Targeting Axl favors an antitumorigenic microenvironment that enhances immunotherapy responses by decreasing Hif-1α levels

Hypoxia is an important phenomenon in solid tumors that contributes to metastasis, tumor microenvironment (TME) deregulation, and resistance to therapies. The receptor tyrosine kinase AXL is an HIF target, but its roles during hypoxic stress leading to the TME deregulation are not well defined. We report here that the mammary gland–specific deletion of Axl in a HER2⁺ mouse model of breast cancer leads to a normalization of the blood vessels, a proinflammatory TME, and a reduction of lung metastases by dampening the hypoxic response in tumor cells. During hypoxia, interfering with AXL reduces HIF-1α levels altering the hypoxic response leading to a reduction of hypoxia-induced epithelial-to-mesenchymal transition (EMT), invasion, and production of key cytokines for macrophages behaviors. These observations suggest that inhibition of Axl generates a suitable setting to increase immunotherapy. Accordingly, combining pharmacological inhibition of Axl with anti–PD-1 in a preclinical model of HER2⁺ breast cancer reduces the primary tumor and metastatic burdens, suggesting a potential therapeutic approach to manage HER2⁺ patients whose tumors present high hypoxic features.

Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in about 20% of breast cancers, representing the HER2-positive (HER2⁺) subtype that is associated with metastasis and poor prognosis (1, 2). While clinical success is partially achieved with the HER2-targeted therapies as a standard of care (e.g., Trastuzumab [Herceptin]), a significant pool of patients are either unresponsive or develop resistance to treatments. The development of new therapeutic approaches exploiting the tumor microenvironment (TME), like immunotherapy, is an attractive modality (3, 4). Indeed, combining immunotherapy and HER2-targeted agents is an emerging idea. Recently, clinical trials combined immune checkpoint point blockade (Pembrolizumab [anti–PD-1]) with Trastuzumab, but so far, it shows modest benefits (5). Indeed, there seems to be considerable roadblocks to hamper exploiting immunotherapies to treat HER2⁺-resistant breast cancer patients. Thus, there is a need to better understand the immune environment of HER2 tumors to develop more effective therapeutic strategies (3). The TME and its associated stromal cells significantly influence therapy responses and affect the metastatic progression that causes the majority of cancer-related deaths (6, 7). Hypoxia is an integral component of the TME and is associated with a poor prognosis and increased risks of metastasis in various types of cancers, including breast cancer (8, 9). The adaptive response to hypoxia involves the up-regulation of a plethora of genes that are under the control of Hypoxia-inducible Factors (HIFs), and collectively, they contribute to angiogenesis, metabolic reprogramming, epithelial-to-mesenchymal transition (EMT), invasion, and immune evasion (10). In solid tumors, low oxygen (O₂) availability promotes cancer cell invasion, protumoral immune responses, and abnormal angiogenesis (8, 11, 12). In particular, the tumors’ abnormal blood vessels are characterized by poor pericyte coverage and a noncontinuous basement membrane (BM) (13). This leads to dysfunctionalities such as poor perfusion and leakiness that accentuate hypoxia, facilitate tumor cell dissemination, and change the immune infiltration profiles (12, 14). Therefore, improving tumor blood vessel functionality, termed vessel normalization, has been suggested as a therapeutic goal (15). Consequently, targeting the TME appears as an interesting avenue to overcome resistance to cancer therapies including chemotherapy, radiotherapy, and immunotherapy (16–18).AXL is a member of the TAM family of receptor tyrosine kinases that is composed of TYRO3, AXL, and MERTK. AXL is broadly expressed in various cancers in which it correlates with poor survival and increased risks of metastasis (19–22). More specifically, its expression in cancer cells supports the acquisition of mesenchymal features and provides advantages including cell invasion and resistance to drugs (19, 23, 24). In HER2⁺ breast cancer cells, we previously reported a physical interaction between AXL and HER2 proteins that promoted invasion and metastasis (20). Functionally, AXL expression correlated with the acquisition of EMT features and was linked to poor patients’ outcome (20). Additionally, AXL has been described as a HIF target and has been shown to act with cMET to mediate the hypoxia-induced invasion of clear cell renal cell carcinoma (25). Nevertheless, the roles of AXL during hypoxic stress and in the TME deregulation remains poorly understood.In this study, we aimed to directly test whether the TME of HER2⁺ tumors can be harnessed toward a therapeutic strategy, more specifically, whether AXL inhibition may favor an immunotherapy response. We found that genetic deletion of Axl in the mammary epithelial cells in a HER2⁺ breast cancer mouse model generated an antitumorigenic TME and reduced metastasis by altering the hypoxic response in tumor cells. Pharmacological inhibition of Axl then enhanced an anti–PD-1 immune checkpoint blockade as supported by reduced primary tumor and metastatic burdens. Collectively, these results indicate that targeting AXL during immunotherapy could be an innovative therapeutic strategy to improve the survival and quality of life of patients afflicted with HER2⁺ cancers that are refractory to current treatments.     

Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

The canonical Wnt signaling pathway plays essential roles in embryonic development and tissue homeostasis (1, 2). In particular, Wnt signaling governs stem cell maintenance and proliferation in many tissues, and its misregulation is a common cause of tumor initiation (3, 4).Wnt ligands bind Frizzled (FZD) receptors and the coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) (5). The activated receptor complexes cluster on Dishevelled (DVL) platforms and are internalized via caveolin into endosomes termed LRP6 signalosomes, which triggers sequential phosphorylation of LRP6 by GSK3β and CK1γ (6–10). LRP6 signalosomes recruit the β-catenin destruction complex, which contains the scaffold proteins AXIN1 and adenomatous polyposis coli, the kinases CK1α and GSK3β, and the E3 ubiquitin ligase β-TrCP (11). This recruitment inhibits GSK3β and releases β-TrCP, which leads to β-catenin stabilization and nuclear translocation in a IFT-A/KIF3A–dependent manner (12–16). LRP6 signalosomes mature into multivesicular bodies, sequestering the Wnt receptors together with GSK3β, thereby maintaining long-term activation of the Wnt pathway and promoting macropinocytosis (14, 17–21). In contrast to Wnt ligands, the Wnt inhibitor Dickkopf-related protein 1 (DKK1) induces the clathrin-dependent internalization and turnover of LRP5/6 and thereby abrogates canonical Wnt signaling (22).LRP6 signalosome formation peaks in mitosis (23, 24). On the one hand, the LRP6 competence to respond to Wnt ligands is promoted during G2/M by a priming phosphorylation at its intracellular domain by CDK14/16 and CCNY/CCNYL1 (24, 25). On the other hand, CDK1 phosphorylates and recruits B-cell CLL/lymphoma 9 (BCL9) to the mitotic LRP6 signalosomes (23). BCL9 protects the signalosome from clathrin-dependent turnover, thereby sustaining basal Wnt activity on the onset of mitosis.Mitotic Wnt signaling not only modulates β-catenin (24) but increasing evidence suggests that it promotes a complex posttranslational program during mitosis (26). For instance, we have shown that mitotic Wnt signaling promotes stabilization of proteins (Wnt/STOP), which is required for cell growth and ensures chromosome segregation in somatic and embryonic cells (23, 26–31). Particularly, basal Wnt/STOP activity maintains proper microtubule plus-end polymerization rates during mitosis, and its misregulation leads to whole chromosome missegregation (31, 32). Furthermore, mitotic Wnt signaling controls the orientation of the spindle (33) and promotes asymmetric division in stem cells through components of the LRP6 signalosome (34). Accordingly, several Wnt components functionally associate with centrosomes, kinetochores, and the spindle during mitosis (25, 33, 35, 36). Consequently, both aberrant up-regulation or down-regulation of Wnt signaling have been associated with chromosome instability (CIN) (31, 32, 35, 37), which is a hallmark of cancer (38). Despite the importance of these processes for tissue renewal and genome maintenance, the targets and specific functions of mitotic Wnt signaling remain largely uncharacterized.Kinesin family member 2A (KIF2A) is a member of the kinesin-13 group (KIF2A,B,C) of minus-end microtubule depolymerases (39–41). KIF2A is essential for the scaling of the spindle during early development (42) and plays critical roles in neurogenesis by modulating both cilium disassembly and neuronal wiring (43–47). In dividing cells, KIF2A was thought to be required for the assembly of a bipolar spindle due to a small interfering RNA (siRNA) off-target effect (48, 49). Current evidence supports a role of KIF2A in microtubule depolymerization at the spindle poles, which can generate pulling forces on attached kinetochores, thereby ensuring the congression, alignment, and segregation of chromosomes (50–56). Genetic depletion of KIF2A in mouse leads to neonatal lethality and to severe brain malformations, including microcephaly (43, 44, 57). KIF2A recruitment to microtubules is tightly coordinated by several protein kinases (45, 47, 50–52, 58–60). For instance, phosphorylation of KIF2A at several sites by Polo-like kinase 1 (PLK1) stimulates its recruitment to and activity at the spindle (45, 58, 61). On the other hand, Aurora kinase A and B inhibit KIF2A activity and restrict its subcellular localization during mitosis (50, 58, 60).Here, we show that mitotic Wnt signaling promotes chromosome congression and alignment in prometaphase by recruiting KIF2A to the spindle in both somatic cells and pluripotent stem cells. We found that KIF2A is recruited by the LRP6 signalosome during mitosis. Mechanistically, we identified that KIF2A clusters with DVL via the N-terminal and motor domains of the depolymerase. We show that Wnt signaling controls KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle poles. We propose that basal Wnt signaling ensures timely chromosome congression and alignment prior cell division by modulating the spindle minus-end depolymerization dynamics through KIF2A.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Modeling putative therapeutic implications of exosome exchange between tumor and immune cells

Development of effective strategies to mobilize the immune system as a therapeutic modality in cancer necessitates a better understanding of the contribution of the tumor microenvironment to the complex interplay between cancer cells and the immune response. Recently, effort has been directed at unraveling the functional role of exosomes and their cargo of messengers in this interplay. Exosomes are small vesicles (30–200 nm) that mediate local and long-range communication through the horizontal transfer of information, such as combinations of proteins, mRNAs and microRNAs. Here, we develop a tractable theoretical framework to study the putative role of exosome-mediated cell–cell communication in the cancer–immunity interplay. We reduce the complex interplay into a generic model whose three components are cancer cells, dendritic cells (consisting of precursor, immature, and mature types), and killer cells (consisting of cytotoxic T cells, helper T cells, effector B cells, and natural killer cells). The framework also incorporates the effects of exosome exchange on enhancement/reduction of cell maturation, proliferation, apoptosis, immune recognition, and activation/inhibition. We reveal tristability—possible existence of three cancer states: a low cancer load with intermediate immune level state, an intermediate cancer load with high immune level state, and a high cancer load with low immune-level state, and establish the corresponding effective landscape for the cancer–immunity network. We illustrate how the framework can contribute to the design and assessments of combination therapies.Immunotherapeutic approaches have recently emerged as effective therapeutic modalities (1) exemplified by immune checkpoint blockade with anti–CTLA-4 to activate T-cells and induce tumor cell killing, which has been shown to be effective for some cancers but not others (2). A better understanding of the intricate interplay between cancer and the immune system, and of mechanisms of immune evasion and of hijacking of the host response by cancer cells, is relevant to the development of effective immunotherapeutic approaches (3–6).The immune-based suppression of tumor development and progression is mediated through nonspecific innate immunity and antigen-specific adaptive immunity (7). However, cancer cells can inhibit the immune response, thus evading suppression in multiple ways (8) (see below for details), and additionally hijack the immune system to their advantage (3, 4). The challenge to understand the tumor–immune interplay stems from the dynamic nature, and complexity and heterogeneity of both the cancer cells and the immune system and their interactions through the tumor microenvironment (9).Here we consider immune cells as consisting of macrophages (10), natural killer cells (11), cytotoxic T cells (12), helper T cells (13), and regulatory T cells (3). These various immune cells are produced, activated, and perform their functions separated by space and time, which contributes to additional complexity (14). Among the immune cells, dendritic cells (DCs) are the most efficient antigen-presenting cells to bridge innate immunity with adaptive immunity (15). DCs also secrete cytokines that promote the antitumor functions of both natural killer cells and macrophages (16, 17). We consider the tumor microenvironment as comprised of a heterogeneous population of cancer cells (18), stromal cells (19), and tumor-infiltrating immune cells (20). The interactions among these cell types contribute to tumor development and progression. Tumor-associated macrophages and cancer-associated fibroblasts regulate tumor metabolism and engender an immune-suppressive environment by secreting TGF-β and other cytokines (21). Fluctuations in energy sources and oxygen within a tumor contribute to malignant progression and cell phenotypic diversity (22, 23).Though secreted factors play critical roles in cell–cell communications, here we focus on the additional role of cell–cell communication mediated by the exchange of special extracellular lipid vesicles called exosomes (24). These nanovesicles of ∼30–200 nm are formed in the multivesicular bodies and then released from the cell into the extracellular space (25). The exosomes carry a broad range of cargo, including proteins, microRNAs, mRNAs, and DNA fragments, to specific target cells at a remote location (26). Membrane markers assign the exosomes to specific targeted cells. Notably, upon entering the target cell, the exosomes induce modulation of cell function and even identity switch (phenotypic, epigenetic, and even genetic) (27). Exosomes have recently emerged as playing an important role in the immune system interaction with tumors (28, 29). Tumor-derived exosomes can promote metastatic niche formation by influencing bone marrow-derived cells toward a prometastatic phenotype through upregulation of c-Met (29). DCs have been shown to induce tumor cell killing through release of exosomes that contain potent tumor-suppressive factors such as TNF and through activation of natural killer cells, cytotoxic T cells, and helper T cells (24, 30–32). However, tumor-derived exosomes (Tex) can directly inhibit the differentiation of DCs in bone marrow (33), which strongly inhibits the dendritic cell-mediated immune response to the tumor. In addition, Tex can also directly inhibit natural killer cells (34).Mathematical models have been devised to study the complex interactions of cancer and immune system, including those that consider spatial heterogeneity (as reviewed in ref. 35) and those that consider spatially homogeneous populations (as reviewed in ref. 36). Cancer–immunity models have been constructed to investigate the effects of therapy (37–39), cancer dormancy (40), and interactions with time delay (41). Other types of modeling methods have also been applied. For example, tumor growth has also been fitted to experimental data by artificial neural networks (42); a detailed network of cancer immune system has been modeled by multiple subset models (43).In this study, we have developed an exosome exchange-based cancer–immunity interplay (ECI) model, to incorporate the special role of DCs and exosome-mediated communications. Distinct from the previous approaches, our modeling strategy is adapted from methodology used in studies of gene regulatory circuits, allowing us to check the multistability features of the system (44). We find that, by including exosome exchange, the cancer–immunity interplay can give rise to three quasi-stable cancer states, which may be associated with the elimination/equilibrium/escape phases proposed in the immunoediting theory (45). The ECI model is also capable of explaining tumorigenesis by considering the time evolution of immune responses. Guided by the treatment simulations, we assess the effectiveness of various therapeutic protocols with and without time delay and noise.     

Generic,phenomenological, on-the-fly renormalized repulsion model for self-limited organization of terminal supraparticle assemblies

Self-limited, or terminal, supraparticles have long received great interest because of their abundance in biological systems (DNA bundles and virus capsids) and their potential use in a host of applications ranging from photonics and catalysis to encapsulation for drug delivery. Moreover, soft, uniform colloidal aggregates are a promising candidate for quasicrystal and other hierarchical assemblies. In this work, we present a generic coarse-grained model that captures the formation of self-limited assemblies observed in various soft-matter systems including nanoparticles, colloids, and polyelectrolytes. Using molecular dynamics simulations, we demonstrate that the assembly process is self-limited when the repulsion between the particles is renormalized to balance their attraction during aggregation. The uniform finite-sized aggregates are further shown to be thermodynamically stable and tunable with a single dimensionless parameter. We find large aggregates self-organize internally into a core–shell morphology and exhibit anomalous uniformity when the constituent nanoparticles have a polydisperse size distribution.The spontaneous formation of uniformly sized aggregates observed in inorganic, biological, and colloidal systems (1–14) is of both fundamental and practical interest. Their formation suggests that a generic mechanism is applicable to those chemically distinct systems, whereas their ability to serve as building blocks for engineering nanostructures at larger length scales is of practical interest. Examples of uniform aggregates include filaments, bundles, and toroids with uniform diameters formed by macromolecules (6–9), and regular domains by like-charged macroions on planar and cylindrical surfaces (5, 11). In a recent study, we observed the assembly of positively charged polydisperse CdSe, PbS, and PbSe nanoparticles (NPs) into monodisperse supraparticles (SPs), which form colloidal crystals at sufficiently high density (12). Spherical SPs with uniform size were also obtained from the assembly of similarly charged protein molecules (cytochrome C) and CdTe nanoparticles (13). It was also demonstrated that SPs can serve as a versatile tool to make nanoscale assemblies with unusual spiky shapes and form several constitutive blocks (14). The fact that the characteristic dimensions of these assemblies is highly uniform over a wide range of monomer concentration suggests that their formation from monomer aggregation is thermodynamically self-limited rather than kinetically arrested.Seminal theoretical studies of uniformly sized aggregates date back to the 1980s, and primarily focused on developing models of aggregation in reactive systems (1–3). An enormous body of theoretical studies that followed has been successful in characterizing the thermodynamic stability of finite-sized aggregates in various systems such as polyelectrolytes (5, 7, 11, 15–19), colloidal suspensions (20–24), and block copolymer and protein solutions (4, 8, 25–30). The optimal size of the assembled clusters is often attributed to the balance of short-ranged attraction and longer-ranged repulsion between the primary building blocks. Nevertheless, uniform clusters are also found in systems where the repulsion is not necessarily long-ranged by nature: it can be energetic (e.g., charge accumulation due to counterions and salt ions on the cluster surfaces as aggregation progresses), entropic (e.g., steric effects due to the nonnegligible size of the counterions and the packing frustration within the aggregates, which prevents counterion penetration), or both (5, 7, 11, 16, 18). Recently, Johnston and coworkers studied the formation of gold colloids in polymeric solutions, showing that the cluster size can be tuned by varying polymer concentration. In this case, the accumulation of the polymeric stabilizers adsorbed on the cluster surfaces provides an effective repulsion between the clusters (29, 30).Coarse-grained models for the effective pairwise interaction between the primary assembling species (macroions, colloids, and nanoparticles) have been developed to elucidate the formation of finite-sized aggregates (18–21, 23, 25). For instance, Zhang et al. derived effective interactions between the macroions and counterions by integrating over the fast modes of the smaller-sized solvent molecules and salt ions using the random phase approximation, showing that the model reproduced finite-sized clusters at dilute concentrations (18). Kung et al. further coarse-grained over the dynamics of the counterions by incorporating their screening effects into the effective NP–NP potential (19). They demonstrated that it is possible for like-charge NPs in a polar solvent to aggregate due to the steric effects and dipolar interactions of the solvent molecules despite the electrostatic repulsion between the NPs themselves. Sciortino et al. showed that an equilibrium cluster phase is stabilized when the cluster–cluster repulsion increases with the cluster sizes (23), in a similar manner to the Dejarguin–Landau–Verwey–Overbeek model for charged colloids in a dielectric medium (26). An equilibrium cluster phase was also confirmed in protein solutions and colloids (4). However, the equilibrium cluster phases reported in previous computational studies were disordered and only stable at low concentrations (4, 23, 31). It remains unclear whether the ordinary pairwise potentials that have been widely used in the colloidal chemistry/physics and coarse-grained molecular simulation literature can yield ordered cluster phases, i.e., colloidal crystals, as observed in experiments.Previous simulation studies typically assume that the effective interaction is identical for all pairs of NPs regardless of whether the NPs are forming an aggregate. This assumption neglects the change in the environment surrounding individual NPs and their charge renormalization (32, 33) during aggregation. In previous work (18, 19, 23), the cluster–cluster interaction was derived from the particle–particle interaction after the clusters had been formed. Only recently have Barros and Luijten proposed a numerical technique to resolve the polarization charge distributions in dynamic dielectric geometries arising during assembly (34). They demonstrated that dynamic polarization indeed alters––both qualitatively and quantitatively––predicted self-assembled structures. In the present study, we develop a generic model in which the repulsive interaction between the primary species (macroions, colloids, and NPs) is renormalized as the aggregates grow during the assembly process. The repulsion renormalization in our model is designed to capture the many-body effects associated with the interaction between the primary particles upon aggregation, which are not present in ordinary pairwise potentials. Such many-body effects are highly relevant for systems where, e.g., the primary particles are similarly charged, yet able to aggregate because of short-ranged attraction (12, 13).The results presented here extend those presented in ref. 12 to consider the effects of repulsion renormalization, to demonstrate SP stability, and to demonstrate that the uniform size of SPs for a given polydispersity of NPs is beyond that expected from statistical considerations. We discuss possible generalization of our model to describe the formation of finite-sized bundles from rod-like polyelectrolytes (7) and of sheets (35) and twisted helical ribbons from colloids (36) and from tetrahedrally shaped NPs (37).     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

Engineering hydrogels with homogeneous mechanical properties for controlling stem cell lineage specification

The extracellular matrix (ECM) is mechanically inhomogeneous due to the presence of a wide spectrum of biomacromolecules and hierarchically assembled structures at the nanoscale. Mechanical inhomogeneity can be even more pronounced under pathological conditions due to injury, fibrogenesis, or tumorigenesis. Although considerable progress has been devoted to engineering synthetic hydrogels to mimic the ECM, the effect of the mechanical inhomogeneity of hydrogels has been widely overlooked. Here, we develop a method based on host–guest chemistry to control the homogeneity of maleimide–thiol cross-linked poly(ethylene glycol) hydrogels. We show that mechanical homogeneity plays an important role in controlling the differentiation or stemness maintenance of human embryonic stem cells. Inhomogeneous hydrogels disrupt actin assembly and lead to reduced YAP activation levels, while homogeneous hydrogels promote mechanotransduction. Thus, the method we developed to minimize the mechanical inhomogeneity of hydrogels may have broad applications in cell culture and tissue engineering.

In tissues, cells reside in a complex extracellular microenvironment whose mechanical properties often vary both in space and in time during regular tissue homeostasis or disease development (1). Growing evidence suggests that changes in local mechanical properties can have a considerable impact on cell fate (2–6, 7–13). For example, the intricate local mechanical environment can strongly affect wound healing and tissue regeneration (14, 15). In synthetic biomaterials (e.g., hydrogels) that are used for cell culture and tissue engineering, the mechanical heterogeneity is also ubiquitous although, in many cases, undesirable (10, 16–22). For hydrogels prepared by the polymerization of monomers, variations in local monomer concentrations and the heat released from the chemical reactions can lead to various defects in the hydrogel network. For hydrogels prepared by the chemical crosslinking of polymers, the broad distribution of the molecular weight of the polymers and their nonuniform mixing before gelation can also cause dramatic variation in the local mechanical properties. Although the effect of the overall mechanical properties of hydrogels on cell behaviors has been widely explored, how mechanical heterogeneity at the nanoscale affects cell behaviors remains poorly understood.A major obstacle in addressing this question is the synthesis of hydrogels with uniformly distributed mechanical properties. By coupling four-armed poly(ethylene glycol) (PEG) macromers with narrowly distributed molecular weights, Sakai and coworkers have shown that it is possible to prepare hydrogels with minimal structural defects (23). The chemical reactions that are widely used for gelation include click chemistry (24, 25), amine-active ester reactions (26), maleimide–thiol conjugation (27, 28), and thiol-ene reactions (29, 30). The maleimide–thiol reaction is of special interest because it takes place under mild conditions, requires no catalysis, and does not generate small-molecule byproducts (27, 31–34). The hydrogels prepared by maleimide–thiol reaction have been widely used for organoid generation (35), protein and cell delivery (36), and controlled release (37). However, this reaction is too fast to allow adequate mixing of the macromer solution, leading to heterogeneous gelation. Because of variation in the crosslinking density, hydrogels often contain microdomains with distinct mechanical properties (38, 39). A few methods have been developed to minimize hydrogel heterogeneity by slowing this reaction, including lowering the gelation pH, changing the local pK_a of thiol, or adding thiol-binding metal ions (38, 40–43). However, these methods often require nonphysiological gelation conditions or lead to only limited improvement.In this work, we introduced host–guest chemistry to slow down the maleimide–thiol reaction for hydrogel preparation. We discovered that maleimide can form a complex with β-cyclodextrin (β-CD), lowering the free maleimide concentrations in the gelation system. We showed that the four-armed PEG hydrogels prepared using this approach possessed fewer network defects and more uniform mechanical properties. Moreover, we revealed that mechanical homogeneity can considerably affect the lineage specification of human embryonic stem cells (hESCs). We proposed that this effect can be attributed to the disruption of the assembly of actin fibers and the subsequent mechanotransduction pathways. We anticipate that this method can greatly improve the mechanical homogeneity of many cell culture systems to better regulate stem cell lineage specification.     

Bioinspired micro/nanomotor with visible light energy–dependent forward,reverse, reciprocating,and spinning schooling motion

In nature, microorganisms could sense the intensity of the incident visible light and exhibit bidirectional (positive or negative) phototaxis. However, it is still challenging to achieve the similar biomimetic phototaxis for the artificial micro/nanomotor (MNM) counterparts with the size from a few nanometers to a few micrometers. In this work, we report a fuel-free carbon nitride (C₃N₄)/polypyrrole nanoparticle (PPyNP)-based smart MNM operating in water, whose behavior resembles that of the phototactic microorganism. The MNM moves toward the visible light source under low illumination and away from it under high irradiation, which relies on the competitive interplay between the light-induced self-diffusiophoresis and self-thermophoresis mechanisms concurrently integrated into the MNM. Interestingly, the competition between these two mechanisms leads to a collective bidirectional phototaxis of an ensemble of MNMs under uniform illuminations and a spinning schooling behavior under a nonuniform light, both of which can be finely controllable by visible light energy. Our results provide important insights into the design of the artificial counterpart of the phototactic microorganism with sophisticated motion behaviors for diverse applications.

In nature , many microorganisms can respond to light and exhibit phototaxis. For instance, green algae can be attracted by weak light and escape from strong light (1–3). Inspired by nature, great efforts have been exerted to develop artificial light–driven and photoresponsive micro/nanomotors (MNMs) (4–8). During the last decade, significant progresses have been made in almost all aspects of the light-driven MNM (9–11). Until now, several different propulsion mechanisms have been established in order to propel the MNMs by light. The efficient propulsion can be realized based on bubbles generated through the photoirradiation-enhanced catalytic reaction (12) or the photoisomerization reactions (13, 14). It can also be achieved based on the Marangoni forces originating from the surface tension gradient caused by the photodoping process or the surfactant released from the photodegraded-light–responsive polymer (15). Recently, tailor-designed external fuel-free light-powered MNMs have been reported, whose propulsion can be achieved via self-diffusiophoresis induced by the photocatalytic reaction (16, 17) or the self-thermophoresis caused by the photothermal effect (18–20).Light (ultraviolet [UV], visible [Vis], or near infrared [NIR], etc.) (21–23) shows great versatility in manipulating the MNM’s movement (including speed and direction) through adjusting its “on” and “off,” wavelength, intensities, etc. (24–27). As a result, light cannot only govern the MNM trajectory in a programmable fashion by changing its incident direction but also enable the independent addressing of specific MNMs (28). These, together with the easily obtainable and remotely controllable features, make the light-driven MNM potentially attractive for various practical applications ranging from the controlled cargo capture, transportation, and release (29), cancer therapy (30), to dynamic assembly (31) or the environmental remediation (32). However, despite these progresses, for the light-driven MNMs, it is still challenging to realize the biomimetic bidirectional phototactic behaviors in water (i.e., positive phototaxis under weak light and negative phototaxis under strong light), as experienced by green algae. Intuitively, a bidirectionally phototactic MNM may be designed by combining two competitive light-driven mechanisms that are both photoresponsive, which could provide additional degree of freedom for motion regulation. Indeed, a 300-μm droplet has recently been reported to have the capability of bidirectional phototaxis in spiropyran’s oil solution (33), which explored the effect of interfacial tension change caused by the spiropyran photoisomerization and the photothermal effect. However, this active droplet cannot be miniaturized to the size of MNM ranging from a few nanometers to a few micrometers (colloidal motor) (34) because its self-propelling velocity sharply decays when reducing its size, which largely limits its application in precision surgery and biomedicine, etc., where MNMs are necessary (25). The demanding medium (spiropyran’s oil solution) where the active droplet operates also confines its performance to a special environment. In addition, the active droplet is unstable due to the droplet fusion, making it unsuitable for the investigation of the fascinating light-controlled collective behaviors of active matter. Therefore, it is very desirable to develop new mechanisms to achieve robust and fuel-free MNMs (active colloids) with the light intensity–dependent bidirectional phototaxis.In this work, we report a fuel-free visible light–driven MNM, which is capable of sensing the light intensity changes and exhibits the biomimetic individual or collective bidirectional phototaxis based on the combined self-diffusiophoresis and self-thermophoresis mechanisms. This MNM consists of binary active components: photocatalytic carbon nitride (C₃N₄) (35) and photothermal polypyrrole nanoparticle (PPyNP) (36). The competition between the local chemical (i.e., self-diffusiophoresis) and thermal (i.e., self-thermophoresis) gradients self-generated due to the photohydrolysis catalyzed by C₃N₄ and the light-absorbing PPyNP, accounts for the light energy–dependent motions. More interestingly, by tuning the light energy, the interplay between the two competitive mechanisms leads to the appearance of the unique reciprocating movement of a single MNM and the emergence of the spinning schooling behavior for a group of MNMs. We believe the unique and versatile motion behaviors of the current MNM can further advance the design and development of the artificial counterpart of the phototactic microorganism for various practical applications.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Hepatitis E virus infects brain microvascular endothelial cells,crosses the blood–brain barrier,and invades the central nervous system

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain–Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood–brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

Hepatitis E virus (HEV), an important emerging human pathogen, infects humans and a plethora of other animal species (1, 2). In humans, HEV primarily causes self-limiting acute viral hepatitis worldwide. It is estimated that there are ∼20 million HEV infections annually, leading to ∼3.4 million clinical cases of hepatitis E and 70,000 hepatitis E-related deaths globally (3). In industrialized countries, clinical cases of hepatitis E are mainly sporadic or clustered in nature, while in developing countries endemic or epidemic hepatitis E is occasionally associated with large outbreaks (4, 5). The main route of HEV transmission is fecal–oral, via contaminated drinking water or consumption of undercooked animal meat products (6). Since the discovery of swine HEV in 1997 from pigs in the United States (7, 8), novel strains of HEV have now been genetically identified from more than a dozen animal species, including domestic and wild pig, rabbit, camel, rat, chick, mongoose, and deer, among others (7, 8). Some of these animal HEVs, such as swine HEV, rat HEV, deer HEV, camel HEV, and rabbit HEV, have been shown to cross species barriers and infect humans (8–10). Hepatitis E is now recognized as an important zoonotic disease with a large number of animal reservoirs, which raises further public health concerns (4, 11).HEV-associated extrahepatic manifestations have increasingly become significant clinical problems (12), including chronic HEV infections in immunosuppressed individuals (13, 14), high mortality in HEV-infected pregnant women (15), and HEV-associated neurological and renal diseases (16–19). HEV-associated nerve root and plexus sequelae, such as Guillain–Barré syndrome and neuralgic amyotrophy, have been reported in a significant proportion of HEV-infected individuals worldwide (20, 21). Additionally, HEV-associated central nervous system (CNS) disorders, such as encephalitis, myelitis, cerebral ischemia, and seizures, have also been reported in HEV-infected patients (22–27). These neurological diseases are almost exclusively associated with zoonotic genotype 3 HEV infection, and to a lesser extent zoonotic genotype 4 HEV infection (28, 29). Although the exact mechanism how HEV infection leads to neurological diseases remains largely unknown, there is evidence suggesting that HEV may cross the blood–brain barrier (BBB) (30–33).HEV is a single-stranded, positive-sense RNA virus in the family of Hepeviridae, which consists of two genera: genus Orthohepevirus (species A, B, C, and D) infecting mammals and avian species and genus Piscihepevirus infecting cutthroat trout (34). Among the eight different HEV genotypes (HEV-1 to HEV-8) in species Orthohepevirus A (35), HEV-3 and HEV-4 are zoonotic, infecting humans and other animal species (8, 36). The genome of HEV consists of three partially overlapping open reading frames (ORFs). ORF1 encodes nonstructural proteins (37). ORF2 encodes the antigenic capsid protein (ORF2^c) that induces neutralizing antibodies, as well as a secreted form of capsid (ORF2^s) that inhibits antibody-mediated neutralization (38). ORF3 encodes a small phosphoprotein that is a functional iron channel involved in virus replication (39). The HEV virion has two forms: The exosome-like membrane-associated quasi-enveloped virions were found in the circulating blood and in cell culture supernatant of HEV-infected cells; the nonenveloped virions were found in feces and bile of infected hosts (40–42). It is known that exosome can migrate relatively freely across the BBB (43, 44). Whether the exosome-like quasi-enveloped HEVs are more permissible to BBB entry requires investigation.In this study, we conducted both in vitro and in vivo studies to delineate the potential mechanism of HEV-associated neuroinvasion. By using an in vitro BBB cell culture model, we first tested the ability and mechanism of membrane-associated quasi-enveloped HEV virions as well as the nonenveloped HEV virions to cross the BBB. Additionally, we also investigated the ability of both quasi-enveloped and nonenveloped HEV virions to invade CNS tissues in experimentally infected pigs. Our results suggest that HEV productively infects brain microvascular endothelial cells, crosses the in vitro BBB, and invades CNS in experimentally infected pigs.     

Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid–protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3–SEC5–SEC6–SEC8 and SEC10–SEC15–EXO70–EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3–EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1–phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein–lipid code for the recruitment of peripheral membrane proteins.

The plasma membrane (PM) of eukaryotic cells is spatially segregated into distinct domains with diverse functions, composition, and scales, a feature essential for many vital processes, including cell polarity regulation, signaling, and interactions with microorganisms (1, 2). Localized exocytosis is a fundamental process contributing to the establishment and maintenance of cellular polarity. An arsenal of small GTPases orchestrates the exocytosis through multiple effectors. Octameric protein complex exocyst is the small GTPase effector that facilitates the fusion of secretory vesicles with the PM (3, 4). The exocyst consists of eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84, that are evolutionarily conserved across eukaryotes (5).The plant exocyst complex is crucial for targeted secretion in cellular processes including the tip growth of root hairs and pollen tubes (6–9); hypocotyl elongation (10); cell wall maturation in xylem, endodermis, and trichomes (11–13); pectin secretion in seed coats (14); recycling of PIN auxin transporters (15); and plant–microbe interactions (16, 17). The exocyst is also important for cell plate initiation and maturation during plant cytokinesis (18–20). At the outer lateral PM of plant epidermal cells, the exocyst controls the secretion of polarly localized cargo proteins (21). Notably, exocyst accumulates at the outer lateral PM in dynamic foci that are distinct from sites of endocytosis (22).While the Exo70 subunit is encoded by a single gene in yeast and animals, many EXO70 isoforms exist in angiosperm plants. Such multiplication enables the existence of diverse, functionally specific exocyst complexes even within one cell (23, 24). Particular EXO70 isoforms are involved in highly localized domain-specific secretion at the PM as documented in pollen tubes and trichomes (8, 13, 25). Some EXO70 isoforms even acquired diverged functions in autophagy regulation (26) or as negative regulators of tip growth (27, 28).In Arabidopsis, housekeeping secretory processes in most sporophytic tissues involve the EXO70 isoform EXO70A1 (29, 30). On the other hand, these processes employ the closely related EXO70A2 isoform in the male gametophyte (9). Mutant plants lacking EXO70A1, unlike other studied EXO70 mutants, are severely morphologically affected and show secretory defects similarly to several mutants in other exocyst subunits (14, 15, 29, 31, 32). Moreover, among the multiple plant EXO70 isoforms, EXO70A1 is sequentially and structurally the most similar to the yeast and animal Exo70 (24, 29, 33). Hence, we focused on the EXO70A1 function in the plant exocyst architecture and PM recruitment in this study.Molecular mechanisms of the exocyst function reside in mediating the first contact of secretory vesicles with the PM and facilitating the subsequent fusogenic SNARE complex formation, leading to a vesicle–PM fusion (34, 35). An emerging model of the exocyst complex based on partially solved structures of several exocyst subunits, protein interaction mapping, fluorescent microscopy, and cryogenic electron microscopy indicates that interlaced rod-like exocyst subunits align longitudinally at the core of the complex with distant parts being flexible and available for concomitant molecular interactions bridging vesicles and the PM (36). In yeast, the Sec15p subunit binds secretory vesicles via interaction with Rab GTPase Sec4p (37), while Sec3p and Exo70p subunits interact with the PM and serve as landmarks for the exocyst recruitment (38–40). Both Sec3p and Exo70p bind the PM-specific phosphatidylinositol 4,5-bisphosphate (PIP₂) (39–41) and protein interactors such as Rho GTPases (42, 43). In animal cells, the exocyst membrane recruitment depends on the direct interaction of Exo70 with PIP₂ (44). In yeast and animals, PIP₂-dependent recruitment represents a general mechanism governing the localization of peripheral membrane proteins to the PM (45). In contrast, other anionic phospholipids, namely phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol 4-phosphate (PI4P), seem to be more important than PIP₂ in constituting the PM phospholipid signature in plants (46–48).In this study, we analyzed the overall Arabidopsis exocyst architecture and described the subunit connectivity map. Although the general architecture of the exocyst is evolutionary conserved, the PM recruitment mechanism represents a unique feature of the plant complex. By combining genetics, live-cell imaging, biochemistry, protein structure modeling, and molecular dynamics simulations, we demonstrated that the EXO70A1 subunit plays an essential role in PM–lipid signature recognition and dominates in the plant exocyst–PM recruitment to the PM.