Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.Preprin

Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions.

International audienceThe invasiveness of cells is correlated with the presence of dynamic actin-rich membrane structures called invadopodia, which are membrane protrusions that are associated with localized polymerization of sub-membrane actin filaments. Similar to focal adhesions and podosomes, invadopodia are cell-matrix adhesion sites. Indeed, invadopodia share several features with podosomes, but whether they are distinct structures is still a matter of debate. Invadopodia are built upon an N-WASP-dependent branched actin network, and the Rho GTPase Cdc42 is involved in inducing invadopodial-membrane protrusion, which is mediated by actin filaments that are organized in bundles to form an actin core. Actin-core formation is thought to be an early step in invadopodium assembly, and the actin core is perpendicular to the extracellular matrix and the plasma membrane; this contrasts with the tangential orientation of actin stress fibers anchored to focal adhesions. In this Commentary, we attempt to summarize recent insights into the actin dynamics of invadopodia and podosomes, and the forces that are transmitted through these invasive structures. Although the mechanisms underlying force-dependent regulation of invadopodia and podosomes are largely unknown compared with those of focal adhesions, these structures do exhibit mechanosensitivity. Actin dynamics and associated forces might be key elements in discriminating between invadopodia, podosomes and focal adhesions. Targeting actin-regulatory molecules that specifically promote invadopodium formation is an attractive strategy against cancer-cell invasion

Adhesion to the bone marrow niche - podosome-like structures in hematopoietic stem cells

Maintenance and homeostasis of the hematopoietic system is enabled by hematopoietic stem and progenitor cells (HSPC). These multipotent cells ensure the replenishment of all hematopoietic cells by giving rise to progeny with the ability to differentiate into respective cells while also self-renewing themselves. A crucial factor to regulate this essential feature of HSPC is their interaction with a specific microenvironment – the hematopoietic stem cell niche in the bone marrow (BM). The interaction of HSPC with their niche provides the signals necessary for the cells to maintain their stemness and to sustain the HSPC population. In order to provide these signals constantly, adhesion of the stem cells to the extracellular matrix (ECM) of the BM niche is of the utmost importance. Yet, the way in which HSPC anchor to their specific microenvironment is not well understood. In the here presented work, the adhesion structures established by HSPC upon contact with fibronectin are investigated in regard to their appearance, maturity state and composition of cell adhesion molecules (CAM). By investigating the spatial distribution of native and mature adhesion structure markers alongside the actin cytoskeleton in adherent HSPC using super resolution microscopy it was found, that HSPC adhere in a dynamic manner and display varying morphologies that can contain protrusions and/or structures resembling podosomes. Further investigation of these podosome-like structures (PLS) revealed that these structures are defined by an F-actin spot strongly co-localizing with phosphorylated PYK2 (pPYK2). Assessment of the spatial distribution of pPYK2, alongside the classic podosome proteins vinculin and paxillin, showed a distribution of these molecules towards the periphery of the F-actin spot. This distribution pattern became more pronounced - resembling classic podosomes more - in cells with round morphology and increased number of PLS. Additionally, examining the differentiation state of HSPC with different morphologies, it was found that the round, PLS containing cells were less differentiated compared to cells with deviating observed morphologies. Together, these findings indicate a highly dynamic adhesion behavior of HSPC, involving PLS. Given the importance of proper stem cell retention in the BM niche environment, for full function of the hematopoietic system, understanding the adhesion processes involved is an essential step in gaining insight into hematopoiesis in health and disease

Transcellular diapedesis is initiated by invasive podosomes

Producción CientíficaDiapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the “transcellular” route) or between them (the “paracellular” route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended “invasive podosomes” to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes

DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates

International audiencePodosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces

Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’

The role of plectin in the regulation of carcinoma cell invasion

The research presented in this thesis endeavours to understand the role of the plakin family of cytoskeletal linker proteins in the migration and invasive potential of epithelial carcinomas. This study focuses on plectin, a plakin family member that has previously been implicated with a role in tissue integrity of the skin and muscle. I investigated the complex gene organisation of the alternative first exons in human plectin, leading to the discovery of a further novel isoform plectin-lk. A panel of colon and breast carcinoma cell lines that vary in their differentiation and metastatic potential were used to investigate the expression of the isoforms at the mRNA and protein levels, showing plectin to be expressed at higher levels in the more invasive cell types. The subcellular localisation of the alternatively spliced plectin isoforms was investigated using green fluorescent protein (GFP) and polyclonal antibodies, revealing isoform specific targeting to different actin structures. Ablation of plectin or vimentin (a major intermediate filament protein of mesenchymal cells that interacts with plectin), by small interfering RNAs suggest these proteins are able to modulate invasion, migration and attachment of the epithelial carcinoma cells. Further investigation into the novel isoform plectin-lk reveals a role in the formation of podosome like adhesion structures in a Rho kinase dependant manner that facilitate migration in the SW480 colon carcinoma cells. The above findings are novel and contribute to the understanding of migration and invasion of cancer cells. Furthermore, this understanding could provide novel targets of cancer cell metastasis

Membrane dynamics in cell migration

Migration of cells is required in multiple tissue-level processes, such as in inflammation or cancer metastasis. Endocytosis is an extremely regulated cellular process by which cells uptake extracellular molecules or internalise cell surface receptors. While the role of endocytosis of focal adhesions (FA) and plasma membrane (PM) turnover at the leading edge of migratory cells is wide known, the contribution of endocytic proteins per se in migration has been frequently disregarded. In this review, we describe the novel functions of the most well-known endocytic proteins in cancer cell migration, focusing on clathrin, caveolin, flotillins and GRAF1. In addition, we highlight the relevance of the macropinocytic pathway in amoeboid-like cell migration