31 research outputs found
The fibril associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix
Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine
Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is similar to 2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.Peer reviewe
Selective targeting of avidin/mannose-6-phosphate receptor chimeras to early or late endosomes
BioImageXD: an open, general-purpose and high-throughput image-processing platform.
BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors
Ion-induced fluorescence imaging of endosomes
10.1016/j.nimb.2012.12.052Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms306113-116NIMB
A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor α2β1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. α2β1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered α2β1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-α2β1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that α2β1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes
Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells
Aims/hypothesis The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry
receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been
implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the
surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar
physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and
monitored the subcellular localisation of the principal isoform within the cells.
Methods Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes
were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoformspecific
antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoformspecific
qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets.
Results An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in
human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected
by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was
the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma
membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a
specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CARSIV
with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This
suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling
and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in
human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine
cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein
recently demonstrated to play a role in insulin granule maturation and trafficking.peerReviewe