The analysis of membrane signalling networks in latex bead- and mycobacterial-phagosomes regulating actin binding and de novo assembly
Final Report Abstract
Two "in vitro" assays are used in this project that monitor A) the "de novo" assembly of actin or B) The binding to F-actin of a defined "in vitro" membrane organelle, the latex bead phagosome (LBP) from macrophages. From recent work two networks of biochemical reactions were found to regulate these independent, and largely competitive processes. Bioinformatic modelling of lipid networks regulating actin assembly by the LBP membrane led to predictions involving ATP synthesis. While the original predictions were mechanistically not supported by data, they led to a new finding of general importance: during some membrane-catalyzed processes of actin assembly phosphorylated lipids (including PI4P, PI4,5P2, cermide-1-P and sphingosine-I-P) open channels that allow ADP to enter the phagosome lumen, or be transported out across the plasma membrane. In both these luminal locations ADP is converted by adenylate kinase to ATP. This ATP is hypothesized to bind and activate ATP-binding purinergic receptor(s) that are directly or indirectly linked to the actin assembly process. On the plasma membrane of raw macrophages the P2x7 receptor has been identified as being the key player from patch clamp analysis. In phagosomes the actin assembly machinery is also regulated by arachidonic acid, prostaglandins leukotrienes and by cAMP/PKA. The LBP-actin binding process is regulated quite differently: many effectors that stimulate actin assembly tend to inhibit binding and vice-versa. The binding assay has an additional complexity over the assembly assay, it requires cytosol- from where actin binding proteins such as myosin V and probably filamin are recruited for binding. Interaction between phagosomes and F-actin could be also regulated by arachidonic acid and PIP2. The experimental goal for both assays is to go deeper in the analysis of the two LBP processes. The challenging goal of the theoretical, bioinformatic analysis in our project was to provide a global systems-level description of all the interactions regulating LBP actin assembly and actin binding including detailed hypotheses that can and will be tested experimentally. Different analytical techniques have to be combined to correctly infer involved cellular interactions. In particular, suitable techniques have to be applied to cope with the limited information available regarding kinetic parameters and to integrate the often only qualitative data, for instance Boolean models. While in the 1st period individual components (lipids, proteins, interactions) were studied on actin assembly and binding in latex bead and mycobacterial phagosomes, the 2nd period focussed on the network effects in phagosomal signalling: The results of the 2nd period have already been translated into a refined description of the phagosome, new algorithms and new insights into the interaction of cytoskeleton and bacterial pathogenicity. However, we could not only establish and refine the network picture of the phagosomal signaling but have now firm indications that a systematic understanding of its signaling can be established dissecting the compartimentalization (cytoplasic surface (outside), phagosomal membrane, phagosomal lumen-inside) and the phagosomal interactome considering the complex role of ATP in the signaling and involved receptors. We studied the phagsomal interactome, including lipid-protein interactions and phagosomal membrane compartimentalization, a key to understand the process.
Publications
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3D reconstruction of the dynactin by single particle image analysis. 2005, Proceedings of the National Academy of Sciences of the USA, Vol. 102. 2005, no. 10, pp. 3667-3672.
Hodgkinson, J.L., Peters, C., Kuznetsov, S.A., W. Steffen
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cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria. Journal of Cell Science, Vol. 119. 2006, Issue 17, pp. 3686-3694.
Kalamidas, S.A., Kuehnel, M.P., Peyron, P., Rybin, V., Rauch, S., Kotoulas, O.B., Houslay, M., Hemmings, B.A., Gutierrez, M.G., Anes, E., Griffiths, G.
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Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages. Cellular Microbiology, Vol. 8. 2006, Issue 6, pp. 939–960.
Anes, E., Peyron, P., Staali, L., Jordao, L., Gutierrez, M.G., Kress, H., Hagedorn, M., Maridonneau-Parini, I., Skinner, M.A., Wildeman, A.G., Kalamidas, S.A., Kuehnel, M., Griffiths, G.
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Integrated network reconstruction, visualization and analysis using YANAsquare. BMC Bioinformatics, Vol. 8. 2007, Issue August:313.
Schwarz R., Liang C., Kaleta C., Kühnel M., Hoffmann E., Kuznetsov S., Hecker M., Griffiths G., Schuster S., Dandekar T.
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Modelling phagosomal lipid networks that regulate actin assembly. BMC Systems Biology, Vol. 2.2006, Issue Dec.: 107.
Kühnel M., Mayorga L.S., Dandekar T., Thakar J., Schwarz R., Anes E., Griffiths G., Reich J.
