵S methionine (lower panel).</p