Trafficking and surface exposure of the metalloprotease MT1-MMP in macrophages

Abstract

Macrophages are immune cells capable of migration and invasion through tissues. They can also associate with metastatic cancer cells and provide support through their matrix remodeling capacities and creation of migration tracks. In both scenarios, localized proteolytic degradation of the extracellular matrix is crucial. The major topic of my PhD thesis was the investigation of trafficking and regulation of the metalloproteinase MT1-MMP, a central regulator of matrix degradation in macrophages. This thesis shows for the first time that MT1-MMP is localized at podosomes, actin-rich matrix-degrading organelles in macrophages. Moreover, podosome dissolution induced formation of MT1-MMP dot-like structures embedded in the plasma membrane called “islets”. They are free of typical podosome-components and persist beyond podosome lifetime. Also, islets constitute a preferential site for reemergence of podosomes, revealing a new function of MT1-MMP as memory device for the reformation of podosomes. This mechanism is completely independent of the proteolytic activity of MT1-MMP. I also showed that the cytoplasmic tail of MT1-MMP and precisely the small peptide LLY573 is responsible for its localization at podosomes. The LLY573 motif ensures this localization thanks to its capacity to bind subcortical actin cytoskeleton. In a second project, I also helped to investigate members of the RabGTPases family controlling cell surface exposure of MT1-MMP, extracellular matrix degradation and invasion of macrophages. We identified Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in human macrophages. Depletion of these RabGTPases as well as overexpression of dominant negative and positive mutants showed that Rab5a, Rab8a and Rab14 do regulate cell surface exposure of MT1-MMP, contact of MT1-MMP-positive vesicles with podosomes, matrix degradation in 2D and also 3D proteolytic cell invasion. These results provide a detailed understanding of MT1-MMP trafficking and of the molecular mechanisms regulating podosome-mediated matrix degradation and proteolytic cell invasion.

Macrophages are immune cells capable of migration and invasion through tissues. They can also associate with metastatic cancer cells and provide support through their matrix remodeling capacities and creation of migration tracks. In both scenarios, localized proteolytic degradation of the extracellular matrix is crucial. The major topic of my PhD thesis was the investigation of trafficking and regulation of the metalloproteinase MT1-MMP, a central regulator of matrix degradation in macrophages. This thesis shows for the first time that MT1-MMP is localized at podosomes, actin-rich matrix-degrading organelles in macrophages. Moreover, podosome dissolution induced formation of MT1-MMP dot-like structures embedded in the plasma membrane called “islets”. They are free of typical podosome-components and persist beyond podosome lifetime. Also, islets constitute a preferential site for reemergence of podosomes, revealing a new function of MT1-MMP as memory device for the reformation of podosomes. This mechanism is completely independent of the proteolytic activity of MT1-MMP. I also showed that the cytoplasmic tail of MT1-MMP and precisely the small peptide LLY573 is responsible for its localization at podosomes. The LLY573 motif ensures this localization thanks to its capacity to bind subcortical actin cytoskeleton. In a second project, I also helped to investigate members of the RabGTPases family controlling cell surface exposure of MT1-MMP, extracellular matrix degradation and invasion of macrophages. We identified Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in human macrophages. Depletion of these RabGTPases as well as overexpression of dominant negative and positive mutants showed that Rab5a, Rab8a and Rab14 do regulate cell surface exposure of MT1-MMP, contact of MT1-MMP-positive vesicles with podosomes, matrix degradation in 2D and also 3D proteolytic cell invasion. These results provide a detailed understanding of MT1-MMP trafficking and of the molecular mechanisms regulating podosome-mediated matrix degradation and proteolytic cell invasion.