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Membrane properties are determined in part by lipid composition, and cholesterol plays a large role in determining these properties. Cellular membranes show a diverse range of cholesterol compositions, the effects of which include alterations to cellular biomechanics, lipid raft formation, membrane fusion, signaling pathways, metabolism, pharmaceutical therapeutic efficacy, and disease onset. In addition, cholesterol plays an important role in non-cellular membranes, with its concentration in the skin lipid matrix being implicated in several skin diseases. In phospholipid membranes, cholesterol increases the tail ordering of neighboring lipids, decreasing the membrane lateral area and increasing the thickness. This reduction in the lateral area, known as the cholesterol condensing effect, results from cholesterol–lipid mixtures deviating from ideal mixing. Capturing the cholesterol condensing effect is crucial for molecular dynamics simulations as it directly affects the accuracy of predicted membrane properties, which are essential for understanding membrane function. We present a comparative analysis of cholesterol models across several popular force fields: CHARMM36, Slipids, Lipid17, GROMOS 53A6L, GROMOS-CKP, MARTINI 2, MARTINI 3, and ELBA. The simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes with varying cholesterol concentrations were conducted to calculate the partial-molecular areas of cholesterol and other condensing parameters, which are compared to the experimental data for validation. While all tested force fields predict small negative deviations from ideal mixing in cholesterol–DOPC membranes, only all-atom force fields capture the larger deviations expected in DMPC membranes. United-atom and coarse-grained models under-predict this effect, condensing fewer neighboring lipids by smaller magnitudes, resulting in too small deviations from ideal mixing. These results suggest that all-atom force fields, particularly CHARMM36 or Slipids, should be used for accurate simulations of cholesterol-containing membranes. [ABSTRACT FROM AUTHOR]

[Display omitted] The partitioning of viral fusion peptides in lipid membranes with varying order was investigated due to the fusion mechanism being a potential therapeutic approach. Using a planar bilayer model and advanced techniques such as neutron reflectometry (NR) and quartz crystal microbalance with dissipation (QCM-D), the structural aspects of peptide-lipid interactions were explored. The study focused on two target membranes: one forming a liquid-ordered domain and the other forming a liquid-disordered domain. Surprisingly, the COVID fusion peptide did not bind significantly to either membrane, as demonstrated by both QCM-D and NR data, suggesting negligible or no interaction with the bilayers. However, the acetylated COVID fusion peptide showed distinct behaviour, indicating a crucial role of N-terminal acetylation in binding to cholesterol-rich liquid-ordered domains. The acetylated peptide induced changes in the structure and thickness of the ordered bilayer with cholesterol whereas proteins and peptides commonly only bind to disordered phases. This study provides valuable insights into the mechanisms of viral membrane fusion and highlights the importance of acetylation in influencing peptide-lipid interactions, laying the groundwork for potential antiviral therapeutic strategies. [ABSTRACT FROM AUTHOR]

Synaptic transmission is mediated by specialized junctions that are characterized by a complex lipid and protein composition. The protein composition of synapses is diverse, and it remains unclear whether the molecular diversity of lipids impacts circuit-specific differences in neurotransmission as well as differential predisposition to synaptic dysfunction in disease. Recent work has shown that rather subtle changes in lipid content can influence synaptic function, but those that depend on molecular interactions between lipids and proteins remain unknown as technical limitations have precluded lipidomic studies at the level of individual synapses. Novel technologies, including mass spectrometry imaging and chemical tagging, now exist to study lipid function with the necessary molecular and spatial detail to resolve the lipidome of single synaptic junctions. The brain is an exceptionally lipid-rich organ with a very complex lipid composition. Lipids are central in several neuronal processes, including membrane formation and fusion, myelin packing, and lipid-mediated signal transmission. Lipid diversity is associated with the evolution of higher cognitive abilities in primates, is affected by neuronal activity, and is instrumental for synaptic plasticity, illustrating that lipids are not static components of synaptic membranes. Several lines of evidence suggest that the lipid composition of synapses is unique and distinct from other neuronal subcompartments. Here, we delve into the nascent field of synaptoneurolipidomics, offering an overview of current knowledge on the lipid composition of synaptic junctions and technological advances that will allow us to study the impact on synaptic function. [ABSTRACT FROM AUTHOR]

Plant-derived nanovesicles (PDVs) as nanodrug delivery carriers have gained recognition due to their satisfactory biosafety. However, there remains a challenge to target tumor sites accurately due to the uncertain membrane protein components on the surface of vesicles. Herein, a composite nanodrug delivery system by encapsulating the chemotherapy drug DOX is establish to efficiently target breast cancer. The novel nanoplatform (LEVBD) is formed by embedding the membrane fragments from breast cancer cell with the lemon-derived nanovesicles (LEVs) as the foundational skeleton. LEVBD reveal wonderful homologous tumor targeting due to fusion of cancer cell membrane components with LEVs, and the encapsulation of hybrid vesicles facilitates the transcellular transport of drugs. After intravenous injection, LEVBD could efficiently and selectively home to homologous tumor sites even under competition from heterologous tumors and significantly inhibit tumor growth without any observable toxic side effects. The doping of homologous cancer cell membranes provides a paradigm for the precise delivery of drug delivery vehicles using plant-derived vesicles as the backbone. [ABSTRACT FROM AUTHOR]

Multivalent DNA probes were creatively designed and decorated on small extracellular vesicles for precise tumor targeting and enhanced synergistic therapy. [Display omitted] • The engineered sEVs achieved precise tumor targeting and synergistic therapy. • Multivalent DNA probes were designed to enhance targeting ability. • Telomerase triggered intracellular nanocarriers aggregation was creatively proposed. • The nanocarrier enables site-specific and photo-controlled drug release. Small extracellular vesicles (sEVs) have gained wide attention as efficient carriers for disease treatment. However, the proclivity of sEVs to be ingested by source cells is insufficient to accurately target specific sites, posing a challenge in realizing controlled targeting treatment. Here, we developed an engineered sEV nanocarrier capable of precise tumor targeting and enhanced synergistic therapy. Multivalent DNA probes, comprising abundant AS1411 aptamers and telomerase primers, were innovatively modified on the sEV membrane (M-D-sEV) for precise tumor targeting. To achieve synergistic therapy, gold nanorod-cerium oxide nanostructures (Au NRs-CeO 2) and manganese dioxide nanosheets-doxorubicin (MnO 2 NSs-DOX) were encapsulated into liposomes (Lip-Mat). Then M-D-sEV and Lip-Mat were fused together through membrane fusion to obtain nanocarriers. Owing to the multivalence of the probes, the surface of the nanocarriers was loaded with numerous aptamers, which greatly enhances their targeting ability and promotes the accumulation of drugs. When nanocarriers were ingested by tumor cells, telomerase and multivalent DNA probes triggered their aggregation, enhancing the therapeutic effect. Furthermore, under laser irradiation, Au NRs-CeO 2 converted light into hyperthermia, thereby inducing the destruction of nanocarriers membrane. This process initiated a series of reactions involving glutathione and H 2 O 2 consumption, as well as DOX release, ultimately achieving synergistic tumor therapy. In vitro and in vivo studies demonstrated the remarkable targeting ability of multivalent DNA probes and excellent therapeutic effect of this strategy. The engineered strategy of sEVs provide a promising approach for precise tumor therapy and hold great potential for the development of efficient, safe, and personalized drug delivery systems. [ABSTRACT FROM AUTHOR]

Odontoblasts, terminally differentiated dentin-producing cells, critically rely on lysosomal functions for intracellular recycling and renewal. Beyond their traditional degradative role, lysosomes actively orchestrate cellular responses to external stimuli through precise and rapid intracellular trafficking and positioning. This study aimed to explore the influence of lysosomal positioning on odontoblast mineralization and the underlying mechanisms implicated in carious inflammation. Human dental pulp stem cells were induced to differentiate into human odontoblast-like cells (hOBLCs). hOBLCs were treated with various doses of LPS (0.1, 1, 5 μg/mL) to mimic carious inflammation. Lysosomal positioning was examined by immunofluorescence staining of lysosomal associated membrane protein 1 in healthy and carious human teeth, LPS-treated hOBLCs, mouse lower incisors at postnatal day 2.5, and mineralization medium cultured human dental pulp stem cells. Lysosomal positioning was manipulated by knockdown or overexpression of SNAPIN or ARL8B. Mineralization was assessed by ARS staining and expression of DSPP and DMP1. Lysosomal exocytosis was examined by detection of lysosomal-plasma membrane fusion, surface exposure of lysosomal associated membrane protein 1 luminal epitopes (1D4B), and extracellularly released lysosomal enzymes. Peripheral lysosomal positioning was markedly increased in odontoblasts within moderate and extensive carious lesions (P <.001) and in hOBLCs following LPS treatment. Increased peripheral dispersion of lysosomes was similarly observed during odontoblastic differentiation in vivo and in vitro. Moreover, peripheral lysosomal positioning promoted mineralization in inflamed hOBLCs, potentially via mTORC1 signaling pathway and lysosomal exocytosis. Inflammatory stimuli prompted a relocation of lysosomes in odontoblasts, redistributing them from perinuclear location toward the cell periphery, which in turn facilitated mineralization, potentially via mTORC1 signaling and lysosomal exocytosis. [ABSTRACT FROM AUTHOR]

Exploration of autophagy in different species has become a hotspot in cell biology in the past decades. Macroautophagy (hereafter, autophagy) is the most widely studied type. One of the hallmarks of autophagy is the fusion of the outer membrane (OM) of a closed double-membrane mature autophagosome (AP) with the lysosomal/vacuolar single membrane. Most researchers in the autophagy field agree upon this description. However, AP-lysosome/vacuole fusion models that do not follow this description frequently appear in the literature, even published in some prestigious journals until now. Some of the misrepresented models are from autophagy laboratories with brilliant publication records. These flaws should be addressed as a public announcement in the autophagy field to avoid spreading misinformation. The editors and reviewers are the guardians to ensure correct models. Abbreviations: AP: autophagosome; IM: inner membrane; OM: outer membrane. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 viral entry requires membrane fusion, which is facilitated by the fusion peptides within its spike protein. These predominantly hydrophobic peptides insert into target membranes; however, their precise mechanistic role in membrane fusion remains incompletely understood. Here, we investigate how FP1 (SFIEDLLFNKVTLADAGFIK), the N-terminal fusion peptide, modulates membrane stability and barrier function across various model membrane systems. Through a complementary suite of biophysical techniques—including electrophysiology, fluorescence spectroscopy, and atomic force microscopy—we demonstrate that FP1 significantly promotes pore formation and alters the membrane's mechanical properties. Our findings reveal that FP1 reduces the energy barrier for membrane defect formation and stimulates the appearance of stable conducting pores, with effects modulated by membrane composition and mechanical stress. The observed membrane-destabilizing activity suggests that, beyond its anchoring function, FP1 may facilitate viral fusion by locally disrupting membrane integrity. These results provide mechanistic insights into SARS-CoV-2 membrane fusion mechanisms and highlight the complex interplay between fusion peptides and target membranes during viral entry. [ABSTRACT FROM AUTHOR]

Interaction with host cell receptors initiates internalization of Kaposi's sarcoma-associated herpesvirus (KSHV) particles. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of glycoproteins H (gH), L (gL), and B (gB), that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed dual-fluorescent recombinant KSHV strains that incorporate fluorescent protein-tagged glycoproteins and capsid proteins. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. We demonstrated lytic reactivation and infectivity of dual-fluorescent KSHV. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. Using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established KSHV fusion kinetics upon infection of different target cells with marked differences in the "time-to-fusion" between cell types. Inhibition of vesicle acidification prevented KSHV particle-cell fusion, implicating low vesicle pH as a requirement. These findings were corroborated by comparison of RRV-YFP wildtype reporter virus and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB and classical fusion assays, these novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion. Author summary: All herpesviruses encode a fusion protein, gB, which fuses the virus membrane with host membranes. Membrane fusion is essential for infection by enveloped viruses. Under conditions where gB proteins from other herpesviruses fuse cells into syncytia, KSHV gB does not readily fuse cells, and KSHV infection was previously shown to be inhibited by substances that raise the pH of endocytotic vesicles. We therefore sought to test whether fusion of KSHV viral particles with cellular membranes is a pH-dependent step. We developed two tools to test this hypothesis. The first tool is a dual-fluorescent KSHV with differently colored protein tags at the viral envelope and the capsid, which lose colocalization upon fusion and release of the capsid from the membrane, but not in the presence of an inhibitor of vesicle acidification. The second tool is a hybrid RRV strain that expresses KSHV gB instead of RRV gB. RRV infected receptor-overexpressing cells in a manner that was not inhibited by an inhibitor of vesicle acidification. The hybrid RRV expressing KSHV gB on the other hand was sensitive to inhibition of vesicle acidification. Together, these findings show that KSHV gB dictates a low-pH, endocytotic route of infection. [ABSTRACT FROM AUTHOR]

The CD225/Dispanin superfamily contains membrane proteins that regulate vesicular transport and membrane fusion events required for neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking has remained unknown. Here we show that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline-rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins; mutations in this region disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins. Synopsis: Proteins of the CD225 superfamily regulate membrane trafficking events in diverse tissues, but the mechanism remains unclear. This study reveals that CD225 proteins contain a conserved SNARE-like motif that binds canonical SNARE proteins and regulates membrane fusion in mammalian cells. Genes in the CD225 family encode an R-SNARE-like motif. Mutations in the R-SNARE-like motif of the CD225 protein PRRT2 disrupt neuronal SNARE binding and are linked to neurological disease. Another CD225 protein, IFITM3, interacts with endosomal SNARE proteins, inhibits homotypic late endosome fusion, and promotes the delivery of endosomal cargo to lysosomes. CD225 proteins regulate membrane transport in mammalian cells by binding SNARE proteins through an R-SNARE-like motif. [ABSTRACT FROM AUTHOR]

Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2-domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a "primary interface" comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers, or helping bridge the membranes, but SNARE complex binding through the primary interface orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. To clarify this paradox, we have used NMR and fluorescence spectroscopy. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I, and that a mutation that impairs spontaneous and Ca2+-triggered neurotransmitter release enhances binding through region I. Moreover, fluorescence assays show that Ca2+ does not induce dissociation of Synaptotagmin-1 from membrane-anchored SNARE complex but causes reorientation of the C2B domain. Based on these results and electrophysiological data described by Toulme et al. (https://doi.org/10.1073/pnas.2409636121), we propose that upon Ca2+ binding the Synaptotagmin-1 C2B domain reorients on the membrane and dissociates from the SNAREs at region I but not region II, acting remotely as a lever that pulls the SNARE complex and facilitates linker zippering or other SNARE structural changes required for fast membrane fusion. [ABSTRACT FROM AUTHOR]

Serine incorporator 5 (SER5) can be incorporated into HIV-1 virions to block viral entry by disrupting the envelope glycoprotein-mediated viral fusion to the plasma membrane. Recent studies suggest that SER5 also inhibits HIV-1 mRNA transcription and the subsequent progeny virion biogenesis. However, the underlying mechanisms through which SER5 antagonizes the viral transcription remain poorly understood. Here, we demonstrate that SER5 inhibits HIV-1 transcription by negatively regulating NF-kB signaling, which is mediated by the retinoic acid-inducible gene I-like receptors, MDA5 and RIG-I. By recruiting TRIM40 as the E3 ubiquitination ligase to promote K48-linked polyubiquitination and proteasomal degradation of MDA5 and RIG-I, SER5 impedes nuclear translocation of the p50/p65 dimer, resulting in repression of HIV-1 LTR-driven gene expression. Hence, our findings strongly support a role for SER5 in restricting HIV-1 replication through inhibition of NF-kB-mediated viral gene expression. [ABSTRACT FROM AUTHOR]

The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus–cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses. [ABSTRACT FROM AUTHOR]

In mammalian reproduction, testis-specific protein IZUMO1 and its receptor JUNO on the oocyte surface are essential for sperm-oocyte recognition, binding, and membrane fusion. However, these factors alone are insufficient to accomplish cytoplasmic membrane fusion. It is believed that other gametic proteins interact with them to facilitate sperm-oocyte interaction on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. In this study, Copper Transport Protein 2 (CTR2) has been identified on the head and mid-tail of rat spermatozoa as well as on the surface of oocytes. CTR2 directly interacts with both IZUMO1 and JUNO, colocalizing with IZUMO1 on the sperm head and with JUNO on the oocyte membrane. Treatment of the capacitated sperm and zona pellucida-free oocytes with anti -CTR2 antibody resulted in a significant decrease in fertilization rates in IVF experiments. These findings suggest that CTR2 plays an important role in mammalian fertilization by interacting with IZUMO1 and JUNO, providing new insights into the molecular mechanisms of mammalian sperm-oocyte adhesion and fusion. [ABSTRACT FROM AUTHOR]

Paramyxovirus membrane fusion requires an attachment protein for receptor binding and a fusion protein for membrane fusion triggering. Nipah virus (NiV) attachment protein (G) binds to ephrinB2 or -B3 receptors, and fusion protein (F) mediates membrane fusion. NiV-F is a class I fusion protein and is activated by endosomal cleavage. The crystal structure of a soluble GCN4-decorated NiV-F shows a hexamer-of-trimer assembly. Here, we used single-molecule localization microscopy to quantify the NiV-F distribution and organization on cell and virus-like particle membranes at a nanometer precision. We found that NiV-F on biological membranes forms distinctive clusters that are independent of endosomal cleavage or expression levels. The sequestration of NiV-F into dense clusters favors membrane fusion triggering. The nano-distribution and organization of NiV-F are susceptible to mutations at the hexamer-of-trimer interface, and the putative oligomerization motif on the transmembrane domain. We also show that NiV-F nanoclusters are maintained by NiV-F-AP-2 interactions and the clathrin coat assembly. We propose that the organization of NiV-F into nanoclusters facilitates membrane fusion triggering by a mixed population of NiV-F molecules with varied degrees of cleavage and opportunities for interacting with the NiV-G/receptor complex. These observations provide insights into the in situ organization and activation mechanisms of the NiV fusion machinery. [ABSTRACT FROM AUTHOR]

Membrane fusion is essential for many cellular physiological functions, which is modulated by highly precise molecular mechanism involving multiple energy barriers. Nanoparticles (NPs), which exhibit immense potential in the field of biomedical applications, can act as fusogen proteins to initiate and regulate membrane fusion. However, the underlying mechanisms of NP-induced membrane fusion and the molecular details involved remain largely elusive. Here, using coarse-grained molecular dynamics simulations, we systematically investigate the NP-induced membrane fusion behaviors and the influences of NP properties (size, hydrophobicity and hydrophilicity). Our results show that the vesicle-bilayer fusion induced by a hydrophobic NP is an intricately state-wise process, involving the approach and local deformation of the vesicle and bilayer bridging by the NP, the flip-flop of lipids from proximal leaflets and the formation of a fusion stalk, as well as further lipid interactions between distal leaflets and complete fusion. Moreover, we find that NP properties have distinct effects on membrane fusion and thus the optimal NP conditions for facilitating membrane fusion are obtained. Our work provides a mechanistic understanding of NP-induced membrane fusion and offers useful insights for efficient and controlled regulation of membrane fusion. [ABSTRACT FROM AUTHOR]

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are the minimal machinery required for vesicle fusion in eukaryotes. Formation of a highly stable four-helix bundle consisting of SNARE motif of these proteins, drives vesicle/membrane fusion involved in several physiological processes such as neurotransmission. Recycling/disassembly of the protein machinery involved in membrane fusion is essential and is facilitated by an AAA+ ATPase, N-ethylmaleimide sensitive factor (NSF) in the presence of an adapter protein, α-SNAP. Here we use single-molecule fluorescence spectroscopy approaches to elucidate the chain of events that occur during the disassembly of SNARE complex by NSF. Our observations indicate two major pathways leading to the sequential disassembly of the SNARE complex: one where a syntaxin separated intermediate state is observed before syntaxin disassembles first, and a second where Vamp disassembles from the other proteins first. These studies uncover two parallel sequential pathways for the SNARE disassembly by NSF along with a syntaxin separated intermediate that couldn't be observed otherwise. SNARE proteins are the minimal machinery required for the vesicle fusion in eukaryotes. Here the authors use single-molecule fluorescence spectroscopy to elucidate the chain of events during the disassembly of SNARE complex by NSF, showing two parallel sequential pathways for SNARE disassembly. [ABSTRACT FROM AUTHOR]

Super‐resolution fluorescence imaging of live cells increasingly demands fluorescent probes capable of multi‐color and long‐term dynamic imaging. Understanding the mechanisms of probe‐target recognition is essential for the engineered development of such probes. In this study, it is discovered that the molecular lipid solubility parameter, Clog P, determines the staining performance of fluorescent dyes on lipid droplets (LDs). Fluorescent dyes with Clog P values between 2.5 and 4 can form buffering pools outside LDs, replacing photobleached dyes within LDs to maintain constant fluorescence intensity in LDs, thereby enabling dynamic super‐resolution imaging of LDs. Guided by Clog P, four different colored buffering LD probes spanning the visible light spectrum have been developed. Using Structured Illumination Microscopy (SIM), the role of LD dynamics have been tracked during cellular ferroptosis with the secretion, storage, and degradation of overexpressed ACSL3 proteins. It is found that LDs serve as storage sites for these proteins through membrane fusion, and further degrade overexpressed proteins via interactions with organelles like lysosomes or through lipophagy, thereby maintaining cellular homeostasis. [ABSTRACT FROM AUTHOR]

As the population ages, the occurrence of osteoporosis is becoming more common. Diabetes mellitus is one of the factors in the development of osteoporosis. Compared with the general population, the incidence of osteoporosis is significantly higher in diabetic patients. Diabetic osteoporosis (DOP) is a metabolic bone disease characterized by abnormal bone tissue structure due to hyperglycemia and insulin resistance, reduced bone strength and increased risk of fractures. This is a complex mechanism that occurs at the cellular level due to factors such as blood vessels, inflammation, and hyperglycemia and insulin resistance. Although the application of some drugs in clinical practice can reduce the occurrence of DOP, the incidence of fractures caused by DOP is still very high. Extracellular vesicles (EVs) are a new communication mode between cells, which can transfer miRNAs and proteins from mother cells to target cells through membrane fusion, thereby regulating the function of target cells. In recent years, the role of EVs in the pathogenesis of DOP has been widely demonstrated. In this article, we first describe the changes in the bone microenvironment of osteoporosis. Second, we describe the pathogenesis of DOP. Finally, we summarize the research progress and challenges of EVs in DOP. [ABSTRACT FROM AUTHOR]

The presence of a guanine quadruplex in the opening reading frame of the messenger RNA coding for the transmembrane serine protease 2 (TMPRSS2) may pave the way to original anticancer and host‐oriented antiviral strategy. Indeed, TMPRSS2 in addition to being overexpressed in different cancer types, is also related to the infection of respiratory viruses, including SARS‐CoV‐2, by promoting the cellular and viral membrane fusion through its proteolytic activity. The design of selective ligands targeting TMPRSS2 messenger RNA requires a detailed knowledge, at atomic level, of its structure. Therefore, we have used an original experimental‐computational protocol to predict the first resolved structure of the parallel guanine quadruplex secondary structure in the RNA of TMPRSS2, which shows a rigid core flanked by a flexible loop. This represents the first atomic scale structure of the guanine quadruplex structure present in TMPRSS2 messenger RNA. [ABSTRACT FROM AUTHOR]

Immunotherapy holds promise as an alternative to antibiotics in treating burn infections. However, the inadequacy of immune response or excessive inflammation both hinder effective bacterial clearance through immunotherapy, therefore necessitating a comprehensive approach that not only enhances immunotherapy against bacteria but also maintains immune homeostasis and promotes skin regeneration. Here, a membrane fusion‐driven combination immunotherapy is reported that mobilizes macrophages to address abovementioned limitations. The core–shell structured membrane fusion‐liposomes (MFL‐Gal‐Mal/Cur) can fuse their functional phospholipid shells with macrophages and bacteria, resulting in the remodeling of targets' surfaces with the galactose‐maltotriose (Gal‐Mal) moieties, and delivering their cores (curcumin‐loaded mesoporous polydopamine, MPDA/Cur) into targets. The embedded Gal‐Mal on membranes enhances macrophages' ability to phagocytize bacteria, as well as increases bacteria's sensitivity to immune cell‐mediated killing. The intracellular MPDA/Cur evade lysosomal degradation, exerting antibacterial effects while also enhancing macrophage lysosomal bactericidal activity through autophagy promotion. This immunotherapy enhances macrophages’ capacity to phagocytize (increase rate for S. aureus: 21%; E. coli: 29%) and eliminate intracellular bacteria (clearance rate for S. aureus: 98%; E. coli: 99%), without exacerbating inflammatory responses. The release of MFL‐Gal‐Mal/Cur from the polysaccharide composite hydrogel can alleviate pain and itching sensations in infected burns, while activating the regeneration of skin appendages. [ABSTRACT FROM AUTHOR]

Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex–membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex–membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology. [ABSTRACT FROM AUTHOR]

The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins, which are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S protein from SARS-CoV-2 variants has revealed this structural adaptation at high resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures, and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Taken together, our data are consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of the S protein. Synopsis: The evolution of SARS-CoV-2 variants has influenced viral Spike (S) protein structure and cleavage by furin, and has been proposed to modulate virion morphology. This study presents a characterization of virion morphology, spike density, furin cleavage and "on virus" spike structure from a range of SARS-CoV-2 variants analysed in parallel. SARS-CoV-2 variants, including B.1, Alpha, Gamma, Delta, Kappa, and Mu variants were imaged by cryo-electron microscopy and cryo-electron tomography. The evolution of more competitive strains has not led to significant changes in the generally spherical morphology of the virions. High-resolution structures of spikes on the virion surface reveal the impact of amino acid variation in the S protein on the protein structure and conformational dynamics throughout the spike. Cryo-electron microscopy and tomography reveal structural variation of Spike protein in intact virions from multiple SARS-CoV2 variants. [ABSTRACT FROM AUTHOR]

The mammalian Apolipoprotein-L families (APOLs) contain several isoforms of membrane-interacting proteins, some of which are involved in the control of membrane dynamics (traffic, fission and fusion). Specifically, human APOL1 and APOL3 appear to control membrane remodeling linked to pathogen infection. Through its association with Non-Muscular Myosin-2A (NM2A), APOL1 controls Golgi-derived trafficking of vesicles carrying the lipid scramblase Autophagy-9A (ATG9A). These vesicles deliver APOL3 together with phosphatidylinositol-4-kinase-B (PI4KB) and activated Stimulator of Interferon Genes (STING) to mitochondrion–endoplasmic reticulum (ER) contact sites (MERCSs) for the induction and completion of mitophagy and apoptosis. Through direct interactions with PI4KB and PI4KB activity controllers (Neuronal Calcium Sensor-1, or NCS1, Calneuron-1, or CALN1, and ADP-Ribosylation Factor-1, or ARF1), APOL3 controls PI(4)P synthesis. PI(4)P is required for different processes linked to infection-induced inflammation: (i) STING activation at the Golgi and subsequent lysosomal degradation for inflammation termination; (ii) mitochondrion fission at MERCSs for induction of mitophagy and apoptosis; and (iii) phagolysosome formation for antigen processing. In addition, APOL3 governs mitophagosome fusion with endolysosomes for mitophagy completion, and the APOL3-like murine APOL7C is involved in phagosome permeabilization linked to antigen cross-presentation in dendritic cells. Similarly, APOL3 can induce the fusion of intracellular bacterial membranes, and a role in membrane fusion can also be proposed for endothelial APOLd1 and adipocyte mAPOL6, which promote angiogenesis and adipogenesis, respectively, under inflammatory conditions. Thus, different APOL isoforms play distinct roles in membrane remodeling associated with inflammation. [ABSTRACT FROM AUTHOR]

Enveloped viruses enter cells by fusing their envelopes to host cell membranes. Vesicular stomatitis virus (VSV) glycoprotein (G) is a prototype for class III fusion proteins. Although structures of the stable pre-and postfusion ectodomain of G are known, its fusogenic intermediates are insufficiently characterized. Here, we incubated VSV virions with late endosome-mimicking liposomes at pH 5.5 and used cryo-electron tomography (cryo-ET) to visualize stages of VSV's membrane fusion pathway, capture refolding intermediates of G, and reconstruct a sequence of G conformational changes. We observe that the G trimer disassembles into monomers and parallel dimers that explore a broad conformational space. Extended intermediates engage target membranes and mediate fusion, resulting in viral uncoating and linearization of the ribonucleoprotein genome. These viral fusion intermediates provide mechanistic insights into class III viral fusion processes, opening avenues for future research and structure-based design of fusion inhibition-based antiviral therapeutics. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is adapting to continuous presence in humans. Transitions to endemic infection patterns are associated with changes in the spike (S) proteins that direct virus-cell entry. These changes generate antigenic drift and thereby allow virus maintenance in the face of prevalent human antiviral antibodies. These changes also fine tune virus-cell entry dynamics in ways that optimize transmission and infection into human cells. Focusing on the latter aspect, we evaluated the effects of several S protein substitutions on virus-cell membrane fusion, an essential final step in enveloped virus-cell entry. Membrane fusion is executed by integral-membrane "S2" domains, yet we found that substitutions in peripheral "S1" domains altered late-stage fusion dynamics, consistent with S1-S2 heterodimers cooperating throughout cell entry. A specific H655Y change in S1 stabilized a fusion-intermediate S protein conformation and thereby delayed membrane fusion. The H655Y change also sensitized viruses to neutralization by S2-targeting fusion-inhibitory peptides and stem-helix antibodies. The antibodies did not interfere with early fusion-activating steps; rather they targeted the latest stages of S2-directed membrane fusion in a novel neutralization mechanism. These findings demonstrate that single amino acid substitutions in the S proteins both reset viral entry—fusion kinetics and increase sensitivity to antibody neutralization. The results exemplify how selective forces driving SARS-CoV-2 fitness and antibody evasion operate together to shape SARS-CoV-2 evolution. Author summary: Most adaptive mutations endowing SARS-CoV-2 with increased human transmissibility and infectivity alter viral spike (S) protein structure and function. Orchestrated structural transitions in these multidomain S proteins mediate cell entry functions. Comparative analyses of adapted SARS-CoV-2 variants identified S domains conducting the rhythm of these transitions. The S1 adaptation H655Y slowed late virus-cell membrane fusion, revealing S1 as a timer for this essential and final cell entry event. The H655Y-directed delay left viruses vulnerable to neutralizing antibodies that target transitional fusion intermediate S conformations. Our findings highlight how mechanistic understandings of S protein structural dynamics illuminate antibody neutralization mechanisms and suggest that SARS-CoV-2 evolutionary pathways are shaped by competing selective pressures to optimize cell entry kinetics and evade neutralizing antibodies. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells. IMPACT STATEMENT: The study presents the unique characteristics of small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) with different lengths of PGlu(DET-Car), revealing the length of PGlu(DET-Car) critically affects the formation of a stable LNP, the cellular uptake, membrane fusion, and gene silencing abilities. [ABSTRACT FROM AUTHOR]

Membrane fusion is an essential process for the survival of eukaryotes and entry of the enveloped viruses. Fusion machines (proteins) and membrane lipids play their intricate role in a concerted way to complete the process with precision and speed. The in vitro study of fusion does not match the kinetics of biological fusion, nonetheless, it provides molecular details of the process. The fusion proteins are more celebrated due to their obvious role in catalyzing the process, but the lipids are also important as they provide the platform for the process. Lipid composition provides the membrane organization, dynamics, and overall physical properties to the membrane, which are crucial for the lamellar to nonlamellar to lamellar reorganization during fusion. The current review discusses the prospective roles of the players in fusion with a special emphasis on lipidic shape, chain unsaturation, membrane physical properties, water percolation at the acyl chain region, and membrane domain. [ABSTRACT FROM AUTHOR]

Membrane fusion is an essential component of the viral lifecycle that allows the delivery of the genetic information of the virus into the host cell. Specialized viral glycoproteins exist on the surface of mature virions where they facilitate fusion through significant conformational changes, ultimately bringing opposing membranes into proximity until they eventually coalesce. This process can be positively influenced by a number of specific cellular factors such as pH, enzymatic cleavage, divalent ions, and the composition of the host cell membrane. In this review, we have summarized how anionic lipids have come to be involved in viral fusion and how the endosomal resident anionic lipid BMP has become increasingly implicated as an important cofactor for those viruses that fuse via the endocytic pathway. [ABSTRACT FROM AUTHOR]

Innervation is closely linked to several biological processes that promote tumor growth, making it an increasingly promising therapeutic target. In this study, biomimetic hollow MnO 2 nanocarriers camouflaged with tumor cell membranes (HMLC) are developed to encapsulate lidocaine, an innervation inhibitor, for effective antineoplastic therapy. This approach aims to suppress nerve fiber growth and induce intracellular redox imbalance. Benefiting from the tumor-homing effect, HMLC accumulates in cancerous tissue during circulation and is endocytosed by tumor cells through homologous membrane fusion. Once inside the cells, MnO 2 can be degraded by the overproduced glutathione and H 2 O 2 , leading to the tumor-specific release of Mn2+ and lidocaine. The Mn2+-mediated Fenton-like reaction promotes the accumulation of reactive oxygen species, and the resulting oxidative stress, combined with glutathione depletion, exacerbates redox imbalance. Simultaneously, the released lidocaine downregulates nerve growth factor and neuronatin. The reduction in nerve growth factor significantly inhibits nerve fiber formation and infiltration in tumor tissue, while the decrease in neuronatin reduces intracellular Ca2+, which helps prevent metastasis. Overall, this strategy highlights the potential of nanoparticle-based tumor innervation disruptors in antineoplastic therapy. Biomimetic manganese-based nanocomposites (HMLC) are developed by the encapsulation of lidocaine (Lid) into hollow mesoporous MnO 2 nanocarriers (H-MnO 2) and subsequent camouflage with tumor cell membrane, aiming at robust antineoplastic therapy through tumor innervation disorder and redox homeostasis disruption. This paradigm is expected to inspire more nanoparticle-based tumor innervation disruptors as adjuvants toward clinical cancer management in the future. [Display omitted] • Biomimetic lidocaine-loaded and manganese-based nanocomposites (HMLC) are developed for antineoplastic therapy. • Tumor-targeted delivery of lidocaine is achieved by HMLC with high efficiency. • HMLC can downregulate multiple neurotrophic factors and decline nerve bundles to render innervation disorder. • The synergy of tumor denervation and redox homeostasis disruption is efficacious in cancer therapy [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) fusion protein (F) facilitates virus-cell membrane fusion, which is critical for viral entry, and cell-cell fusion. In contrast to many type I fusion proteins, RSV F must be proteolytically cleaved at two distinct sites to be fusogenic. Cleavage at both sites results in the release of a 27 amino-acid fragment, termed Pep27. We examined proteolytic processing and the role of Pep27 for RSV F from both RSV A2 and RSV B9320 laboratory-adapted strains, allowing important comparisons between A and B clade F proteins. F from both clades was cleaved at both sites, and pulse-chase analysis indicated that cleavage at both sites occurs early after synthesis, most likely within the secretory pathway. Mutation of either site to alter the furin recognition motif blocked cell-cell fusion activity. To assess the role of Pep27 in F processing and expression, we deleted the Pep27 fragment, but preserved the cleavage sites. Deletion of Pep27 reduced F surface expression and cell-cell fusion. Two conserved N-linked glycosylation sites within Pep 27 are present in both the RSV A2 and RSV B9320 F. Randomization of the Pep27 sequence, while conserving the two N-liked glycosylation sites, did not significantly change surface expression, and only modestly reduced cell-cell fusion. However, the disruption of either Pep27 glycosylation site reduced cell-cell fusion. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within Pep27 play in the biological function of F. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are crucial for maintaining lipid and energy homeostasis within cells. LDs are highly dynamic organelles that present a phospholipid monolayer rich in neutral lipids. Additionally, LDs are associated with structural and nonstructural proteins, rapidly mobilizing lipids for various biological processes. Lipids play a pivotal role during viral infection, participating during viral membrane fusion, viral replication, and assembly, endocytosis, and exocytosis. SARS-CoV-2 infection often induces LD accumulation, which is used as a source of energy for the replicative process. These findings suggest that LDs are a hallmark of viral infection, including SARS-CoV-2 infection. Moreover, LDs participate in the inflammatory process and cell signaling, activating pathways related to innate immunity and cell death. Accumulating evidence demonstrates that LD induction by SARS-CoV-2 is a highly coordinated process, aiding replication and evading the immune system, and may contribute to the different cell death process observed in various studies. Nevertheless, recent research in the field of LDs suggests these organelles according to the pathogen and infection conditions may also play roles in immune and inflammatory responses, protecting the host against viral infection. Understanding how SARS-CoV-2 influences LD biogenesis is crucial for developing novel drugs or repurposing existing ones. By targeting host lipid metabolic pathways exploited by the virus, it is possible to impact viral replication and inflammatory responses. This review seeks to discuss and analyze the role of LDs during SARS-CoV-2 infection, specifically emphasizing their involvement in viral replication and the inflammatory response. [ABSTRACT FROM AUTHOR]

Viral infections are a major global health concern, affecting millions of people each year. Viral entry is one of the crucial stages in the infection process, but its details remain elusive. Enveloped viruses are enclosed by a lipid membrane that protects their genetic material and these viruses are linked to various human illnesses, including influenza, and COVID‐19. Due to the advancements made in the field of molecular simulation, significant progress has been made in unraveling the dynamic processes involved in viral entry of enveloped viruses. Simulation studies have provided deep insight into the function of the proteins responsible for attaching to the host receptors and promoting membrane fusion (fusion proteins), deciphering interactions between these proteins and receptors, and shedding light on the functional significance of key regions, such as the fusion peptide. These studies have already significantly contributed to our understanding of this critical aspect of viral infection and assisted the development of effective strategies to combat viral diseases and improve global health. This review focuses on the vital role of fusion proteins in facilitating the entry process of enveloped viruses and highlights the contributions of molecular simulation studies to uncover the molecular details underlying their mechanisms of action.

BACKGROUND: Viral infection elicits the type I interferon (IFN-I) response in host cells and subsequently inhibits viral infection through inducing hundreds of IFN-stimulated genes (ISGs) that counteract many steps in the virus life cycle. However, most of ISGs have unclear functions and mechanisms in viral infection. Thus, more work is required to elucidate the role and mechanisms of individual ISGs against different types of viruses. RESULTS: Herein, we demonstrate that poliovirus receptor-like protein4 (PVRL4) is an ISG strongly induced by IFN-I stimulation and various viral infections. Overexpression of PVRL4 protein broadly restricts growth of enveloped RNA and DNA viruses, including vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whereas deletion of PVRL4 in host cells increases viral infections. Mechanistically, it suppresses viral entry by blocking viral-cellular membrane fusion through inhibiting endosomal acidification. The vivo studies demonstrate that Pvrl4-deficient mice were more susceptible to the infection of VSV and IAV. CONCLUSION: Overall, our studies not only identify PVRL4 as an intrinsic broad-spectrum antiviral ISG, but also provide a candidate host-directed target for antiviral therapy against various viruses including SARS-CoV-2 and its variants in the future.

In vitro embryo production (IVP) technology has been increasingly applied to beef cattle breeding. In vitro maturation (IVM) technology is the basis of IVP. However, the quality of in vitro-generated mature oocytes is still poor. Mitochondria are the energy factories of oocytes, so they are crucial for oocyte quality. OPA1 is a protein located on the mitochondrial inner membrane, and its main function is to mediate mitochondrial inner membrane fusion. This work demonstrated that OPA1 is expressed at different stages of meiosis in bovine oocytes. The inhibition of OPA1 activity resulted in a reduced rate of first polar body excretion from bovine oocytes and disruption of the spindle structure. OPA1 deficiency impacted mitochondria by leading to mitochondrial dysfunction, promoting mitochondrial fission, and inducing mitophagy through the PINK1/Parkin pathway. Taken together, our findings suggest that OPA1 is essential for bovine oocyte maturation and that OPA1 deficiency leads to mitochondrial dysfunction and promotes mitochondrial fission as well as mitophagy. • OPA1 deficiency decreases bovine oocytes quality. • OPA1 deficiency causes mitochondrial dysfunction in bovine oocytes. • OPA1 deficiency induces mitochondrial fission in bovine oocytes. • OPA1 deficiency activates mitophagy through the PINK1/Parkin pathway. [ABSTRACT FROM AUTHOR]

Transmembrane Serine Protease 2 (TMPRSS2), known primarily for its role as a protease, has emerged as a critical receptor for microbial agents such as human coronavirus HKU1 and exotoxin TcsH. HKU1 utilizes both sialoglycan and TMPRSS2 for cellular entry, where sialoglycan primes the spike protein for TMPRSS2 binding. TMPRSS2 undergoes autocleavage to enhance its affinity for the HKU1 spike, facilitating viral membrane fusion postcleavage. Interestingly, TMPRSS2's catalytic function is dispensable for both HKU1 and TcsH interactions, suggesting alternative roles in pathogenesis. Structural insights highlight potential therapeutic targets against viral infections and cancers, leveraging TMPRSS2 interactions for drug development. Understanding the interplay between TMPRSS2 and microbes opens new avenues for targeting TMPRSS2 in developing treatments for infections. [ABSTRACT FROM AUTHOR]

ABSTRACT Mitochondria are dynamic and plastic, undergoing continuous fission and fusion and rearrangement of their bioenergetic sub‐compartments called cristae. These fascinating processes are best understood in animal and fungal models, which are taxonomically grouped together in the expansive Opisthokonta supergroup. In opisthokonts, crista remodelling and inner membrane fusion are linked by dynamin‐related proteins (DRPs). Animal Opa1 (optical atrophy 1) and fungal Mgm1 (mitochondrial genome maintenance 1) are tacitly considered orthologs because their similar mitochondria‐shaping roles are mediated by seemingly shared biochemical properties, and due to their presence in the two major opisthokontan subdivisions, Holozoa and Holomycota, respectively. However, molecular phylogenetics challenges this notion, suggesting that Opa1 and Mgm1 likely had separate, albeit convergent, evolutionary paths. Herein, we illuminate disparities in proteolytic processing, structure, and interaction network that may have bestowed on Opa1 and Mgm1 distinct mechanisms of membrane remodelling. A key disparity is that, unlike Mgm1, Opa1 directly recruits the mitochondrial phospholipid cardiolipin to remodel membranes. The differences outlined herein between the two DRPs could have broader impacts on mitochondrial morphogenesis. Outer and inner membrane fusion are autonomous in animals, which may have freed Opa1 to repurpose its intrinsic activity to remodel cristae, thereby regulating the formation of respiratory chain supercomplexes. More significantly, Opa1‐mediated crista remodelling has emerged as an integral part of cytochrome c‐regulated apoptosis in vertebrates, and perhaps in the cenancestor of animals. By contrast, outer and inner membrane fusion are coupled in budding yeast. Consequently, Mgm1 membrane‐fusion activity is inextricable from its role in the biogenesis of fungal lamellar cristae. These disparate mitochondrial DRPs ultimately may have contributed to the different modes of multicellularity that have evolved within Opisthokonta. [ABSTRACT FROM AUTHOR]

Membrane fusion is an essential biological process that merges two separate lipid bilayers into a whole one. Membrane fusion proteins facilitate this process by bringing lipid bilayers in close proximity to reduce the repulsive energy between membranes. Along with their interactions with membranes, the structures and dynamics of membrane fusion proteins are key to elucidating the mechanisms of membrane fusion. Solid‐state NMR (SSNMR) spectroscopy has unique advantages in determining the structures and dynamics of membrane fusion proteins in their membrane‐bound states. It has been extensively applied to reveal conformational changes in intermediate states of viral membrane fusion proteins and to characterize the critical lipid–membrane interactions that drive the fusion process. In this review, we summarize recent advancements in SSNMR techniques for studying membrane fusion proteins and their applications in elucidating the mechanisms of membrane fusion. [ABSTRACT FROM AUTHOR]

Peptide‐based drugs hold great potential for cancer treatment, and their effectiveness is driven by mechanisms on how peptides target cancer cells and escape from potential lysosomal entrapment post‐endocytosis. Yet, the mechanisms remain elusive, which hinder the design of peptide‐based drugs. Here hendeca‐arginine peptides (R11) are synthesized for targeted delivery in bladder carcinoma (BC), investigated the targeting efficiency and elucidated the mechanism of peptide‐based delivery, with the aim of refining the design and efficacy of peptide‐based therapeutics. It is demonstrated that the over‐activated Piezo1/integrin β1 (ITGB1) signaling axis significantly facilitates tumor‐targeted delivery of R11 peptides via macropinocytosis. Furthermore, R11 peptides formed hydrogen bonds with integrin β1, facilitating targeting and penetration into tumor cells. Additionally, R11 peptides protected integrin β1 from lysosome degradation, promoting its recycling from cytoplasm to membrane. Moreover, this findings establish a positive feedback loop wherein R11 peptides activate Piezo1 by increasing membrane fusion, promoting Ca2+ releasing and resulting in enhanced integrin β1‐mediated endocytosis in both orthotopic models and clinical tissues, demonstrating effective tumor‐targeted delivery. Eventually, the Piezo1/integrin β1 signaling axis promoted cellular uptake and transport of peptides, establishing a positive feedback loop, promoting mechanical delivery to cancer and offering possibilities for drug modification in cancer therapy. [ABSTRACT FROM AUTHOR]

As life extended into eukaryota, a great host of strategies emerged in the pursuit of cellular life. Some cells have been successful in solitude, some moved into cooperatives (i.e., multicellular organisms), but one additional strategy emerged. Throughout eukaryotes, many of the diverse multicellular cooperatives took life in partnership one step further. These cells came together and lost their singularity in the expanse of syncytial life. Recently in our search for this elusive “how”, we discovered the intriguing peculiarity of a nuclear, RNA‐binding protein living a second life as a fusion manager at the surface of developing osteoclasts, ushering them into syncytia 1. It is from here that we will develop several thoughts about the advantages of multinucleated cells and discuss how these fusing cells pass through several hallmarks of cell death. We will propose that cell fusion shares much with cell death because cell fusion is a death of sorts for the cells that undergo it – a death of the life that was and the beginning of new life in a community without borders. [ABSTRACT FROM AUTHOR]

The VCPIP1‐P97/VCP (Valosin‐Containing Protein) complex is required for post‐mitotic Golgi cisternae reassembly and maintenance in interphase. However, the organization and mechanism of this complex in regulating Golgi membrane fusion is still elusive. Here, the cryo‐electron microscopy (cryo‐EM) structures of the human VCPIP1‐P97/VCP complex are presented. These studies reveal that three independent VCPIP1 molecules sit over the C‐terminal substrate exit tunnel formed by P97/VCP homo‐hexamer, resulting in an unusual C3 to C6 symmetric barrel architecture. The UFD1 (unknown function domain 1) from VCPIP1, but not the N‐terminal OTU domain and the C‐terminal UBL domain, docks to the two adjacent D2 domains of P97/VCP, allosterically causing the cofactors binding domain‐NTDs (N‐terminal domains) of P97/VCP in a "UP" and D1 domain in an ATPase competent conformation. Conversely, VCPIP1 bound P97/VCP hexamer favors the binding of P47, and thus the intact SNARE complex, promoting Golgi membrane fusion. These studies not only reveal the unexpected organization of humanVCPIP1‐P97/VCP complex, but also provide new insights into the mechanism of VCPIP1‐P97/VCP mediated Golgi apparatus reassembly, which is a fundamental cellular event for protein and lipid processing. [ABSTRACT FROM AUTHOR]