LI 925/3-2 Macrophage podosomes as integ

Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 170445358
Teilprojekt zu SPP 1464: Principles and evolution of actin-nucleator complexes

In this project, we use macrophage podosomes as a model system to study mechanisms of actin nucleation at highly localized subcellular sites. A particular emphasis is placed on the role of formin members in the formation and turnover of actin filaments at podosomes, and the mechanisms that allow integration of Arp2/3 complex activity with formin-dependent actin nucleation. Podosomes are highly dynamic, actin-based adhesion structures in a variety of cell types, and especially monocytic cells such as primary macrophages constitutively form numerous podosomes. The F-actin-rich core of podosomes is essential for their formation, and WASp-Arp2/3 complex-dependent actin nucleation has been shown to play a major role in podosome regulation. However, the recent discovery of substructures within the actin core (inner core and surrounding “cap”) suggest the co-existence of different modes of actin nucleation at podosomes. Purification of podosome-enriched cell fractions and subsequent mass spectrometry analysis led us to the identification of several formins at podosomes, which are now gradually verified by localization of respective fusion proteins. Importantly, podosomes show different forms of dynamics such as de novo formation, growth, steady state (accompanied by internal actin turnover), and fission, and it will be extremely interesting to determine if these stages are differentially regulated by the identified nucleators. These points will be addressed by a combination of biochemical and microscopic methods, including a synchronized podosome reformation assay. In sum, podosomes present as an excellent and accessible model to determine subcellular recruitment and time-dependent activation of actin nucleators. The existence of different substructures within the actin core, as well as the multi-layered dynamics of podosomes will thus allow new insights into the spatiotemporal interplay not only between different classes (Arp2/3 and formins) but also between different isoforms (formins) of actin nucleators.