Tissue infiltration of macrophages, although critical for innate immunity, is also involved in pathologies, such as chronic inflammation and cancer. In vivo, macrophages migrate mostly in a constrained three-dimensional (3D) environment. However, in vitro studies, mainly focused on two dimensions, do not provide meaningful clues about the mechanisms involved in 3D macrophage migration. In contrast, tumor cell 3D migration is well documented. It comprises a protease-independent and Rho kinase (ROCK)-dependent amoeboid migration mode and a protease-dependent and ROCK-independent mesenchymal migration mode. In this study, we examined the influence of extracellular matrix (composition, architecture, and stiffness) on 3D migration of human macrophages derived from blood monocytes (MDMs). We show that: 1) MDMs use either the amoeboid migration mode in fibrillar collagen I or the mesenchymal migration mode in Matrigel and gelled collagen I, whereas HT1080 tumor cells only perform mesenchymal migration; 2) when MDMs use the mesenchymal migratory mode, they form 3D collagenolytic structures at the tips of cell protrusions that share several markers with podosomes as described in two dimensions; 3) in contrast to tumor cells, matrix metalloproteinase inhibitors do not impair protease-dependent macrophage 3D migration, suggesting the involvement of other proteolytic systems; and 4) MDMs infiltrating matrices of similar composition but with variable stiffness adapt their migration mode primarily to the matrix architecture. In conclusion, although it is admitted that leukocytes 3D migration is restricted to the amoeboid mode, we show that human macrophages also perform the mesenchymal mode but in a distinct manner than tumor cells, and they naturally adapt their migration mode to the environmental constraints.

SummaryMycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P1-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

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Cancer cells use different modes of migration, including integrindependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contractionbased amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement.uring metastasis, tumor cells encounter various extracellular matrix (ECM) environments with distinct composition and architecture, including basement membrane (BM) and interstitial collagen networks (1-3). Although basic mechanisms of cell motility on 2D substrata are generally well understood, much less is known regarding the mechanics of cell migration in 3D matrix environments (1, 4-7). Recent evidence supports the conclusions that 2D and 3D migration can differ considerably and that different types of 3D motility exist depending on the biophysical properties of the 3D environment (viscoelasticity, confinement, porosity) (8-15). Understanding the mechanics of cancer cell migration in these different 3D environments is therefore of paramount importance.Schematically, two modes of 3D migration of invasive cells have been described; the models differ by the dependency on actomyosin contractility, requirement for integrin adhesion, and matrix remodeling. In the mesenchymal mode, which has some parallel with 2D migration of fibroblasts (1), invasive cells are elongated and require pericellular matrix proteolysis for extending Racdependent F-actin-based leading pseudopodia in an integrindependent manner (16)(17)(18)(19)(20). In contrast, some carcinoma cells invade with a low-adhesion amoeboid mode of migration, characterized by a round morphology, no stable intrinsic cell polarity, and a required high level of RhoA/ROCK-driven actomyosin contractility (17, 18). Amoeboid cell motility, which is typically associated with bleb formation, is independent of proteases, and it is generally thought that by forming blebs, tumor cells can squeeze themselves through preexisting voids in the matrix (5, 21). In vivo, bleb-based motility has been implicate...

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

Proteolytic degradation of the extracellular matrix (ECM) is one intrinsic property of metastatic tumor cells to breach tissue barriers and to disseminate into different tissues. This process is initiated by the formation of invadopodia, which are actin-driven, finger-like membrane protrusions. Yet, little is known on how invadopodia are endowed with the functional machinery of proteolytic enzymes [1, 2]. The key protease MT1-MMP (membrane type 1-matrix metalloproteinase) confers proteolytic activity to invadopodia and thus invasion capacity of cancer cells [3-6]. Here, we report that MT1-MMP-dependent matrix degradation at invadopodia is regulated by the v-SNARE TI-VAMP/VAMP7, hence providing the molecular inventory mediating focal degradative activity of cancer cells. As observed by TIRF microscopy, MT1-MMP-mCherry and GFP-VAMP7 were simultaneously detected at proteolytic sites. Functional ablation of VAMP7 decreased the ability of breast cancer cells to degrade and invade in a MT1-MMP-dependent fashion. Moreover, the number of invadopodia was dramatically decreased in VAMP7- and MT1-MMP-depleted cells, indicative of a positive-feedback loop in which the protease as a cargo of VAMP7-targeted transport vesicles regulates maturation of invadopodia. Collectively, these data point to a specific role of VAMP7 in delivering MT1-MMP to sites of degradation, maintaining the functional machinery required for invasion.

Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck ؊/؊ mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck ؊/؊ BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocytelimited expression with a role in 3D migration, could be a target for new antiinflammatory and antitumor molecules. IntroductionPhagocytes constitute the first line of host defense against microorganisms. 1,2 To reach an infectious site, they transmigrate through the endothelial wall, basal membranes, and connective tissues and infiltrate the damaged organ to affect host defense and tissue repair. 3 Nevertheless, phagocytes have not only friend but also foe functions. 4,5 In several pathologic states, including chronic inflammatory 6,7 and neurodegenerative diseases 8 or atherosclerosis, 9-11 phagocyte-dependent tissue lesions often occur. In addition, it has been established that the presence of macrophages within tumors is a sign of a poor prognosis as they enhance angiogenesis and metastases (see Mantovani et al 12 ; Condeelis and Pollard 13 ; and Balkwill et al 14 for reviews). In contrast, T-cell infiltration into tumors is often associated with a more positive prognosis. 15 Therefore it is becoming a challenge to specifically control tissue infiltration of macrophages without affecting lymphocyte migration. By targeting macrophage migration-related molecules, new anti-inflammatory and antitumorbased drugs could be developed. 16,17 However, the cellular and molecular mechanisms involved in macrophage migration are poorly understood.Phagocyte migration has been studied mostly in vitro in 2 dimensions (2D) in response to chemotactic factors. In these experiments, cells are plated on either plastic or glass coverslips, coated or not with matrix proteins. However, in vivo, phagocyte transendothelial migration and infiltration through tissue involve primarily 3-dimensional (3D) regulation. Transend...

It is generally assumed that cells interrogate the mechanical properties of their environment by pushing and pulling on the extracellular matrix (ECM). For instance, acto-myosin-dependent contraction forces exerted at focal adhesions (FAs) allow the cell to actively probe substrate elasticity. Here, we report that a subset of long-lived and flat clathrin-coated structures (CCSs), also termed plaques, are contractility-independent mechanosensitive signaling platforms. We observed that plaques assemble in response to increasing substrate rigidity and that this is independent of FAs, actin and myosin-II activity. We show that plaque assembly depends on αvβ5 integrin, and is a consequence of frustrated endocytosis whereby αvβ5 tightly engaged with the stiff substrate locally stalls CCS dynamics. We also report that plaques serve as platforms for receptor-dependent signaling and are required for increased Erk activation and cell proliferation on stiff environments. We conclude that CCSs are mechanotransduction structures that sense substrate rigidity independently of cell contractility.