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.     

Plectin deposition at podosome rings requires myosin contractility

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.     

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C     

Macrophage podosomes assemble at the leading lamella by growth and fragmentation

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.     

The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.     

Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein

Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.     

Dendritic Cell Podosome Dynamics Does Not Depend on the F-actin Regulator SWAP-70

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.     

The circle of life: Phases of podosome formation,turnover and reemergence

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.     

MEK/ERK pathway mediates PKC activation‐induced recruitment of PKCζ and MMP‐9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13‐dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP‐9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation‐induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu‐induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)‐inhibited PDBu‐induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP‐9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu‐induced podosomes by influencing the recruitment of PKCζ and MMP‐9 to podosomes. J. Cell. Physiol. 228: 416–427, 2013. © 2012 Wiley Periodicals, Inc.     

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.     

L-Plastin Nanobodies Perturb Matrix Degradation,Podosome Formation,Stability and Lifetime in THP-1 Macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.     

Phosphorylation of cortactin by p21-activated kinase

Cortactin is an F-actin binding protein that is enriched in dynamic cytoskeletal organelles such as podosomes, membrane ruffles, and lamellipodia. We have shown previously that Src-phosphorylation of cortactin is not required for its translocation to phorbol-ester induced podosomes in A7r5 aortic smooth muscle cells, but may be important for stability and turnover of podosomes. However, little is known of the role of Ser/Thr kinases in the regulation of cortactin. Here, we report that p21-associated kinase (PAK), which plays a crucial role in the formation of podosome and membrane ruffles, is able to phosphorylate cortactin in vitro. The predominant phosphorylation site is located at Ser113 in the first actin-binding repeat. Phosphorylation by PAK is not required for the translocation of cortactin to podosomes, lamellipodia, or membrane ruffles in A7r5 smooth muscle cells. However, binding of cortactin to F-actin is significantly reduced by PAK-phosphorylation. Taken together, these results suggest a role for PAK-phosphorylation of cortactin in the regulation of the dynamics of branched actin filaments in dynamic cytoskeletal organelles.     

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.     

The matricellular protein CCN5 regulates podosome function via interaction with integrin αvβ3

CCN proteins play crucial roles in cell motility, matrix turnover, and proliferation. In particular, CCN5 plays a role in cell motility and proliferation in several cell types; however, no functional binding proteins for CCN5 have been identified. In this study we report that CCN5 binds to the cell surface receptor integrin α_vβ₃ in vascular smooth muscle cells. Furthermore, this interaction takes place in podosomes, organelles known to degrade matrix and mediate motility. We show that CCN5 regulates the ability of podosomes to degrade matrix, but does not affect podosome formation. The level of CCN5 present in a podosome negatively correlates with its ability to degrade matrix. Conversely, knockdown of CCN5 greatly enhances the matrix-degrading ability of podosomes. These findings suggest that the antimotility effects of CCN5 may be mediated through the direct interaction of CCN5 and integrin α_vβ₃ in podosomes and the concomitant suppression of matrix degradation that is required for cell migration.     

Lasp-1 regulates podosome function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.     

Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes

Gamma-aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta2 subunit-containing GABAA receptors, in the brain.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Self-organized podosomes are dynamic mechanosensors

Podosomes are self-organized, dynamic, actin-containing structures that adhere to the extracellular matrix via integrins [1-5]. Yet, it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors, like focal adhesions [6-9]. We show here that myosin-II proteins form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin-II-dependent tension dissipated podosome actin rings before dissipating the myosin-ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate and were abolished when myosin-light-chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions, and they increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a nonmotile, nonrotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial for regulating these self-organized structures in living cells.     

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.