In this section, we focus on how membrane microdomains participate in cell migration and subsequent fusion at the OC precursor (pOC) stage and how these microdomains mediate the osteolytic and secretory functions of OCs during the mOC stage.
Migration of pOCs: membrane microdomains serve as platforms
During OC culture in vitro, lamellipodia are often observed; they represent the direction of cell extension and are considered the hallmark structure of early OC development.32,33,34 Here, we clarify the basic functions of these membrane microstructures structures and the key proteins that constitute them.
When pOCs migrate, the structure at the leading edge of the cell dynamically extends and retracts.35 OCs without podosomes have spicule-like structures, which are referred to as lamellipodia, as observed after knockout of Cortactin.36 This structure has also been reported in the literature by Akisaka et al.34 The activation and formation of lamellipodia are generally believed to be induced by the Arp2/3 complex consisting of microfilamentous nucleation factors, in which the nucleation-promoting factors Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) play important roles.34,35 Previous findings suggest that lamellipodia are actin-based structures and that their formation depends on regulatory factors.
Lamellipodia are common in migratory cells such as fibroblasts, and their general characteristics include the broad protrusion of the leading edge and an edge that can roll back from the membrane ruffle.37,38 Whether lamellipodia function similarly in fibroblasts and OCs is unclear. Through the use of transmission electron microscopy (TEM), Domon T et al.33 showed that the morphology of migrating OCs was irregular and flat, and they confirmed that these cells have lamellipodia, indicating that via this migratory structure, OCs can move on dentin.33 Mature osteoclasts (mOCs) also appear to have lamellipodia on their membrane. mOCs cultured in vitro exhibited stretched out lamellipodia that can mechanically decompose substrates and bring the substrates to the surface of the cell body via retraction of the lamellipodia.39 Although lamellipodium-like structures were observed in this study, the specific differences between lamellipodia in the pOC and mOC stages remain unclear. Notably, we initially focus our attention on lamellipodia during the migratory phase.
Lamellipodia determine the direction of cell migration, and lamellipodium stretching requires the actin network; therefore, we need to identify the scaffolding and regulatory proteins that determine the OC membrane structure.40 Structurally, focal adhesion anchors the cell to the matrix and thus provides the mechanical force needed for actin contraction.37 Additionally, cells migrate not only by extending lamellipodia, which are formed by scaffolds via an actin network but also by extending filopodia, which contain only part of the actin bundle and aggregate to form lamellipodia.39,41 Hence, matrix anchoring and actin movement are the two critical components of pseudopod formation and movement.37 We conclude that matrix anchoring is dependent mainly on membrane proteins to form the first structural platform, while the actin that is recruited forms the second platform and mediates contractile motility based on regulatory factors to establish actin flow.37,42,43
In OCs, the initiation site of the lamellipodium membrane microdomain may be composed of integrins or other adhesion receptors (e.g., other receptor tyrosine kinases (RTKs) such as colony-stimulating factor 1 receptor, also known as c-FMS44) that activate downstream regulatory proteins, including GTPases, protein kinases, and phosphatases, to induce the ARP2/3-related actin network and thus form the “second platform”.45,46 Considering the work of Boujemaa–Paterski, Rajaa et al., Geiger, Benjamin et al. and Fukunaga, Tomohiro et al., we propose a model based on integrin adhesion in OCs. (1) Integrins recruit and activate vinculin via talin to form nascent adhesions. (2) Vinculin is recruited and binds highly branched F-actin networks and contracts to establish actin flow, at which point the adhesions mature. (3) Centripetal actin flow at 1–3 μm·min−1 may stimulate the maturation of other nascent adhesions. (4) Eventually, many adhesions accumulate, enhancing mechanical resistance and ultimately leading to expansion of the leading edge of the lamellar pseudopod (Fig. 3).42,43,47 Among the integrins highly expressed in OCs, αvβ348 but not other integrin subunits has been shown to colocalize with vinculin, talin, and arp2/3. RTKs, such as epidermal growth factor receptor (EGFR), can also form the first platform based on its regulation of downstream PI3K, SRC, RAS, and RAC expression, and the modulation of the ARP2/3 complex and WASP affects the formation of the actin network and lamellipodia.32,49,50,51,52 Although evidence to support a role for RTKs in lamellipodium formation in OCs is insufficient, the inhibitory effect of RTK inhibitors on osteoclasts suggests that RTKs may be involved, which warrants further study.53,54 Other adhesion-related proteins, including cadherin, also remain to be investigated as regulators of lamellipodium formation.55,56,57 Notably, many lattice-like protein sheets have been found at the edge of filamentous pseudopodia.58 Although not colocalized with the OC actin network, this protein lattice is tightly bound to the apatite surface and may act as an adhesive rather than an endocytic agent. Therefore, it remains unclear whether this part of the protein lattice can serve as a scaffold for establishing the structural domain of the lamellar pseudopod membrane.
In conclusion, the identification of essential assembly sites in the microstructural domain of the lamellar pseudopod membrane may facilitate the development of locally acting regulators of early OC polarization. Although it remains unclear whether adhesion receptors in addition to integrins are involved in the assembly process, targeted regulation of the “first platform” and “second platform” in lamellipodium formation may facilitate the selective regulation of OC functions.
OC fusion: membrane microdomain interactions with the actin cytoskeleton
The actin cytoskeleton of OCs is a dynamic structure that changes rapidly during cell migration, fusion, and resorption. The membrane microdomains of OCs need to be supported by the actin cytoskeleton, and when cortical actin is reconstructed, the cell membrane structure changes accordingly.
During the fusion phase of the OC life cycle, the actin cytoskeleton promotes the extension of filopodia between cells or actin flow, which results in the formation of the characteristic TNT membrane domain structure (Fig. 4) or ZLS (Fig. 5) to trigger fusion.59,60 In the early stage of fusion, monocytes rely on their TNTs to fuse with another monocyte and thus generate multinucleated cells.60 The later stage is dominated by the formation of ZLSs between multinucleated cells and their fusion partners.61
TNT-associated membrane domains, not filopodia, are the keys to early fusion
The conventional view is that at the early stage of OC fusion, pOCs filopodia protrude to initiate fusion with partners. Although the role of filopodia has been demonstrated, many questions, such as how the nucleus is delivered and how filopodia trigger fusion, remain unanswered.59,62 Recently, a TNT, which is a very thin membrane tube, was found at the head of a filopodium where two cells contacted each other.60 Therefore, TNTs, not filopodia, may directly participate in the connection between fusion partners and drive material transport. TNTs are thought to be important in cell communication among bone marrow-derived cells (including macrophages, OCs, and dendritic cells) and in the fusion of macrophages and pOCs. Therefore, to clarify the role of TNT-related membrane domains in OC fusion, we need to define which TNTs can mediate fusion and the key mechanisms through which TNT-related membrane domains are involved in fusion.31,63
How do TNT-associated membrane microdomains form?
First, as described by McCoy–Simandle, Kessler et al., a TNT is identified according to the following three phenotypic criteria: (1) it connects at least two cells, (2) it contains F-actin, and (3) it does not attach to the matrix but extends from filopodia. This definition can be used to distinguish a TNT from any other F-actin-rich structure, and a TNT may be considered a special membrane microdomain.64 A TNT is generated in two situations: when filopodia protrude between fusion partners and when two cells located next to each other are separated under the action of chemokines.65 In these cases, a tube is formed in the plasma membranes where the fused cells are connected and cellular components such as organelles are transported (Fig. 4a).
Which TNTs participate in OC fusion?
TNTs are designated closed or open depending on whether they are connected to the target cell.65 Previous studies have mainly suggested that closed-end TNTs mediate gap junction formation, but after their conversion into open-ended TNTs, TNTs are known to participate in a process similar in virus‒cell membrane fusion or cell‒cell fusion.66,67 In addition, TNTs are classified into two functionally distinct types according to their size: (1) those with a diameter less than 5 µm are thin TNTs and contain only F-actin, and (2) those with a diameter ranging from 5 to 20 µm are thick TNTs and contain both F-actin and microtubules. Previous studies have revealed that large organelles, including lysosomes, mitochondria, and even nuclei, can be transferred only through thick TNTs (Fig. 4b).60,65
Therefore, although TNT-associated membrane microdomains spanning pOCs at the fusion stage have been observed,60 it is thought that TNTs participate in OC fusion only when their diameter is in a specific range (5–20 µm) and when there is intercommunication within the tunneling tube.
How do TNT-associated membrane microdomains participate in OC fusion?
M-Sec is a key factor in the formation of a TNT; its expression is upregulated during osteoclastogenesis, and M-Sec depletion significantly inhibits OC fusion by inhibiting TNT formation.31,68 Nonetheless, the specific mechanism through which TNT-associated membrane microdomains mediate cell fusion remains to be elucidated. pOCs recognize distant fusion partners through long intercellular F-actin structures. When two cells approach each other, thin and short actin protrusions (approximately 10 μm) can be observed on the leading edge of the cells.69 Nuclei have also been observed in these structures,70 suggesting that the nucleus may be transported through tubes formed by the actin cytoskeleton, which may trigger prophase fusion.
Based on these findings, we asked the following question: What is required for TNTs to mediate fusion? (Fig. 4c).
Membrane proteins, the surface proteins in the membrane domain of TNTs, can recruit the actin cytoskeleton. Two types of membrane proteins involved in TNT function have been found in Ocs: DC-STAMP,71 which shows transport activity, and connexins, including CD3672 and CX-43.73 Although these proteins have been identified, the components of the TNT shell have not been fully characterized, and the exact mechanisms underlying TNT functions remains unclear.
Actin-related regulatory proteins: Myosin is critical for providing power to F-actin and is often recruited to the membrane domain. Myosin 10 (MyoX) has been identified as a molecular motor that regulates TNT formation. This unconventional myosin is specifically expressed in OCs.74 As shown through in vitro experiments, pOCs remained in a monocyte state after MyoX expression was reduced by shRNA. This result was largely obtained to MyoX binding to microtubules through its MyTH4 tail domain, regulating F-actin cytoskeleton dynamics to promote the formation of an ordered TNT. Moreover, DC-STAMP, a transmembrane protein in the structural domain of the TNT membrane, penetrates other precursor cells by further interacting with the F-actin backbone to achieve migration through TNTs.31
Interactions between actin and the perinuclear cytoskeleton: The nucleus is sometimes located within the microtubule-actin network, which mediates its transport, and the microtubule–actin filaments usually originate from the perinuclear region, which suggests that the nucleus and F-actin are closely related. Moreover, some regulatory proteins play irreplaceable roles in nucleus-related F-actin dynamics. The actin-binding ARP2/3 complex stabilizes bent and branched actin structures, whereas c-Src and cortactin colocalize with F-actin at the cell periphery, which suggests that the latter may participate in the rearrangement and stabilization of bent and branched F-actin networks.70 In addition, c-Src, cortactin, cofilin, and actin can accumulate around the nucleus, suggesting that their involvement in nuclear movement might partially involve the regulation of nucleus delivery via thick TNTs.70,75,76
Thus, TNT-associated membrane microdomains facilitate the transport of substances, including nuclei, and this process requires signal recognition mediated by surface molecules, including DC-STAMP, and interactions between F-actin and the perinuclear cytoskeleton. However, only a fraction of the relevant proteins in a TNT have been identified, and the proposed structural domain of the TNT membrane suggests that the scaffolding proteins in this structure not only include marker proteins of intercellular connections but also bind the intracellular actin cytoskeleton to the perinuclear frame. Here, we summarize only some of the components involved in these intercellular linkages, as their specific relation to nuclear transport events via natural scaffolding proteins remains to be discovered.
ZLS-associated membrane microdomains are key for multinucleated cell fusion
What are ZLS-associated membrane microdomains?
After single-nucleated precursor cells fuse to form multinucleated cells, they still need to combine with other fusion partners to form multinucleated OCs with more than three nuclei and podosome belts.59,61,77 The experiments conducted by Takito, Jiro et al. revealed that the F-actin cytoskeleton of multinucleated cells agglomerates form a zipper-like F-actin structure when in contact with other multinucleated cells, which has also been shown to be the basic manner through which multinucleated cells fuse.59,78 Therefore, elucidation of the ZLS membrane microdomain is extremely important to clarify the life cycle of OCs.
Formation and function of the ZLS
A ZLS and its associated membrane microdomains have attracted our interest. Membrane proteins in a TNT may recruit actin-related regulatory proteins by downstream signaling to then associate with cortical actin in the perinuclear area. In contrast to TNT-related membrane microdomains, ZLSs appear to mediate the closeness between two precursor OCs through a complex composed of F-actin and regulatory proteins. Force may be critical in directly promoting fusion events. When mononuclear and multinuclear cells collide with actin rings, the cell membranes at the collision site move in response to actin flow, leaving the plasma membranes close together, and F-actin condenses on the plasma membrane to form a cluster of ZLSs (Fig. 5).78 The formation of this structure is the basis for subsequent cell membrane fusion events. Subsequently, when plasma membranes are fused via actin flow, the ZLSs are reconstituted, and cortical actin is cleaved to form a foot vesicle band. Thus, these multinucleated cells fused by ZLSs can give rise to larger OCs (Fig. 5).59,78 Clarifying the mechanism through which the membrane microdomain and F-actin cytoskeleton induce ZLSs will help us elucidate the key mechanisms underlying the later stages of OC fusion.
Motility forces of the actin cytoskeleton and ZLS-associated membrane microdomains
Published studies have not clarified how E-cadherin and integrin β3 on the surface of ZLSs regulate actin flow or stability of the F-actin cytoskeleton. Dufrançais, Ophélie et al. found that these structures are not involved in the early fusion process but may stabilize adhesion points, promote migration, or induce protein hydrolysis in the later fusion phase.60 In contrast, migration and adhesion between multinucleated cells and fusion partners may be facilitated by binding between membrane proteins, which induces downstream signaling and establishes actin flow. Accordingly, we focused on the potential role of the actin cytoskeleton and intracellular motility forces (Fig. 5).
Actin backbone: Arp2/3 and cortactin are colocalized with actin at the center of a ZLS, and the core framework of a ZLS is based on F-actin and nonmuscle myosin IIA. The periphery of the structure is covered with paxillin and vinculin to regulate its traveling wave motion.78 The cell contact surface is also covered with fusion-related proteins, including zyxin, E-cadherin, CD47-SIRPα, and integrin β3, forming a composite structure consisting of the intracellular cytoskeleton and plasma membrane proteins.79,80
Actin flow: Actin within a single podosome “foot” undergoes vertical oscillatory motion, which in turn forms a traveling wave. An analysis of the spatiotemporal location of podosomes revealed that the vertical motion is based on two factors, namely, regulatory protein comovement patterns (vinculin and talin show similar vertical oscillations) and actin aggregation and assembly in the podosome core.81 In turn, this traveling wave triggered by the overall vertical oscillation of the actin cytoskeleton moves in such a way that neighboring cells squeeze against each other. Additionally, the distribution of the F-actin bundle at the OC podosome overlaps with that of myoIIA, the activation of which leads to the generation of circumferential forces and helps maintain a balancing effect on actin wave motion.78
Similar to TNTs, ZLSs contain and regulate actin proteins such as cortactin, paxillin, and vinculin.78 Moreover, the ZLS membrane microdomain similarly lacks a backbone protein that integrates the membrane protein component mediating contact recognition with the actin regulator of traveling wave formation. Importantly, the mechanism through which stomatin is bound by contact partners via exosomes and further mediates contact adhesion suggests that the structural domain microfusion mechanism that we propose may be activated at this stage; this hypothesis was assessed in previous studies, and further investigation of the mechanism underlying ZLS membrane microdomain formation is needed.22
Bone resorption and secretory lysosomes
The classical structural signature of mature OCs is the formation of F-actin-rich adhesion structures on the ventral membrane contacting the bone surface, i.e., the ruffled border.1,58 This membrane is called a ruffled border (RB) due to the large number of folds.1,82 Although RBs have been shown to be involved in the process of OC bone resorption, it remains unclear exactly how it functions.
The RB is enclosed by a sealing zone composed of integrin αvβ3 as the core protein and V-ATPase, a transporter protein (such as CLC-7), a small GTPase, and lysosome-associated membrane proteins (LAMP1, 2).83,84,85,86 However, Mika T K Mulari et al. showed that the RB is also divided into an uptake region and a release region.1,85 The release zone mediates vesicle entry into a cell that stores enzymes and acidic ions, thus allowing the release of lysosomal contents into the bone matrix enclosed by the sealed zone, followed by internalization of the osteolysis products into vesicles in the resorption zone, which are then released to the functional secretory domain (FSD) at the tip of the cell via transcytosis (Fig. 6).87,88 In this process, the RB acts as a “transit station”, mediating the output and input of vesicles.
Hence, we aim to elucidate the mechanism underlying RB generation and the occurrence of bone resorption by focusing on secretory vesicles.
Although we currently do not know whether transcytosis is necessary for the osteolytic function of mature OCs, the output of vesicles is considered crucial for osteolysis, which is why we focus first on these secretory vesicles.89,90 Notably, another reason for this focus on secretory vesicles is that the RB is equivalent to a transit station that mediates secretory lysosome release and vesicle transcytosis, which means that the membrane microdomains are somewhat similar among these three structures, and therefore, it is necessary to elucidate the membrane microdomains of secretory vesicles.
In contrast to other cells, OCs have evolved specific lysosome-associated organelles (LROs), which are acidic vesicles that specifically secrete osteolytic proteins. These secretory vesicles are thus called secretory lysosomes.91,92 Research on the unique characteristics of the membrane microdomains of secretory lysosomes is worth considering.
Based on recent literature, we found that V-ATPase performed both scaffolding and recruitment roles in the membrane microdomain, and these functions are closely related to different V-ATPase subunits (Table 2).93,94,95 We propose that the V-ATPase a and d subunits, which recruit downstream signaling factors and affect cellular localization, are critical for V-ATPase scaffolding action.89,96,97
The kinetic marker and scaffolding protein V-ATPase
The membrane microdomain of secretory lysosomes in OCs has some specific features. V-ATPase is a key structure mediating lysosomal transport, and the heterogeneity of its subunits determines their distribution and subcellular locations in different cells. Knockdown of the a3 subunit inhibits its plasma membrane-targeting ability, leading to the suppression of bone resorption. Previous studies have suggested that OC bone resorption requires V-ATPase containing the a3 subunit.95,98 In addition, a3 subunit assembly involves assembly of the d subunit (the d2 isoform) heterodimer, which is expressed at fourfold higher levels in OCs than the d1 subunit prevalent in other cells, suggesting that V-ATPase consisting of the a3 and d2 subunits is essential for OC membrane behaviors.89,99,100
The membrane microdomains of secretory lysosomes in OCs are composed mainly of ATPase as the core protein because ATPase not only maintains acidity inside the lysosome but also supplies energy to enable interactions with Rab family members and regulate the Rab protein guanine nucleotide binding for targeted lysosome transport.94,101 This finding indicates that V-ATPase is not only a membrane marker of secretory lysosomes but also the driving force for their function (Fig. 6).
Based on our understanding of V-ATPase, we have additional questions to answer: How does V-ATPase respond to signaling that drives the transport of secretory lysosomes, and how is V-ATPase oriented relative to the RB?
In response to the first question, V-ATPase can bind to small GTPases and regulatory proteins in a vesicular pH-dependent manner, which implies that V-ATPase plays a role in not only promoting an acidic pH environment but also in transmitting acid-dependent signaling.102 The a and c subunits of the V0 complex may play primary roles in linking these activities. Although these mechanisms are not fully understood, we can conclude that the maturation of pH-related signaling in secretory lysosomes likely initiates the bone resorption process.
Movement and release: Membrane microdomain components in secretory lysosomes
We explored the role of the membrane microdomains in the movement of secretory lysosomes, including membrane-bound transport and membrane-bound attachment (Fig. 7).
Membrane-bound transport with CD68 as a marker and Rab7 as a motility driver
Rab7 (the GDP-bound form) binds to the a3 isoform of V-ATPase in the lysosomal membrane.103 After activation, Rab7 (the GTP-bound form) binds motor proteins and their adapters to move inward along microtubules.103,104 CD68 is a lysosomal marker at this stage that colocalizes with lysosomes and the OC plasma membrane.105
Membrane-bound attachment with CD63 as a marker and Rab27 as a motility driver
Rab27 is involved in the fusion of the lysosomal membrane with the plasma membrane, and Rab27a knockdown suppresses the binding of the CD63-labeled intracellular compartment to the plasma membrane. Moreover, when Rab27a binds to GTP and is activated, the effector proteins Slp4 and Rab27a can colocalize with a structural compartment containing CD63 to enhance the stability of secretory lysosomes after binding to the plasma membrane.106
Furthermore, secretory lysosomes are predicted to bind to Rab7 and Rab27a through the a3 subunit of V-ATPase, which activates their downstream effectors to maintain their stability and plasma membrane attachment. Thus, the identification of Rab effectors that regulate the cell polarity-directed transport of secretory lysosomes in OCs may help clarify the specific transport patterns of secretory lysosomes.
In summary, mOCs undergo targeted transport and binding as well as show polarity through the actions of plasma membrane and the membrane microdomain of secretory lysosomes that recruit different Rab proteins with different kinetic properties as drivers for transport at different stages. Therefore, identifying the critical scaffolding proteins or complexes that integrate energy exchange factors is expected to enable the elucidation of the complete mechanism underlying secretory lysosome movement in the OC-mediated osteolysis process.
The ruffled border and transcytosis
Based on the aforementioned information, we conclude that RB is closely related to the vesicle cycle in OCs. On the one hand, secretory lysosomes in OCs are targeted to the plasma membrane based on the V-ATPase-associated membrane microdomains and are incorporated into it via membrane fusion. The key membrane proteins from secretory lysosomes, such as CCL, remain on the plasma membrane, expanding the membrane area, and then, the plasma membrane bends to form a ruffled border (Table 3).1 During the formation of a RB, small connecting bands are observed on the outer side of a forming RB, and they are gradually assembled into an RB, and therefore, the peripheral region of the ruffled border is a key “site” for membrane fusion107 (Table 4).
On the other hand, when OCs undergo the bone resorption process at one site, they endocytose the corresponding metabolites into a resorption pit into the center of the RB. Then, vesicles are formed and transport metabolites to the FSD at the top of the cell and release them. This process, referred to as transcytosis, is a critical mechanism of OC secretory function in bone.85,88,90,108,109,110,111
We discussed the key membrane domains of secretory lysosomes in the previous section, but the role of secretory vesicles in transcytosis has not been addressed. The secretory lysosomal components in an RB have been identified. Whether these components are utilized by transcytotic vesicles remains to be explored. It also remains unclear whether V-ATPase and other specific Rab molecules are crucial proteins that participate in bone secretion. These gaps in knowledge have not been explained in the literature to date, but we believe that the membrane microdomain of secretory lysosomes will provide a paradigm for further investigation into the key structures of secretory lysosomes. The process of V-ATPase vesicle formation involving actin has been described in Han, Guanghong et al.112