Phosphatidylserine orchestrates Myomerger membrane insertions to drive myoblast fusion.

Muscle cell fusion is a multistep process where the final step of the reaction drives progression beyond early hemifusion events to complete fusion. This step requires activity of the muscle-specific fusogen Myomerger, a single-pass transmembrane protein containing 84 amino acids with an ectodomain that includes two α-helices. Previous studies have demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. An obvious question is how such destabilizing activity might be regulated to avoid membrane and cellular damage, and how the two juxtaposed helices cooperate in fusion. Using cellular fusion assays and in vitro liposome assays, we report that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We demonstrate that externalized phosphatidylserine (PS), a lipid previously implicated in myoblast fusion, has a determinant role in the regulation of Myomerger activity. The membrane-proximal, amphipathic Helix-1 is normally disordered and its α-helical structure is induced by PS, making membrane interactions more efficacious. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and augments the membrane-stressing effects of Helix-1. These data reveal that Myomerger fusogenic activity is an exquisitely orchestrated event involving its two ectodomain helices, which are controlled by membrane lipid composition, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final step of myoblast fusion.

Flagging fusion: Phosphatidylserine signaling in cell-cell fusion.

Formations of myofibers, osteoclasts, syncytiotrophoblasts, and fertilized zygotes share a common step, cell-cell fusion. Recent years have brought about considerable progress in identifying some of the proteins involved in these and other cell-fusion processes. However, even for the best-characterized cell fusions, we still do not know the mechanisms that regulate the timing of cell-fusion events. Are they fully controlled by the expression of fusogenic proteins or do they also depend on some triggering signal that activates these proteins? The latter scenario would be analogous to the mechanisms that control the timing of exocytosis initiated by Ca2+ influx and virus-cell fusion initiated by low pH- or receptor interaction. Diverse cell fusions are accompanied by the nonapoptotic exposure of phosphatidylserine at the surface of fusing cells. Here we review data on the dependence of membrane remodeling in cell fusion on phosphatidylserine and phosphatidylserine-recognizing proteins and discuss the hypothesis that cell surface phosphatidylserine serves as a conserved "fuse me" signal regulating the time and place of cell-fusion processes.

Cell-surface phosphatidylserine regulates osteoclast precursor fusion.

Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans, the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

Insights into the localization and function of myomaker during myoblast fusion.

Multinucleated skeletal muscle fibers form through the fusion of myoblasts during development and regeneration. Previous studies identified myomaker (Tmem8c) as a muscle-specific membrane protein essential for fusion. However, the specific function of myomaker and how its function is regulated are unknown. To explore these questions, we first examined the cellular localization of endogenous myomaker. Two independent antibodies showed that whereas myomaker does localize to the plasma membrane in cultured myoblasts, the protein also resides in the Golgi and post-Golgi vesicles. These results raised questions regarding the precise cellular location of myomaker function and mechanisms that govern myomaker trafficking between these cellular compartments. Using a synchronized fusion assay, we demonstrated that myomaker functions at the plasma membrane to drive fusion. Trafficking of myomaker is regulated by palmitoylation of C-terminal cysteine residues that allows Golgi localization. Moreover, dissection of the C terminus revealed that palmitoylation was not sufficient for complete fusogenic activity suggesting a function for other amino acids within this C-terminal region. Indeed, C-terminal mutagenesis analysis highlighted the importance of a C-terminal leucine for function. These data reveal that myoblast fusion requires myomaker activity at the plasma membrane and is potentially regulated by proper myomaker trafficking.

Survival motor neuron protein deficiency impairs myotube formation by altering myogenic gene expression and focal adhesion dynamics.

While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility.

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus.

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.

Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.

Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 µM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1-2 µM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10-20 µM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration. This Ca(2+) signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca(2+) chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) inhibited both Ca(2+) spikes and peptide entry, suggesting that an increase in intracellular Ca(2+) precedes and is required for peptide entry. One of the hallmarks of Ca(2+) signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles.

Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin.

Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-cell fusion in development, tissue homoeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. In the present study, we explored the fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of LPC (lysophosphatidylcholine) that reversibly blocks membrane merger. After 16 h, we removed LPC and observed cell fusion events that would normally develop within 16 h develop instead within 30-90 min. Thus, whereas osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes, but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion, revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes.

Protein-driven membrane stresses in fusion and fission.

Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cells and release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and in most cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling.

Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

FORMATION OF MULTINUCLEATED OSTEOCLASTS DEPENDS ON AN OXIDIZED SPECIES OF CELL SURFACE ASSOCIATED LA PROTEIN.

The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells - generated by an increased number of cell fusion events - have higher resorptive activity. We find that osteoclast fusion and bone-resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La's unique regulatory role in osteoclast multinucleation and function is controlled by a ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.

RANKL inhibition reduces lesional cellularity and Gαs variant expression and enables osteogenic maturation in fibrous dysplasia.

Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gαs variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease.TRIAL REGISTRATION: ClinicalTrials.gov NCT03571191.

Cationic cell-penetrating peptide binds to planar lipid bilayers containing negatively charged lipids but does not induce conductive pores.

Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 µM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 µM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes.

Viral and developmental cell fusion mechanisms: conservation and divergence.

Membrane fusion is a fundamental requirement in numerous developmental, physiological, and pathological processes in eukaryotes. So far, only a limited number of viral and cellular fusogens, proteins that fuse membranes, have been isolated and characterized. Despite the diversity in structures and functions of known fusogens, some common principles of action apply to all fusion reactions. These can serve as guidelines in the search for new fusogens, and may allow the formulation of a cross-species, unified theory to explain divergent and convergent evolutionary principles of membrane fusion.

An inducible explant model of osteoclast-osteoprogenitor coordination in exacerbated osteoclastogenesis.

Elucidating a basic blueprint of osteoclast-osteoblast coordination in skeletal remodeling and understanding how this coordination breaks down with age and disease is essential for addressing the growing skeletal health problem in our aging population. The paucity of simple, activatable, biologically relevant models of osteoclast-osteoblast coordination has hindered our understanding of how skeletal remolding is regulated. Here, we describe an inducible ex vivo model of osteoclast-osteoblast progenitor coordination. Induction activates the release of osteoclastogenic factors from osteoprogenitors, which elicits the differentiation and fusion of neighboring preosteoclasts. In turn, multinucleated osteoclasts release soluble coupling factors, RANK+ extracellular vesicles and promote osteoprogenitor proliferation, recapitulating aspects of perturbed coordination in diseases underpinned by excessive osteoclast formation. We expect this model to expedite the investigation of cell-cell fusion, osteoclast-osteoblast progenitor coordination, and extracellular vesicle signaling during bone remodeling and offer a powerful tool for evaluating signaling cascades and novel therapeutic interventions in osteoclast-linked skeletal disease.

Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesis.

Multinucleated osteoclasts, essential for skeletal remodeling in health and disease, are formed by the fusion of osteoclast precursors, where each fusion event raises their bone-resorbing activity. Here we show that the nuclear RNA chaperone, La protein has an additional function as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. As fusion begins, La reappears as a low molecular weight species at the osteoclast surface, where it promotes fusion. La's role in promoting osteoclast fusion is independent of canonical La-RNA interactions and involves direct interactions between La and Annexin A5, which anchors La to transiently exposed phosphatidylserine at the surface of fusing osteoclasts. Disappearance of cell-surface La, and the return of full length La to the nuclei of mature, multinucleated osteoclasts, acts as an off switch of their fusion activity. Targeting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting La's potential as a therapeutic target.

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion.

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.

AFF-1, a FOS-1-regulated fusogen, mediates fusion of the anchor cell in C. elegans.

Cell fusion is fundamental for reproduction and organ formation. Fusion between most C. elegans epithelial cells is mediated by the EFF-1 fusogen. However, fusion between the anchor cell and the utse syncytium that establishes a continuous uterine-vulval tube proceeds normally in eff-1 mutants. By isolating mutants where the anchor-cell fails to fuse, we identified aff-1. AFF-1 ectopic expression results in fusion of cells that normally do not fuse in C. elegans. The fusogen activity of AFF-1 was further confirmed by its ability to fuse heterologous cells. AFF-1 and EFF-1 differ in their fusogenic activity and expression patterns but share eight conserved predicted disulfide bonds in their ectodomains, including a putative TGF-beta-type-I-Receptor domain. We found that FOS-1, the Fos transcription factor ortholog that controls anchor-cell invasion during nematode development, is a specific activator of aff-1-mediated anchor-cell fusion. Thus, FOS-1 links cell invasion and fusion in a developmental cascade.

Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.