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Osteoclasts are highly motile cells that depend on rapid changes in their actin cytoskeleton to complete cycles of movement and attachment during bone resorption. Attachment and migration of osteoclasts occur through dot-like F-actin-enriched adhesive structures called podosomes. Each podosome consists of a central core of actin filaments surrounded by a diffuse actin cloud comprising of filamentous and monomeric actin. A variety of signaling and actin-binding proteins (nucleating, severing, and bundling) are present in these structures. This article presents an overview of the organization of podosomes and the possible roles of actin-binding and signaling molecules in the dynamic regulation of actin organization during migration and bone resorption of osteoclasts.

Bone resorption by activated osteoclasts occurs in three successive steps: attachment of the osteoclasts to the bone surface to form the sealing zone, bone mineral solubilization, and organic matrix degradation. In the last two decades, great advances have been made in understanding the mechanisms underlying osteoclast-mediated bone resorption. Mature osteoclasts on the bone surface become polarized, a process regulated by small G proteins, which leads to the formation of the sealing zone, which is the highly compact superstructure formed by podosomes. Integrins, which mediate both podosome formation and osteoclast-bone interaction, transduce pivotal signaling for osteoclast activation. Osteoclasts generate an isolated extracellular microenvironment between themselves and the bone known as the sealing zone surrounding the ruffled membrane to release hydrogen ions (H+) and chloride ions (Cl−) through the V-type ATPase proton pump and the CLC chloride channel into the resorption lacuna to reach an acidic pH which favors bone resorption. V-ATPase proton pump subunit Atp6i and chloride channels subunit ClC-7 are essential for osteoclast-mediated extracellular acidification. Cathepsin K was revealed to be the lysosomal protease that is essential for organic matrix degradation in bone resorption. In humans, mutations of Atp6i and ClC-7, and Cathepsin K lead to malignant osteopetrosis and pycnodysostosis respectively. In this article, we summarize recent advances in understanding the mechanisms of osteoclast-mediated bone resorption and signaling that regulates osteoclast activation, polarization, acidification, and matrix degradation. Further understanding of the mechanisms underlying osteoclast activation and the signaling that regulates osteoclast polarization, acidification, lysosome trafficking, and bone resorption will provide unprecedented therapeutic strategies to treat osteolytic diseases such as osteoporosis, periodontal disease, and rheumatoid arthritis.

Summary: Bone-resorbing osteoclasts play a central role in bone remodeling and its pathology. To digest bone, osteoclasts re-organize both F-actin, to assemble podosomes/sealing zones, and membrane traffic, to form bone-facing ruffled borders enriched in lysosomal membrane proteins. It remains elusive how these processes are coordinated. Here, we show that ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain-containing protein 1) fulfills this function. At podosomes/sealing zones, ARAP1 is part of a protein complex where its RhoGAP domain regulates actin dynamics. At endosomes, ARAP1 interacts with AP-3 adaptor complexes where its Arf-GAP domain regulates the Arf1-dependent AP-3 binding to membranes and, consequently lysosomal membrane protein transport to ruffled borders. Accordingly, ARAP1 or AP-3 depletion in osteoclasts alters their capacity to digest bone in vitro. and AP-3δ-deficient mocha mice, a model of the Hermansky-Pudlak storage pool syndrome, develop osteoporosis. Thus, ARAP1 bridges F-actin and membrane dynamics in osteoclasts for proper bone homeostasis. : Cell Biology; Organizational Aspects of Cell Biology; Functional Aspects of Cell Biology Subject Areas: Cell Biology, Organizational Aspects of Cell Biology, Functional Aspects of Cell Biology

Osteoclasts are bone resorbing cells acting as key mediators of bone disorders. Upon adhesion to bone, osteoclasts polarize and reorganize their cytoskeleton to generate a ring-like F-actin-rich structure, the sealing zone, wherein the osteoclast's resorptive organelle, the ruffled border, is formed. The dynamic self-organization of actin-rich adhesive structures, the podosomes, from clusters to belts is crucial for osteoclast-mediated bone degradation. Mice lacking the protein SWAP-70 display an osteopetrotic phenotype due to defective bone resorption caused by impaired actin ring formation in Swap-70−/− osteoclasts. To further elucidate the mechanisms underlying this defect, we investigated the specific function of SWAP-70 in the organization and dynamics of podosomes. These detailed studies show that the transition from podosome clusters to rings is impaired in Swap-70−/− osteoclasts. Live cell imaging of dynamic F-actin turnover and SWAP-70 localization during podosome patterning indicate that SWAP-70 is dispensable for cluster formation but plays a key role in F-actin ring generation. Our data provide insights in the role of SWAP-70's F-actin binding domain and pleckstrin homology (PH) domain in the proper localization of SWAP-70 and formation of a peripheral podosome belt, respectively. Ex vivo bone analyses revealed that SWAP-70-deficient osteoclasts exhibit defective ruffled border formation and V-ATPase expression. Our findings suggest an important role of membrane binding of SWAP-70 for the regulation of actin dynamics, which is essential for podosome patterning, and thus for the resorptive activity of osteoclasts., Highlights • SWAP-70 controls dynamic podosome patterning but not assembly of podosomes. • PIP3 and F-actin binding are required for proper subcellular localization of SWAP-70. • SWAP-70-deficient osteoclasts are impaired in ruffled border formation.

In order for the cell to function well within a multicellular system, the mechanical properties of the plasma membrane need to meet two different requirements: cell shape maintenance and rearrangement. To achieve these goals, phosphoinositides play key roles in the regulation of the cortical actin cytoskeleton. PI(4,5)P2 is the most abundant phosphoinositide species in the plasma membrane. It maintains cell shape by linking the actin cortex to the membrane via interactions with Ezrin/Radixin/Moesin (ERM) proteins and class I myosins. Although the role of D3-phosphoinositides, such as PI(3,4,5)P3, in actin-driven cell migration has been a subject of controversy, it becomes evident that the dynamic turnover of the phosphoinositide by the action of metabolizing enzymes, such as 5-phosphatases, is necessary. Recent studies have revealed an important role of PI(3,4)P2 in podosome/invadopodia formation, shedding new light on the actin-based organization of membrane structures regulated by phosphoinositide signaling. This article is part of a Special Issue entitled Phosphoinositides.

The human body contains approximately 10 trillion human cells, enough cells to cover approximately 800,000 football fields and many more cells in each of us than there are human beings on Earth. Yet, each cell in a single individual contains the same genome. Not only undifferentiated stem cells, but also partially and terminally differentiated cells of the epithelial and endothelial layers, germ and nurse cells, brain cells, metabolic and storage cells, muscle cells, immune cells, and cells of connective tissues, all contain the exact same genes in the same individual. Hence this unbelievable cellular diversity within a single individual cannot be explained by genetic variations among cells. Indeed, one just has to observe the truly fascinating diversity of phenotypes displayed by clonal, “normal” cells, stemming from the same single mother cell and plated on a uniform culture dish to immediately appreciate how incredibly heterogeneous cellular phenotypes can be. Within just a few generations, isogenic cells display multifold variations in not only cellular and nuclear size, but also in cellular and nuclear shape, cell motility parameters, the ability of cells to secrete soluble and insoluble factors and to adhere to each other and to their extracellular matrix, cytoskeletal organization, and in spatial and temporal distribution of organelles.

Summary Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force., Graphical Abstract, Highlights • Nontransformed fibroblasts on RGD membranes form podosome-like protrusions • Nanopatterned RGD membranes enable traction force, suppressing protrusion formation • Local activation of PI3K transforms prepodosomal-like RGD clusters • PIP3-bound RhoA GAP ARAP3 is recruited at the protrusion and downregulates RhoA-GTP, Outside-in integrin activation plays a critical role in cell adhesion and migration. Yu, Sheetz, and colleagues now report that plating cells onto fluid RGD membranes, thus inhibiting the development of traction force, causes the formation of podosome-like protrusions. The podosome-like adhesions on RGD membranes lack matrix metalloproteinases but share many other molecular components with classic podosomes in macrophages. The authors demonstrate that podosome-like protrusion formation is mediated by a PIP3-dependent pathway and is further aided by ARAP3-mediated RhoA inactivation.

Migration is essential for immune system function, from the establishment of the hematopoietic system in the bone marrow through to the capture and killing of invading pathogens. This reliance on migration is demonstrated by the existence of a subset of inherited immunodeficiencies that are a result of mutations that impact on migratory processes. Much of our understanding of the importance of migration in the immune response comes from the study of these immunodeficiencies. Wiskott–Aldrich syndrome is the first described and best studied migratory immunodeficiency, and this article will focus on this disorder, and its wide ranging impact on immune function. We will also give an overview of other migration defects that lead to immunodeficiency, including their genetic, biochemical, and cell biological basis.

Focal adhesions, and other related contacts, are dynamic multiprotein complexes that anchor extracellular-matrix receptors of the integrin family with the actin cytoskeleton, thereby mediating a robust cell–matrix adhesion. In this article we first describe focal adhesions from a structural perspective, outlining information obtained over the last four decades from different microscopy techniques. With this basis, we then overview the functional and molecular diversity of integrin adhesions, including focal adhesions and related cell–matrix adhesion sites such as focal complexes, fibrillar adhesions, and podosomes. Finally, we discuss the influence of actomyosin contractility on these sites and the mechanisms underlying the mechanosensitivity of focal adhesions.

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3- positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.

Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration. Furthermore, we outline detailed procedures to examine focal adhesion assembly and disassembly using time-lapse fluorescent microscopy. Finally, we provide methods for the analysis of podosomes, which are highly dynamic adhesive structures that form in immune cells and invasive cancer cells.

This chapter reviews the biological features of osteoclast development. Osteoclasts are hematopoietic cells that have bone-resorbing activity, and participate in bone remodeling and bone marrow formation. Mature functional osteoclasts are large multinuclear cells consisting of multiple osteoclasts fused with each other. Since embryonic stem (ES) cells have the potential to differentiate into all cell lineages, it should be possible to derive any cell lineage by appropriate induction of ES cells in culture. Osteoclasts, derived from hematopoietic stem cells, participate in bone remodeling and form bone marrow cavities through their bone resorbing activity. The precursors share their characteristics with the precursors of monocytic lineage cells, such as macrophages and dendritic cells. In one-step culture, a wider range of cell lineages could be observed in a dish compared with cloned stromal cell (OP9) cultures. In addition to hematopoietic lineages, at least endothelial cells, osteoblasts, myocardial cells, melanocytes, and pigmented epithelial cells are observed. Osteoblasts build the bone, endothelial cells invade the bone, and osteoclasts resorb the bone and make bone marrow cavity. These two lineages of cells are closely associated and located as concentric circles.

Publisher Summary This chapter discusses the osteoclast cytoskeleton. Eukaryotic cells contain three major classes of cytoskeletal fibers: 7 nm diameter actin microfilaments, 24 nm diamenter microtubules (MTs), and 10 nm diameter intermediate filaments. All of these fibers are formed by finely regulated polymerization of protein subunits. These cytoskeletal elements play a fundamental role not only in determining the steady-state organization of the cytoplasm, but also in facilitating selective delivery between spatially segregated organelles. The actin and MT cytoskeletal systems are closely interrelated both structurally and functionally in cells. Both filament systems commonly coexist within domains of the cytoplasm, and factors that cause redistribution of one often lead to a change in the distribution of the other. Cell motility is a cytoskeleton-dependent function. At the leading edge of migrating mouse fibroblasts, a biased assembly of actin is involved in the formation and stabilization of protrusions, while the perinuclear tail region exhibits a net disassembly of actin fibers. The osteoclast can be in a secretory or in a migratory state depending on the phase of its activity. The more recent findings on cytoskeleton involvement in these processes can very possibly apply to this very peculiar cell. Podosomes are highly dynamic and can assemble and disassemble in minutes. They are also linked to integrin receptors, endowed with the capability of linking several proteins of the extracellular matrix. The number and distribution of podosomes is related to osteoclastic resorptive activity. During bone resorption, they are thickly packed in a ring-like structure that surrounds the resorption lacuna, so closely assembled to resemble, at low magnification, a continuous actin ring. In nonresorbing osteoclasts, they can be observed in small groups scattered on the ventral membrane, or in ring-like fashion, but singularly identifiable.

Publisher Summary This chapter describes the methods for examining osteoclast development and function. Cytochemical staining for tartrate-resistant acid phosphatase (TRAP) is widely used for identifying osteoclasts in vivo and in vitro. Osteoclasts are shown to possess abundant calcitonin receptors. Expression of calcitonin receptors is one of the most reliable markers for identifying osteoclasts. For autoradiography of 125 I-labeled calcitonin, cultures are performed on plastic coverslips placed in 24-well culture plates. Cocultures of primary osteoblastic cells with bone marrow cells or spleen cells produce more osteoclasts than bone marrow cultures do. Osteoclasts formed on plastic culture dishes cannot be detached by treatment with either trypsin-EDTA or bacterial collagenase. To obtain functionally active osteoclasts formed in cocultures, a collagen gel culture is developed. Osteoclasts adhere to the bone surface through specialized discrete structures called podosomes in the clear zone, which consist mainly of dots containing F-actin. Therefore, the ringed structure of podosomes (actin ring) formed in osteoclasts is a characteristic of polarized osteoclasts. The actin rings are visualized by staining F-actin with rhodamine-conjugated phalloidin.

Publisher Summary Initial steps in smooth-muscle-cell differentiation start in very early stages. The different types of cardiovascular cells originate from the ventrolateral mesoderm (Xenopus) or the splanchnic mesoderm (Aminotes). This ventrolateral mesoderm can be regionalized using smooth muscle α-actin as a marker. Unlike the endothelial cells and cardiomyocytes, smooth muscle cells remain positive for smooth muscle α-actin expression. Smooth muscle cells are not unique in expressing smooth muscle α-actin. Other cells, most probably also from ventrolateral mesoderm origin, express smooth muscle α-actin as well, suggesting that smooth muscle α-actin marks a certain differentiation in state of cells of ventrolateral mesoderm origin or perhaps a functional state required in many cells during development. Actin expression in relation to the developing tunica media is associated with remodeling of the vascular system. There are, however, still actin patterns that are not well understood. They may be related to different influences on smooth-muscle differentiation in the vascular wall because until now, it is not known exactly which cells contribute to the development of vascular smooth muscle cells. In this respect, the role of neural crest cells is not clear as well. Some of the neural crest cells differentiate into smooth muscle cells, but others may have an inductive role.

Publisher Summary This chapter discusses growth factor-induced actin reorganization in Swiss 3T3 cells. Several factors that induce membrane ruffling are also known as “chemoattractants”; the cells subsequently polarize in response to such factors and become motile. The leading edge of motile cells often displays similar membrane ruffling structures to those seen early on in the response. In addition to actin reorganization at the plasma membrane, factors can also alter the density or the organization of actin filaments in the cell interior. In fibroblasts, stress fibers are the principal actin structures observed in the cytosol. Swiss 3T3 cells are well suited to analyzing growth factor-induced changes in actin organization because they show strong contact inhibition of growth and density arrest at a low density as a fiat monolayer of cells. Several extracellular factors stimulate the formation of stress fibers and membrane ruffles in Swiss 3T3 cells. The small GTP-binding proteins Rho and Rac regulate the factor-induced formation of stress fibers and membrane ruffles, respectively.

Publisher Summary This chapter describes the cell motility assays based on phagokinetic and scattering activities. Phagokinetic activity is estimated by measuring cell tracks formed by the phagocytosis of colloidal gold particles due to migrating cultured cells, and scattering activity is estimated by observing the scattering of the epithelial cells growing in colonies by phase contrast microscopy. The chapter describes the methods for microinjection of Rho– GDP-Dissociation Inhibitor (GDI) or C3, both of which inhibit cell motility by the cell motility assays. Cell motility is essential for inflammatory reactions, tissue repair, and immune system interactions in normal cells. Cell motility is essential for malignant cancer cells to infiltrate surrounding tissues and for smooth muscle cells in the media to migrate to the intima at the atherosclerotic region of vascular cells. The actomyosin system is involved in cell motility. In cultured cells, actin filaments exist principally in three types of structures: the cortical actin network, actin stress fibers, and cell surface protrusions, including membrane ruffling and microspikes. Membrane ruffling, which is caused by the polymerization of actin at the inner surface of the plasma membrane, is associated with cell motility in cultured cells.

Publisher Summary Single proteins, either α-actinin or talin, form a bridge between actin and integrin. Interaction of actin with these two proteins may serve different functions in focal adhesions. In situ, there are structures that appear to be homologous, with focal adhesions, such as the dense plaques of smooth muscle, the myotendinous junctions of skeletal muscle, and the adhesions made by platelets and many other cells, interacting with the extracellular matrix. During development, cell interactions with the extracellular matrix provide cues for cellular differentiation, migration, and growth characteristics. Cells grown in tissue culture, such as fibroblasts, endothelial cells, and epithelial cells, develop focal adhesions and provide a model system to study the interaction of cells with the extracellular matrix. Much of the research on focal adhesions is directed toward understanding the proteins involved in linking actin filaments to the plasma membrane. This chapter describes the current knowledge of the organization of focal adhesions and presents data that suggest two alternative mechanisms for the attachment of actin to the membrane at these sites. Additional linkages to the membrane at focal adhesions almost certainly exist. This is suggested by the existence of other focal adhesion components that have been detected by different methods and also by the low affinity of some of the interactions described in the chapter that may require additional stabilization in the cell. The chapter discusses the interaction between α-actinin and β 1 integrin and the interaction between talin and actin.

Publisher Summary It is required that to form metastases the tumor cells must penetrate the vascular wall to enter the interstitial stroma. Such penetration is thought to be an active process rather than a passive bursting of the vessel because of growth pressure. The two components of the vascular wall, which are mechanical barriers to tumor cell penetration, are the endothelium and the ntinuous basement membrane (BM). Ultrastructural studies indicate that the tumor cells may traverse the endothelium by moving between the endothelial cell bodies or by destroying the entire endothelial cell. Two types of cells, inflammatory cells, and malignant cells are known to possess the biochemical machinery for actively migrating through the blood vessel walls. Cellular extravasation is a complex cascade of the events involving attachment to endothelium or basement membrane followed by active invasion and migration of cells through vascular BM and adjacent perivascular connective tissue stroma. The mechanism for extravasation of the inflammatory cells and the tumor cells may be similar. The properties of the malignant cells that help them move across tissue boundaries include decreased cell adhesiveness, increased cell motility, angiogenesis factors, and enzymatic activities. The dense network of collagen fibers of the extracellular matrix is impenetrable to most cells. Invasive cells, such as inflamnatory cells and tumor cells, produce matrix degrading enzymes to cross tissue boundaries. Destruction of the extracellular matrix during tumor invasion has been observed histologically and ultrastructurally. A further application of the collagenase antibodies could be the localization of clinically silent micrometastases in the patients. Appropriate radionuclide scanning techniques have already been developed to localize the monoclonal antibodies injected into patients. Collagenase inhibitors could also play a future role in the therapy of tumor invasion.