- Meeting abstract
- Open Access
PW03-027 - CASP1 variants and live cell imaging
© Rabe et al; licensee BioMed Central Ltd. 2013
- Published: 8 November 2013
- Live Cell Imaging
- Febrile Episode
- Receptor Interact Protein
- Proximity Ligation Assay
- Sterile Inflammation
Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite reduced secretion of IL-1ß. As previously demonstrated by our group CASP1 variants lead to reduced enzymatic activity of procaspase-1 by destabilizing the tertiary structure of the caspase-1 tetramer. A possible explanation for an alternative pro-inflammatory pathway has been provided by Lamkanfi and colleagues indicating an association between enzymatically inactive procaspase-1 and receptor interacting protein kinase 2 (RIP2) leading to NF-kB activation.
The objective of this project is the identification of possible subcellular mechanisms how CASP1 variants interfere with the IL-1β production or release and lead to the activation of alternative pro-inflammatory pathways.
Using confocal microscopy, in vivo live cell imaging and an in situ proximity ligation assay we analyzed the subcellular distribution of procaspase-1 wildtype and mutants as well as the interaction with RIP2 in naïve or virally transduced THP-1 cells.
THP-1 cells were virally transduced with GFP- or mCherry fusion proteins of procaspase-1 wildtype and variant CASP1-L 265S. Procaspase-1 activation, initiated by the assembly of multiprotein complexes (inflammasomes), was induced by stimulation with LPS and Nigericin. First results suggest disturbed microvesicle shedding from CASP1-L 265S expressing cells after administration of Nigericin. In addition to live cell imaging, the interaction of procaspase-1 and RIP2 has been studied in vitro in naïve THP-1 cells using antibody labeling and proximity ligation assay showing a time dependency after LPS stimulation.
This result suggests a possible influence of procaspase-1 variants on plasma membrane properties, pyroptosis and the release of microvesicles.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.