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Dissertation zugänglich unter
Discovery and analysis of novel podosome components in human macrophages
Entdeckung und Analyse neuer Podosomenkomponenten in menschlichen Makrophagen
(2018) European Journal of Cell Biology (2012); Adhesion Protein Protocols, Methods in Molecular Biology, vol. 1046 (2013); Nature Communications (2018)
Dokument 1.pdf (21.911 KB)
Freie Schlagwörter (Englisch):
macrophage , actin , cytoskeleton , podosome
Linder, Stefan (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Macrophages are important cells of the innate immune system. They are highly specialized in the phagocytosis of pathogens and contribute to tissue homeostasis, remodelling and tumor progression or resolution. To address all these functions, macrophages have developed a very typical and highly dynamic cytoskeleton, which notably sustains cell locomotion and allows extracellular matrix degradation and remodelling. Essential structures of the macrophage actin cytoskeleton are podosomes. They are multipurpose organelles with the key abilities to adhere, degrade extracellular matrix by releasing proteolytic enzymes and sense the stiffness of the surrounding environment, all of which allows the cell to adapt to different tissues, from bones to brain. Like other cell-matrix contact, podosomes comprise several hundreds of proteins which contribute to their structure, dynamics and functions. However, no attempt has been made to map the whole, or even a substantial part of their proteome, in contrast to studies performed on focal adhesions, spreading initiation centres or invadopodia. In the first part of my PhD project I tried to fill this gap, providing a list of 203 proteins, of which 33 were already known as podosome components, and that likely represents a good approximation of the real podosome proteome. By comparison with other adhesion structure, we found that podosomes have an in-termediate position in terms of functions and molecular machineries, in addition to a set of 136 new potential candidates not shared with any other cell-matrix adhesion structure.
From an initial screening, we could already characterize some proteins as new podosome components and among them, in the second part of my PhD project, I decided to focus on lymphocyte-specific protein 1 (LSP1) for further evaluation. We found that LSP1 localizes at a newly discovered substructure of the podosome, the so-called cap, and that it regulates podosome dynamics and mechanosensing.
Interestingly, LSP1 dysregulation can lead to severe defects in immune cell locomotion, such as neutrophil actin dysfunction (NAD47/89), where LSP1 overexpression causes immobility of neutrophils and, as consequence, recurrent infections in patients.
In particular, we discovered that LSP1 can moderately activate myosin IIA and compete with supervillin, a myosin hyper activating protein, for binding of myosin regulators and actin isoforms, especially beta-actin. Moreover, we found that actin isoform shows specific gradients of distribution in macrophages and that such subcellular patterns form the molecular basis for the differential recruitment of two actomyosin regulators, namely LSP1 and supervillin, that significantly contribute to actomyosin symmetry breaking by supporting different levels of myosin activity.
In the last part of the thesis, I summarize the principles for creating an algorithm to semi-automatically detect podosomes in still images or time lapse videos of macrophages stained for F-actin. By exploiting specific tools for image processing is thus possible now to gather large numbers and robust statistics of certain podosome parameters, such as absolute number and density, which are very important to investigate the impact of certain components, including LSP1, on podosome dynamics.