Multivalent Binding Research Articles

The N-terminal region of DNMT3A engages the nucleosome surface to aid chromatin recruitment

AbstractDNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.

Systematic Evaluation of Macromolecular Carbohydrate-Lectin Recognition Using Precision Glycopolymers.

The precise modulation of protein-carbohydrate interactions is critical in glycobiology, where multivalent binding governs key cellular processes. As such, synthetic glycopolymers are useful for probing these interactions. Herein, we developed precision glycopolymers (PGPs) with unambiguous local chemical composition and well-defined global structure and systematically evaluated the effect of polymer length, hydrophobicity, and backbone hybridization as well as glycan density and identity on the binding to both mammalian and plant lectins. Our studies identified glycan density as a critical factor, with PGPs below 50% grafting density showing significantly weaker lectin interactions. Coarse-grained molecular dynamics simulations suggest that the observed phenomena may be due to a decrease in carbohydrate-carbohydrate interactions in fully grafted PGPs, leading to improved solvent accessibility. In functional assays, these PGPs reduced the cell viability and migration in 4T1 breast cancer cells. Our findings establish a structure-activity relationship in glycopolymers, providing new strategies for designing synthetic glycomacromolecules for a myriad of applications.

Intelligent Modular DNA Lysosome-Targeting Chimera Nanodevice for Precision Tumor Therapy.

Lysosome targeting chimeras (LYTACs) have emerged as a powerful modality that can eliminate traditionally undruggable extracellular tumor-related pathogenic proteins, but their low bioavailability and nonspecific distribution significantly restrict their efficacy in precision tumor therapy. Developing a LYTAC system that can selectively target tumor tissues and enable a modular design is crucial but challenging. We here report a programmable nanoplatform for tumor-specific degradation of multipathogenic proteins using an intelligent modular DNA LYTAC (IMTAC) nanodevice. We employ circular DNA origami to integrate predesigned modular multitarget protein binding sites and pH-responsive protein degradation promoters that specifically recognize cell-surface lysosome-shuttling receptors in tumor tissues. By precisely manipulating the stoichiometry and modularity of promoters and ligands targeting diverse proteins, the IMTAC nanodevice enables accurate localization and delivery into tumor tissues, where the acidic tumor microenvironment triggers degradation switch activation, multivalent binding, and efficient degradation of various prespecified proteins. The tissue-specificity and multiple ligands in IMTACs significantly improve the drug utilization rate while reducing off-target effects. Importantly, this system demonstrates the capability of collabo-rative degradation of EGFR and PDL1 in tumor tissue for combined targeting and immunity therapy of hepatocellular carcinoma (HCC), resulting in obvious tumor necrosis and inhibition of tumor growth in vivo even at low concentrations. This study presents a unique strategy for building a general, intelligent, modular, and simple encoded nanoplatform for designing precision medicine degraders and developing proprietary antitumor drugs.

Spatially Controlled DNA Frameworks for Sensitive Detection and Specific Isolation of Tumor Cells.

High-affinity, specific, and sensitive probes are crucial for the specific recognition and identification of tumor cells from complex matrices. Multivalent binding is a powerful strategy, but the irrational spatial distribution of the functional moieties may reduce the probe performance. Here, we constructed a Janus DNA triangular prism nanostructure (3Zy1-JTP-3) for sensitive detection and specific isolation of tumor cells. Benefiting from spatial features of the triangular prism, the fluorescence intensity induced by 3Zy1-JTP-3 was almost 4 times that of the monovalent structure. Moreover, the DNA triangular prisms were connected to form hand-in-hand multivalent DNA triangular prism structures (Zy1-MTP), in which the fluorescence intensity and affinity were increased to 9-fold and 10-fold of 3Zy1-JTP-3, respectively. Furthermore, 3Zy1-JTP-3 and Zy1-MTP were combined with magnetic beads, and the latter showed higher capture efficiency (> 90%) in whole blood. This work provides a new strategy for the efficient capture of rare cells in complex biological samples.

Spatially Controlled DNA Frameworks for Sensitive Detection and Specific Isolation of Tumor Cells

High‐affinity, specific, and sensitive probes are crucial for the specific recognition and identification of tumor cells from complex matrices. Multivalent binding is a powerful strategy, but the irrational spatial distribution of the functional moieties may reduce the probe performance. Here, we constructed a Janus DNA triangular prism nanostructure (3Zy1‐JTP‐3) for sensitive detection and specific isolation of tumor cells. Benefiting from spatial features of the triangular prism, the fluorescence intensity induced by 3Zy1‐JTP‐3 was almost 4 times that of the monovalent structure. Moreover, the DNA triangular prisms were connected to form hand‐in‐hand multivalent DNA triangular prism structures (Zy1‐MTP), in which the fluorescence intensity and affinity were increased to 9‐fold and 10‐fold of 3Zy1‐JTP‐3, respectively. Furthermore, 3Zy1‐JTP‐3 and Zy1‐MTP were combined with magnetic beads, and the latter showed higher capture efficiency (> 90%) in whole blood. This work provides a new strategy for the efficient capture of rare cells in complex biological samples.

Intranasal Delivery of Pure Nanodrug Loaded Liposomes for Alzheimer's Disease Treatment by Efficiently Regulating Microglial Polarization.

The activated M1-like microglia induced neuroinflammation is the critical pathogenic event in Alzheimer's disease (AD). Microglial polarization from pro-inflammatory M1 toward anti-inflammatory M2 phenotype is a promising strategy. To efficiently accomplish this, amyloid-β (Aβ) aggregates as the culprit of M1 microglia activation should be uprooted. Interestingly, this study finds out that the self-reassembly of curcumin molecules into carrier-free curcumin nanoparticles (CNPs) exhibits multivalent binding with Aβ to achieve higher inhibitory effect on Aβ aggregation, compared to free curcumin with monovalent effect. Based on this, the CNPs loaded cardiolipin liposomes are developed for efficient microglial polarization. After intranasal administration, the liposomes decompose to release CNPs and cardiolipin in response to AD oxidative microenvironment. The CNPs inhibit Aβ aggregation and promote Aβ phagocytosis/clearance in microglia, removing roadblock to microglial polarization. Subsequently, CNPs are endocytosed by microglia and inhibit TLR4/NF-κB pathway for microglia polarization (M1→M2). Meanwhile, cardiolipin is identified as signaling molecule to normalize microglial dysfunction to prevent pro-inflammatory factors release. In AD transgenic mice, neuroinflammation, Aβ burden, and memory deficits are relieved after treatment. Through combined attack by extracellularly eradicating roadblock of Aβ aggregation and intracellularly inhibiting inflammation-related pathways, this nanotechnology assisted delivery system polarizes microglia efficiently, providing a reliable strategy in AD treatment.

Multifunctional Dendrimer-Peptide Conjugates for MET Receptor-specific Imaging of Cancer Cells

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is frequently upregulated or mutated in various cancers. Targeting MET signaling pathway has been utilized as a treatment for cancer, as since MET overexpression is often associated with poor prognosis. Selective imaging of MET-overexpressing tumor cells would thus provide a high diagnostic value; however, it remains elusive due to a lack of targeted imaging contrast agents. Herein, we have developed a multifunctional diagnostic dendrimer-peptide conjugate (DPC) system with a strong avidity to MET-expressing cancer cells. The system was prepared by conjugating MET-inhibiting peptides (C7) to generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers. Due to the dendrimer-mediated multivalent binding effect, the DPCs exhibited a significantly stronger binding to the human MET protein than free C7, as measured using surface plasmon resonance. Confocal microscopy revealed increased binding of the DPCs to the MET-expressing EBC-1 and UW-Lung-21 cells, whereas a MET knock-out cell line showed negligible interactions with the DPCs. The DPCs were then conjugated with Zirconium-89 for positron emission tomography and computed tomography (PET/CT) scanning, demonstrating their selective accumulation to MET-expressing tumors in vivo. Additionally, the plasma half-life of the DPCs was measured at ~53hours, which was significantly longer than free C7. These results collectively suggest that this DPC system has potential as a targeted imaging platform specific to MET-expressing tumors, which would be applicable to various cancer types.

Red Blood Cell‐Derived Small Extracellular Vesicles Inhibit Influenza Virus through Surface‐Displayed Sialic Acids

Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell‐derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface‐displayed SAs to interact with IAV through multivalent HA‐SA interactions. This high‐avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose‐dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti‐IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti‐IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics.

Red Blood Cell-Derived Small Extracellular Vesicles Inhibit Influenza Virus through Surface-Displayed Sialic Acids.

Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell-derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface-displayed SAs to interact with IAV through multivalent HA-SA interactions. This high-avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose-dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti-IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti-IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics.

A Visual Distance-Based Capillary Immunoassay Using Biomimetic Polymer Nanoparticles for Highly Sensitive and Specific C-Reactive Protein Quantification.

The low-cost daily monitoring of C-reactive protein (CRP) levels is crucial for screening acute inflammation or infections as well as managing chronic inflammatory diseases. In this study, we synthesized novel 2-Methacryloyloxy ethyl phosphorylcholine (MPC)-based biomimetic nanoparticles with a large surface area to develop a visual CRP-quantification assay using affordable glass capillaries. The PMPC nanoparticles, synthesized via reflux precipitation polymerization, demonstrated multivalent binding capabilities, enabling rapid and specific CRP capture. In the presence of CRP, PMPC nanoparticles formed sandwich structures with magnetic nanoparticles functionalized with CRP antibodies, thereby enhancing detection sensitivity and specificity. These sandwich complexes were magnetically accumulated into visible and quantifiable stacks within the glass capillaries, allowing for the rapid, sensitive, and specific quantification of CRP concentrations with a detection limit of 57.5 pg/mL and a range spanning from 0 to 5000 ng/mL. The proposed visual distance-based capillary biosensor shows great potential in routine clinical diagnosis as well as point-of-care testing (POCT) in resource-limited settings.

Sulfoglycodendron Antivirals with Scalable Architectures and Activities.

Many viruses initiate their cell-entry by binding their multisubunit receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multiprotein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed the detailed nature of binding between these antivirals and viral protein receptors and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.

Balancing thermodynamic stability, dynamics, and kinetics in phase separation of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are prevalent participants in liquid-liquid phase separation due to their inherent potential for promoting multivalent binding. Understanding the underlying mechanisms of phase separation is challenging, as phase separation is a complex process, involving numerous molecules and various types of interactions. Here, we used a simplified coarse-grained model of IDPs to investigate the thermodynamic stability of the dense phase, conformational properties of IDPs, chain dynamics, and kinetic rates of forming condensates. We focused on the IDP system, in which the oppositely charged IDPs are maximally segregated, inherently possessing a high propensity for phase separation. By varying interaction strengths, salt concentrations, and temperatures, we observed that IDPs in the dense phase exhibited highly conserved conformational characteristics, which are more extended than those in the dilute phase. Although the chain motions and global conformational dynamics of IDPs in the condensates are slow due to the high viscosity, local chain flexibility at the short timescales is largely preserved with respect to that at the free state. Strikingly, we observed a non-monotonic relationship between interaction strengths and kinetic rates for forming condensates. As strong interactions of IDPs result in high stable condensates, our results suggest that the thermodynamics and kinetics of phase separation are decoupled and optimized by the speed-stability balance through underlying molecular interactions. Our findings contribute to the molecular-level understanding of phase separation and offer valuable insights into the developments of engineering strategies for precise regulation of biomolecular condensates.

Ferritin-based disruptor nanoparticles: A novel strategy to enhance LDL cholesterol clearance via multivalent inhibition of PCSK9-LDL receptor interaction.

Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

Label-Free Mapping of Multivalent Binding Pathways with Ligand-Receptor-Anchored Nanopores.

Understanding single-molecule multivalent ligand-receptor interactions is crucial for comprehending molecular recognition at biological interfaces. However, label-free identifications of these transient interactions during multistep binding processes remains challenging. Herein, we introduce a ligand-receptor-anchored nanopore that allows the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, we clarify two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent. Real-time kinetic analysis at the single-monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (-13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. Our single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.

Synthetic polypeptides inhibit nucleic acid-induced inflammation in autoimmune diseases by disrupting multivalent TLR9 binding to LL37-DNA bundles.

Complexes of extracellular nucleic acids (NAs) with endogenous proteins or peptides, such as LL37, break immune balance and cause autoimmune diseases, whereas NAs with arginine-enriched peptides do not. Inspired by this, we synthesize a polyarginine nanoparticle PEG-TK-NPArg, which effectively inhibits Toll-like receptor-9 (TLR9) activation, in contrast to LL37. To explore the discrepancy effect of PEG-TK-NPArg and LL37, we evaluate the periodic structure of PEG-TK-NPArg-NA and LL37-NA complexes using small-angle X-ray scattering. LL37-NA complexes have a larger inter-NA spacing that accommodates TLR9, while the inter-NA spacing in PEG-TK-NPArg-NA complexes mismatches with the cavity of TLR9, thus inhibiting an interaction with multiple TLR9s, limiting their clustering and damping immune induction. Subsequently, the inhibitory inflammation effect of PEG-TK-NPArg is proved in an animal model of rheumatoid arthritis. This work on how the scavenger-NA complexes inhibit the immune response may facilitate proof-of-concept research translating to clinical application.

Sulfoglycodendron Antivirals with Scalable Architectures and Activities.

Many viruses initiate their cell-entry by binding their multi-protein receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multi-protein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed detail nature of binding between these antivirals and viral protein receptors, and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.

General quasi-equilibrium multivalent binding model to study diverse and complex drug-receptor interactions of biologics.

Pharmacokinetics and pharmacodynamics of many biologics are influenced by their complex binding to biological receptors. Biologics consist of diverse groups of molecules with different binding kinetics to its receptors including IgG with simple one-to-one drug receptor bindings, bispecific antibody (BsAb) that binds to two different receptors, and antibodies that can bind to six or more identical receptors. As the binding process is typically much faster than elimination (or internalization) and distribution processes, quasi-equilibrium (QE) binding models are commonly used to describe drug-receptor binding kinetics of biologics. However, no general QE modeling framework is available to describe complex binding kinetics for diverse classes of biologics. In this paper, we describe novel approaches of using differential algebraic equations (DAE) to solve three QE multivalent drug-receptor binding (QEMB) models. The first example describes the binding kinetics of three-body equilibria of BsAb that binds to 2 different receptors for trimer formation. The second example models an engineered IgG variant (Multabody) that can bind to 24 identical target receptors. The third example describes an IgG with modified neonatal Fc receptor (FcRn) binding affinity that competes for the same FcRn receptor as endogenous IgG. The model parameter estimates were obtained by fitting the model to all data simultaneously. The models allowed us to study potential roles of cooperative binding on bell-shaped drug exposure-response relationships of BsAb, and concentration-depended distribution of different drug-receptor complexes for Multabody. This DAE-based QEMB model platform can serve as an important tool to better understand complex binding kinetics of diverse classes of biologics.

Mechanics and thermodynamics of multivalent-binding induced shrinkage of hydrogels

Understanding the fundamental mechanism of shrink hydrogel sensors necessitates a complete comprehension of analyte-centered multivalent binding that occurs within their salt-rich microenvironments. However, the mechanics and thermodynamics governing this phenomenon remain insufficiently understood. Here, we aim to derive a theoretical framework that examines the impact of temporary cross-link formation on the hydrogel shrinkage due to specific binding interaction between the fixed receptors and the multivalent analytes. As a highlight of our theory, we mathematically quantify the hydrogels’ permanent and temporary cross-links using statistical thermodynamics to describe the multivalent complexation with different binding degrees while accounting for molecular-level transport factors when predicting the sensor’s shrinking characteristics. Consequently, our theory unveils the upper bounds set by the external analyte concentration and analyte binding valency onto the actuation sensitivity of these sensors, whereby tuning the receptor density permits further modulation of their performances. These findings tightly correlate the microscopic properties of the analyte and hydrogel to the macroscopic behaviors of shrink sensors, facilitating a structured design regime for advanced biomedical applications.

Electrocatalytic Glycerol Upgrading into Glyceric Acid on Ni3Sn Intermetallic Compound.

Electrochemical glycerol oxidation features an attractive approach of converting bulk chemicals into high-value products such as glyceric acid. Nonetheless, to date, the major product selectivity has mostly been limited as low-value C1 products such as formate, CO, and CO2, due to the fast cleavage of carbon-carbon (C-C) bonds during electro-oxidation. Herein, the study develops an atomically ordered Ni3Sn intermetallic compound catalyst, in which Sn atoms with low carbon-binding and high oxygen-binding capability allow to tune the adsorption of glycerol oxidation intermediates from multi-valent carbon binding to mono-valent carbon binding, as well as enhance *OH binding and subsequent nucleophilic attack. The Ni3Sn electrocatalyst exhibits one of the highest glycerol-to-glyceric acid performances, including a high glycerol conversion rate (1199µmol h-1) and glyceric acid selectivity (62 ± 3%), a long electrochemical stability of > 150 h, and the capability of direct conversion of crude glycerol (85% purity) into glyceric acid. The work features the rational design of highly ordered catalytic sites for tailoring intermediate binding and reaction pathways, thereby facilitating the efficient production of high-value chemical products.

Low-affinity LFA1-dependent outside-in signaling mediates avidity modulation via the Rabin8-Rab8 axis.

Lymphocyte interactions mediated by leukocyte integrin lymphocyte function-associated antigen 1 (LFA1) and intercellular adhesion molecules (ICAMs) are important for lymphocyte trafficking and antigen recognition. Integrins are regulated by the modulation of ligand-binding affinity and avidity (valency). Although the mechanism underlying high-affinity LFA1 binding has been investigated extensively, the molecular mechanisms by which low-affinity multivalent binding initiates adhesion remain unclear. We previously showed that ICAM1 and monoclonal antibodies that recognize specific LFA1 conformations induce the accumulation of LFA1 at the contact surface. In this study, we found that the small GTPase Rab8 is critical for intracellular transport and accumulation of LFA1 at cell contact areas mediated by low-affinity LFA1-dependent outside-in signaling. Super-resolution microscopy revealed that Rab8 co-localized with LFA1 in small vesicles near the contact membrane. Inactivation of Rab8 decreased ICAM1-dependent adhesion and substantially reduced LFA1 density on the contact membrane. The GTP-bound active form of Rab8 increased cell adhesiveness and promoted LFA1 accumulation at the contact area through co-trafficking with LFA1. Rab8 activation was induced by low-affinity conformation-dependent outside-in signaling via the guanine exchange factor Rabin8, which induced Rab8 activation at the cell contact area independent of Rap1. Single-molecule imaging of ICAM1 on a supported planner lipid bilayer demonstrated that Rab8 increased the frequency of LFA1-ICAM1 interactions without affecting their binding lifetime, indicating that Rab8 is mainly involved in the modulation of LFA1 avidity rather than LFA1 affinity. The present findings underscore the importance of low-affinity conformation-dependent outside-in signaling via the Rabin8-Rab8 axis leading to the initiation of LFA1 transport to the contact area.