Genetic engineering of Hoxb8-immortalized hematopoietic progenitors - a potent tool to study macrophage tissue migration.

Tumor-associated macrophages (TAMs) are detrimental in most cancers. Controlling their recruitment is thus potentially therapeutic. We previously found that TAMs perform protease-dependent mesenchymal migration in cancer, while macrophages perform amoeboid migration in other tissues. Inhibition of mesenchymal migration correlates with decreased TAM infiltration and tumor growth, providing rationale for a new cancer immunotherapy specifically targeting TAM motility. To identify new effectors of mesenchymal migration, we produced ER-Hoxb8-immortalized hematopoietic progenitors (cells with estrogen receptor-regulated Hoxb8 expression), which show unlimited proliferative ability in the presence of estrogen. The functionality of macrophages differentiated from ER-Hoxb8 progenitors was compared to bone marrow-derived macrophages (BMDMs). They polarized into M1- and M2-orientated macrophages, generated reactive oxygen species (ROS), ingested particles, formed podosomes, degraded the extracellular matrix, adopted amoeboid and mesenchymal migration in 3D, and infiltrated tumor explants ex vivo using mesenchymal migration. We also used the CRISPR/Cas9 system to disrupt gene expression of a known effector of mesenchymal migration, WASP (also known as WAS), to provide a proof of concept. We observed impaired podosome formation and mesenchymal migration capacity, thus recapitulating the phenotype of BMDM isolated from Wasp-knockout mice. Thus, we validate the use of ER-Hoxb8-immortalized macrophages as a potent tool to investigate macrophage functionalities.

Trim33 is essential for macrophage and neutrophil mobilization to developmental or inflammatory cues.

Macrophages infiltrate and establish in developing organs from an early stage, often before these have become vascularized. Similarly, leukocytes, in general, can quickly migrate through tissues to any site of wounding. This unique capacity is rooted in their characteristic amoeboid motility, the genetic basis of which is poorly understood. Trim33 (also known as Tif1-γ), a nuclear protein that associates with specific DNA-binding transcription factors to modulate gene expression, has been found to be mainly involved in hematopoiesis and gene regulation mediated by TGF-ß. Here, we have discovered that in Trim33-deficient zebrafish embryos, primitive macrophages are unable to colonize the central nervous system to become microglia. Moreover, both macrophages and neutrophils of Trim33-deficient embryos display a reduced basal mobility within interstitial tissues, and a profound lack of a response to inflammatory recruitment signals, including local bacterial infections. Correlatively, Trim33-deficient mouse bone marrow-derived macrophages display a strongly reduced three-dimensional amoeboid mobility in fibrous collagen gels. The transcriptional regulator Trim33 is thus revealed as being essential for the navigation of macrophages and neutrophils towards developmental or inflammatory cues within vertebrate tissues.

Rho/ROCK pathway inhibition by the CDK inhibitor p27(kip1) participates in the onset of macrophage 3D-mesenchymal migration.

Infiltration of macrophages into tissue can promote tumour development. Depending on the extracellular matrix architecture, macrophages can adopt two migration modes: amoeboid migration--common to all leukocytes, and mesenchymal migration--restricted to macrophages and certain tumour cells. Here, we investigate the initiating mechanisms involved in macrophage mesenchymal migration. We show that a single macrophage is able to use both migration modes. Macrophage mesenchymal migration is correlated with decreased activity of Rho/Rho-associated protein kinase (ROCK) and is potentiated when ROCK is inhibited, suggesting that amoeboid inhibition participates in mechanisms that initiate mesenchymal migration. We identify the cyclin-dependent kinase (CDK) inhibitor p27(kip1) (also known as CDKN1B) as a new effector of macrophage 3D-migration. By using p27(kip1) mutant mice and small interfering RNA targeting p27(kip1), we show that p27(kip1) promotes mesenchymal migration and hinders amoeboid migration upstream of the Rho/ROCK pathway, a process associated with a relocation of the protein from the nucleus to the cytoplasm. Finally, we observe that cytoplasmic p27(kip1) is required for in vivo infiltration of macrophages within induced tumours in mice. This study provides the first evidence that silencing of amoeboid migration through inhibition of the Rho/ROCK pathway by p27(kip1) participates in the onset of macrophage mesenchymal migration.

The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

Extracellular proteolysis in macrophage migration: losing grip for a breakthrough.

Macrophage tissue infiltration is a hallmark of several pathological situations including cancer, neurodegenerative disorders and chronic inflammation. Hence, deciphering the mechanisms of macrophage migration across a variety of tissues holds great potential for novel anti-inflammatory therapies. Leukocytes have long been thought to migrate through tissues by using the amoeboid (protease-independent) migration mode; however, recent evidence indicates that macrophages can use either the amoeboid or the mesenchymal (protease-dependent) migration mode depending on the environmental constraints. Proteolytic activity is required for several key processes including cell migration. Paradoxically, the role of proteases in macrophage migration has been poorly studied. Here, by focusing on the best characterized extracellular protease families - MMPs, cathepsins and urokinase-type plasminogen activator - we give an overview of their probable involvement in macrophage migration. These proteases appear to play a role in all of the situations encountered by migrating macrophages, i.e. diapedesis, 2D and 3D migration. Migration of macrophages across tissues seems to proceed through an integrative analysis of numerous environmental clues allowing the cells to adapt their migration mode (amoeboid/mesenchymal) and secrete dedicated proteases to ensure efficient tissue infiltration, as discussed in this review. The role of proteases in macrophage migration is an emerging field of research, which deserves further work to allow a more precise understanding.

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis.

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 phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules.

Matrix architecture dictates three-dimensional migration modes of human macrophages: differential involvement of proteases and podosome-like structures.

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.

HIV-1 Nef triggers macrophage fusion in a p61Hck- and protease-dependent manner.

Macrophages are a major target of HIV-1 infection. HIV-1-infected macrophages form multinucleated giant cells (MGCs) using poorly elucidated mechanisms. In this study, we show that MGC formation was reduced when human macrophages were infected with nef-deleted HIV-1. Moreover, expression of Nef, an HIV-1 protein required in several aspects of AIDS, was sufficient to trigger the formation of MGCs in RAW264.7 macrophages. Among Nef molecular determinants, myristoylation was dispensable, whereas the polyproline motif was instrumental for this phenomenon. Nef has been shown to activate hematopoietic cell kinase (Hck), a Src tyrosine kinase specifically expressed in phagocytes, through a well-described polyproline-SH3 interaction. Knockdown approaches showed that Hck is involved in Nef-induced MGC formation. Hck is expressed as two isoforms located in distinct subcellular compartments. Although both isoforms were activated by Nef, only p61Hck mediated the effect of Nef on macrophage fusion. This process was abolished in the presence of a p61Hck kinase-dead mutant or when p61Hck was redirected from the lysosome membrane to the cytosol. Finally, lysosomal proteins including vacuolar adenosine triphosphatase and proteases participated in Nef-induced giant macrophage formation. We conclude that Nef participates in HIV-1-induced MGC formation via a p61Hck- and lysosomal enzyme-dependent pathway. This work identifies for the first time actors of HIV-1-induced macrophage fusion, leading to the formation of MGCs commonly found in several organs of AIDS patients.

[Phagocyte migration: an overview]. / La migration des phagocytes: tour d'horizon.

Phagocytes are the first line of host defense thanks to their capacity to infiltrate infected and wounded tissues, where they exert their bactericidal and tissue repair functions. However, tissue infiltration of phagocytes also stimulates the progression of pathologies such as cancer and chronic inflammatory diseases. It is therefore necessary to identify the molecular and cellular mechanisms that control this process to identify new therapeutic targets. Phagocytes leave the blood stream by crossing the vascular wall and infiltrate interstitial tissues, a three-dimensional environment. A state-of-the-art of the different steps of phagocyte tissue recruitment in vivo and of the different in vitro models is developed in this synthesis. We focus on recent data concerning the migration of phagocytes in three-dimensional environments. The use of two different migration modes, amoeboid and mesenchymal, by macrophages and the role of podosomes and proteases in the mesenchymal migration are discussed.

Hematopoietic cell kinase (Hck) isoforms and phagocyte duties - from signaling and actin reorganization to migration and phagocytosis.

The activity of hematopoietic cell kinase (Hck), a member of the Src family kinases, is modulated by regulatory mechanisms leading to distinct protein conformations with gradual levels of activity. Hck is mostly expressed in phagocytes as two isoforms, p59Hck and p61Hck, which show distinct subcellular localizations and trigger distinct phenotypes when expressed ectopically in fibroblasts. Hck has been reported to be involved in phagocytosis, adhesion and migration, and to regulate formation of membrane protrusions, lysosome exocytosis, podosome formation, and actin polymerization. The present review focuses on the mechanisms regulating Hck activity as well as on the functions of Hck isoforms in phagocytes, and presents selected examples of Hck substrates and/or adaptors shown to interact with the kinase in myeloid cells. Deciphering Hck signaling pathways is a challenge to progress in the understanding of innate immune responses and pathologies involving phagocytes such as inflammatory diseases, leukemia, and human immunodeficiency virus-1 (HIV-1) infection.

The Protease-Dependent Mesenchymal Migration of Tumor-Associated Macrophages as a Target in Cancer Immunotherapy.

Macrophage recruitment is essential for tissue homeostasis but detrimental in most cancers. Tumor-associated macrophages (TAMs) play a key role in cancer progression. Controlling their migration is, thus, potentially therapeutic. It is assumed that macrophages use amoeboid motility in vivo like other leukocytes. However, it has not yet been explored. We examined TAM migration using intravital microscopy in mouse tumors and by monitoring ex vivo tissue infiltration in human surgical samples. We demonstrated that TAMs perform protease-dependent and ROCK-independent mesenchymal migration inside mouse fibrosarcoma and breast cancer explants using their own matrix metalloproteases (MMP). In contrast, macrophages use ROCK-dependent and protease-independent amoeboid migration inside inflamed ear derma and in connective tissue at the tumor periphery. We also showed that inhibition of mesenchymal migration correlates with decreased TAM recruitment and tumor growth. In conclusion, this study elucidates how macrophages migrate in vivo, and it reveals that the MMP-dependent migration mode of TAMs provides a rationale for a new strategy in cancer immunotherapy: to target TAMs specifically through their motility. Cancer Immunol Res; 6(11); 1337-51. ©2018 AACR.

Re-arrangements of podosome structures are observed when Hck is activated in myeloid cells.

Podosomes are adhesion structures with an extracellular matrix-degrading capacity mostly found in monocyte-derived cells. We have previously shown that the protein tyrosine kinase Hck, a member of the Src family, triggers the de novo formation of podosome rosettes in a lysosome-dependent manner when expressed in its constitutively active form. Hck is specifically expressed in myeloid cells. In human monocyte-derived macrophages (MDMs) it is present at podosomes. Here we addressed whether its activation by lipopolysaccharide and interferon-gamma has an effect on podosome organization in MDMs. Several structures were observed evolving from individual podosomes to clusters, aggregates and rosettes. In chronic myeloid leukemia cells, Hck is constitutively activated by the fusion protein Bcr-Abl and podosome-like structures were present. Finally, in monocyte-derived osteoclasts, Hck was found to accumulate at podosome belts. In conclusion, in monocyte-derived cells, it is likely that Hck could play a role in podosome re-arrangements.

The human macrophage mannose receptor is not a professional phagocytic receptor.

The macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full-length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full-length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR-mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin-coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further.

HIV-1 Infection of T Lymphocytes and Macrophages Affects Their Migration via Nef.

The human immunodeficiency virus (HIV-1) disseminates in the body and is found in several organs and tissues. Although HIV-1 mainly targets both CD4(+) T lymphocytes and macrophages, it has contrasting effects between these cell populations. HIV-1 infection namely reduces the viability of CD4(+) T cells, whereas infected macrophages are long-lived. In addition, the migration of T cells is reduced by the infection, whereas HIV-1 differentially modulates the migration modes of macrophages. In 2-dimensions (2D) assays, infected macrophages are less motile compared to the control counterparts. In 3D environments, macrophages use two migration modes that are dependent on the matrix architecture: amoeboid and mesenchymal migration. HIV-1-infected macrophages exhibit a reduced amoeboid migration but an enhanced mesenchymal migration, via the viral protein Nef. Indeed, the mesenchymal migration involves podosomes, and Nef stabilizes these cell structures through the activation of the tyrosine kinase Hck, which in turn phosphorylates the Wiskott-Aldrich syndrome protein (WASP). WASP is a key player in actin remodeling and cell migration. The reprogramed motility of infected macrophages observed in vitro correlates in vivo with enhanced macrophage infiltration in experimental tumors in Nef-transgenic mice compared to control mice. In conclusion, HIV infection of host target cells modifies their migration capacity; we infer that HIV-1 enhances virus spreading in confined environments by reducing T cells migration, and facilitates virus dissemination into different organs and tissues of the human body by enhancing macrophage mesenchymal migration.

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.

Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments.

Leukocytes migrate through most tissues in the body, a process which takes place in 3D environments. We have previously shown that macrophages use the amoeboid migration mode in porous matrices such as fibrillar collagen I and the mesenchymal mode involving podosomes and matrix proteolysis in dense matrices such as Matrigel. Whether such a plasticity may apply to other leukocytes and to all subsets of macrophages is unknown. Here, we therefore provide a comparative analysis of the in vitro 3D migration modes adopted by primary human leukocytes. Blood-derived monocytes, neutrophils and T lymphocytes were found to use the amoeboid mode in a porous fibrillar collagen I matrix but were unable to infiltrate dense Matrigel and to form podosomes. M2-polarized macrophages and elicited peritoneal macrophages formed podosome rosettes, degraded the ECM and infiltrated both matrices. In contrast, M1 macrophages were motionless in 2D and 3D environments, whilst resident macrophages, devoid of podosomes, were only able to use the amoeboid mode. Thus, we conclude that whereas all leukocytes use the amoeboid mode to migrate through porous matrices, it is only certain macrophages that can adopt the mesenchymal mode that permits migration through dense matrices. Interestingly, the acquisition of mesenchymal migration capacity by macrophages correlates with the presence of podosomes and with their capacity to organize those as rosettes, which appears to be modulated by their differentiation and polarization states. As a perspective, specific control of the mesenchymal migration would be a potential target for therapeutic approaches aiming at decreasing macrophage tissue infiltration.

Macrophage podosomes go 3D.

Macrophage tissue infiltration is a critical step in the immune response against microorganisms and is also associated with disease progression in chronic inflammation and cancer. Macrophages are constitutively equipped with specialized structures called podosomes dedicated to extracellular matrix (ECM) degradation. We recently reported that these structures play a critical role in trans-matrix mesenchymal migration mode, a protease-dependent mechanism. Podosome molecular components and their ECM-degrading activity have been extensively studied in two dimensions (2D), but yet very little is known about their fate in three-dimensional (3D) environments. Therefore, localization of podosome markers and proteolytic activity were carefully examined in human macrophages performing mesenchymal migration. Using our gelled collagen I 3D matrix model to obligate human macrophages to perform mesenchymal migration, classical podosome markers including talin, paxillin, vinculin, gelsolin, cortactin were found to accumulate at the tip of F-actin-rich cell protrusions together with ß1 integrin and CD44 but not ß2 integrin. Macrophage proteolytic activity was observed at podosome-like protrusion sites using confocal fluorescence microscopy and electron microscopy. The formation of migration tunnels by macrophages inside the matrix was accomplished by degradation, engulfment and mechanic compaction of the matrix. In addition, videomicroscopy revealed that 3D F-actin-rich protrusions of migrating macrophages were as dynamic as their 2D counterparts. Overall, the specifications of 3D podosomes resembled those of 2D podosome rosettes rather than those of individual podosomes. This observation was further supported by the aspect of 3D podosomes in fibroblasts expressing Hck, a master regulator of podosome rosettes in macrophages. In conclusion, human macrophage podosomes go 3D and take the shape of spherical podosome rosettes when the cells perform mesenchymal migration. This work sets the scene for future studies of molecular and cellular processes regulating macrophage trans-migration.

The oncogenic activity of the Src family kinase Hck requires the cooperative action of the plasma membrane- and lysosome-associated isoforms.

Hck is a phagocyte specific proto-oncogene of the Src family expressed as two isoforms, p59Hck and p61Hck. It plays a critical role in Bcr/Abl-chronic myeloid leukaemia and is able to transform fibroblasts in vitro. However, the tumourigenic activity of Hck and the respective oncogenic functions of Hck isoforms have not been examined. Tet-Off fibroblasts expressing constitutively active mutants of p59Hck and p61Hck together or individually were used. In contrast to cells expressing p59Hck(ca) or p61Hck(ca) alone, cells expressing both isoforms were transformed in vitro and induced tumour formation in 90% of nude mice within 2 weeks. This is the first demonstration of (i) the tumourigenic activity of Hck in mice, (ii) the cooperative action of the two Hck isoforms in vitro and in vivo. To our knowledge, this is the first example of a transforming activity 'split' in two requisite isoforms.

Tyrosine-phosphorylated STAT5 accumulates on podosomes in Hck-transformed fibroblasts and chronic myeloid leukemia cells.

In chronic myeloid leukemia (CML), the transforming activity of Bcr/Abl involves constitutive activation of the phagocyte specific Src-family tyrosine kinase Hck, which in turn directly activates the signal transducer and activator of transcription 5 (STAT5). The effect of Hck on STAT5 was first explored independently of Bcr/Abl by expressing the constitutively active Hck mutant (Hck(ca)) in MEF3T3-TetOff fibroblasts. As previously reported, Hck(ca)-expressing cells form podosomes which are actin-rich structures involved in trans-tissular cell migration and found in the few cell types able to cross anatomic boundaries. We demonstrated that in these cells, the tyrosine-phosphorylated form of STAT5 (PY-STAT5) increased and preferentially localized on podosomes together with Hck, instead of translocating to the nucleus as observed with conventional stimuli such as IFNgamma. To examine whether similar results were obtained in the presence of Bcr/Abl, the CML cell line K562 was used. We observed that (i) podosomal structures are present in these cells in contrast to Bcr/Abl-negative leukemic cells, (ii) podosome formation was inhibited by Bcr/Abl- and Src-kinase inhibitors, and (iii) PY-STAT5 mainly colocalized with Hck on these structures. The presence of podosomes was not sufficient to trap STAT5 since in normal macrophages which spontaneously form podosomes and express regulated Hck, PY-STAT5 is in the nucleus. In conclusion, this is the first report showing that PY-STAT5 associates to podosomes in a process dependent on constitutive activation of Hck. We propose that STAT5, previously classified as a transcription factor, could play another role outside the nucleus, elicited by the Bcr/Abl-Hck transforming pathway.

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes.

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.