Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.

A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and three-dimensional invasion of macrophages.

The matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth.

The Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved.

Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence.

Podosomes are dynamic cell adhesions that are also sites of extracellular matrix degradation, through recruitment of matrix-lytic enzymes, particularly of matrix metalloproteinases. Using total internal reflection fluorescence microscopy, we show that the membrane-bound metalloproteinase MT1-MMP is enriched not only at podosomes but also at distinct "islets" embedded in the plasma membrane of primary human macrophages. MT1-MMP islets become apparent upon podosome dissolution and persist beyond podosome lifetime. Importantly, the majority of MT1-MMP islets are reused as sites of podosome reemergence. siRNA-mediated knockdown and recomplementation analyses show that islet formation is based on the cytoplasmic tail of MT1-MMP and its ability to bind the subcortical actin cytoskeleton. Collectively, our data reveal a previously unrecognized phase in the podosome life cycle and identify a structural function of MT1-MMP that is independent of its proteolytic activity. MT1-MMP islets thus act as cellular memory devices that enable efficient and localized reformation of podosomes, ensuring coordinated matrix degradation and invasion.

Podosomes in space: macrophage migration and matrix degradation in 2D and 3D settings.

Migration of macrophages is a key process for a variety of physiological functions, such as pathogen clearance or tissue homeostasis. However, it can also be part of pathological scenarios, as in the case of tumor-associated macrophages. This review presents an overview of the different migration modes macrophages can adopt, depending on the physical and chemical properties of specific environments, and the constraints they impose upon cells. We discuss the importance of these environmental and also of cellular parameters, as well as their relative impact on macrophage migration and on the formation of matrix-lytic podosomes in 2D and 3D. Moreover, we present an overview of routinely used and also newly developed assays for the study of macrophage migration in both 2D and 3D contexts, their respective advantages and limitations, and also their potential to reliably mimic in vivo situations.

The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes.

Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA-induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures.

Functional dissection of an intrinsically disordered protein: understanding the roles of different domains of Knr4 protein in protein-protein interactions.

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded C-terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two-hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N-terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C-terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or "hubs" are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein-protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.