Podosomes are multifunctional organelles of invasive cells that combine several key abilities, including adhesion, matrix degradation and mechanosensing. The necessary spatiotemporal fine-tuning of podosome structure, turnover and function implies the existence of an intricate network of proteins, comparable to other integrin-based adhesions. However, no systematic effort has yet been made to map the podosome proteome. Here, we describe the purification of podosome-enriched fractions from primary human macrophages, labelled with isotopically stable amino acids, and the subsequent mass spectrometric analysis of these fractions. We present a consensus list of 203 proteins, comprising 33 known podosome proteins and 170 potential novel components. We also present second-level analyses of the podosome proteome, as well as proof-of-principle experiments by showing that the newly identified components WDR1/AIP-1 and hnRNP-K localise to the core structure of macrophage podosomes. Comparisons with other adhesion structure proteomes confirm that the podosome proteome shares components with focal adhesions and invadopodia, but also reveal an extensive overlap with spreading initiation centres (SICs). We suggest that the consensus list comprises a significant part of the podosome proteome and will be helpful for future studies on podosome structure, composition and function, and also for detailed classification of adhesion structure subtypes.
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