Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence

Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies.     

Biological responses of human mesenchymal stem cells to titanium wear debris particles

Wear debris-induced osteolysis is a major cause of orthopedic implant aseptic loosening, and various cell types, including macrophages, monocytes, osteoblasts, and osteoclasts, are involved. We recently showed that mesenchymal stem/osteoprogenitor cells (MSCs) are another target, and that endocytosis of titanium (Ti) particles causes reduced MSC proliferation and osteogenic differentiation. Here we investigated the mechanistic aspects of the endocytosis-mediated responses of MSCs to Ti particulates. Dose-dependent effects were observed on cell viability, with doses >300 Ti particles/cell resulting in drastic cell death. To maintain cell viability and analyze particle-induced effects, doses <300 particles/cell were used. Increased production of interleukin-8 (IL-8), but not IL-6, was observed in treated MSCs, while levels of TGF-β, IL-1β, and TNF-α were undetectable in treated or control cells, suggesting MSCs as a likely major producer of IL-8 in the periprosthetic zone. Disruptions in cytoskeletal and adherens junction organization were also observed in Ti particles-treated MSCs. However, neither IL-8 and IL-6 treatment nor conditioned medium from Ti particle-treated MSCs failed to affect MSC osteogenic differentiation. Among other Ti particle-induced cytokines, only GM-CSF appeared to mimic the effects of reduced cell viability and osteogenesis. Taken together, these results strongly suggest that MSCs play both responder and initiator roles in mediating the osteolytic effects of the presence of wear debris particles in periprosthetic zones.     

Structural Requirements for Modulating 4-Benzylpiperidine Carboxamides from Serotonin/Norepinephrine Reuptake Inhibitors to Triple Reuptake Inhibitors

In this study, we determined the effect of 24 different synthetic 4-benzylpiperidine carboxamides on the reuptake of serotonin, norepinephrine, and dopamine (DA), and characterized their structure–activity relationship. The compounds with a two-carbon linker inhibited DA reuptake with much higher potency than those with a three-carbon linker. Among the aromatic ring substituents, biphenyl and diphenyl groups played a critical role in determining the selectivity of the 4-benzylpiperidine carboxamides toward the serotonin transporter (SERT) and dopamine transporter (DAT), respectively. Compounds with a 2-naphthyl ring were found to exhibit a higher degree of inhibition on the norepinephrine transporter (NET) and SERT than those with a 1-naphthyl ring. A docking simulation using a triple reuptake inhibitor 8k and a serotonin/norepinephrine reuptake inhibitor 7j showed that the regions spanning transmembrane domain (TM)1, TM3, and TM6 form the ligand binding pocket. The compound 8k bound tightly to the binding pocket of all three monoamine reuptake transporters; however, 7j showed poor docking with DAT. Co-expression of DAT with the dopamine D₂ receptor (D₂R) significantly inhibited DA-induced endocytosis of D₂R probably by reuptaking DA into the cells. Pretreatment of the cells with 8f, which is one of the compounds with good inhibitory activity on DAT, blocked DAT-induced inhibition of D₂R endocytosis. In summary, this study identified critical structural features contributing to the selectivity of a molecule for each of the monoamine transporters, critical residues on the compounds that bound to the transporters, and the functional role of a DA reuptake inhibitor in regulating D₂R function.     

Avian Reovirus σB Interacts with Caveolin-1 in Lipid Rafts during Dynamin-Dependent Caveolae-Mediated Endocytosis

Caveolin-1 (Cav-1) is the basic component of caveolae, a specialized form of lipid raft that plays an essential role in endocytic viral entry. However, the evidence of direct involvement of caveolae and Cav-1 in avian reovirus (ARV) entry remains insufficient. In this study, the membrane lipid rafts were isolated as detergent-resistant microdomains (DRMs) by sucrose gradient centrifugation, and the capsid protein σB of ARV was found to associate with Cav-1 in DRMs fractions. Additionally, the interaction between ARV σB protein and Cav-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays. Furthermore, we found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors, while it is insensitive to clathrin inhibitors. In conclusion, these results indicate that the ARV σB protein interacts with Cav-1 during dynamin-dependent caveolae-mediated endocytosis for the entry of ARV.     

Cancer targeted drug delivery using active low-density lipoprotein nanoparticles encapsulated pyrimidines heterocyclic anticancer agents as microtubule inhibitors

Recently, nanomedicine had the potential to increase the delivery of active compounds to specific cell sites. Nano-LDL particles are recognized as an excellent active nano-platform for cancer-targeted delivery. Loading of therapeutic agents into nano-LDL particles achieved by surface loading, core loading, and apolipoprotein-B100 interaction. Therefore, loading nano-LDL particles’ core with pyrimidine heterocyclic anticancer agents will increase cancer cytotoxic activity targeting tubulin protein. First, by mimicking the native LDL particle''s metabolic pathway, and second the agent’s chemical functional groups like the native amino acids cytosine and thymine structures will not be recognized as a foreign entity from the cell’s immune system. Nano-LDL particles will internalize through LDL-receptors endocytosis and transport the anticancer agent into the middle of the cancer cell, reducing its side effects on other healthy cells. Generally, the data revealed that pyrimidine heterocyclic anticancer agents’ size is at the nano level. Agents’ morphological examination showed nanofibers, thin sheets, clusters, and rod-like structures. LDL particles’ size became bigger after loading with pyrimidine heterocyclic anticancer agents and ranged between 121.6 and 1045 nm. Then, particles were tested for their cytotoxicity against breast (MDA468) and prostate (DU145) cancer cell lines as surrogate models with dose-response study 10, 5, 1 µM. The IC₅₀ values of the agents against DU145 and MDA468 possessed cell growth inhibition even at the 1 µM concentration ranges of 3.88 ± 1.05 µM and 3.39 ± 0.97 µM, respectively. In sum, nano-LDL particles proved their efficiency as active drug delivery vehicles to target tubulin in cancer cells.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse

Ligation of T cell receptor (TCR) to peptide–MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC–TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.

The fundamental molecular interactions responsible for regulating the adaptive immune response occur within a nanoscale gap between T cells and antigen-presenting cells (APCs) termed the immunological synapse (IS). IS formation is induced upon T cell receptor (TCR) interactions with agonist peptide-Major Histocompatibility Complex (pMHC) on the surface of APCs (1, 2). This process can be recapitulated by antigen presentation on supported lipid bilayers (SLBs), a minimal system composed of a mobile lipid phase configured to present relevant ligands at physiological densities (3, 4). Such IS formation on SLBs allows for microscopic analysis of the receptor–ligand interactions and membrane trafficking events underlying the initiation and effector functions of the adaptive immune system.During activation, the components of the IS rearrange in the opposing lipid membranes to form a characteristic bull’s-eye pattern consisting of three primary domains (5, 6). The bull’s-eye itself is termed the central supramolecular activation cluster (cSMAC) and is dominated by TCR and its ligands in a synaptic cleft. This area is surrounded by an adhesive ring defined by Lymphocyte Function-associated Antigen-1 (LFA-1) on the T cell side associated with intercellular adhesion molecule-1 (ICAM-1) on the SLB termed the peripheral supramolecular activation cluster (pSMAC). The outer edge of the contact is defined by an F-actin–rich sensory compartment termed the distal supramolecular activation cluster (dSMAC). TCR engagement is initiated in microclusters that arise from filopodia in the dSMAC and retain protrusive activity even as they traffic through the pSMAC toward the cSMAC (7–11). This is accompanied by CD3 tyrosine phosphorylation, recruitment of Zeta-associated protein of 70 kDa (ZAP-70), and phosphorylation of Linker of Activated T cells (LAT) (12) and recruitment of SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) (13). These then form condensates organized by LAT, traversing concentric actin networks en route to the cSMAC (14).Formation of the cSMAC by helper T cells has been shown to require recognition of ubiquitinated TCR by Tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT) (15). Then, following TSG101-dependent TCR sorting, the ESCRT-associated ATPase VPS4 mediates scission of TCR loaded extracellular vesicles termed synaptic ectosomes, which bud directly from the plasma membrane into the synaptic cleft (16). This is mechanistically similar to formation of the intraluminal vesicles (ILVs) of multivesicular endosomes and budding of HIV virions from the plasma membrane (17, 18). Prior to action of TSG101, the ESCRT component Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) recognizes ubiquitinated cargo and recruits clathrin, which assembles into flat lattices to mediate recruitment of subsequent ESCRT machinery (19, 20). Clathrin- and HRS-positive vesicles have also been shown to polarize toward the IS during T cell activation, and they have been implicated in recruiting F-actin there (21). However, it is not known whether clathrin is involved in the formation of synaptic ectosomes.Clathrin is primarily known for its role in endocytosis, where it is recruited to the plasma membrane by adaptor proteins such as Epsin-1 (EPN1) and Adaptor Protein complex-2 (AP-2) (22, 23). Together, these proteins facilitate deformation and invagination of the membrane which is ultimately pinched off as a clathrin-coated endocytic vesicle (24) by the large GTPase dynamin (25–27). Previous reports have shown that this mechanism is engaged to internalize TCR during constitutive TCR recycling (28, 29) and to internalize nonactivated bystander TCR during T cell activation (30). It has also been shown that TCR triggering leads to phosphorylation of clathrin heavy chain (CHC), (31) and TCR has been observed in clathrin-coated pits following antibody activation (32). However, conflicting evidence has indicated that clathrin is not involved in internalization of triggered TCR in the Jurkat cell line (30, 33, 34). Hence, the role of clathrin in endocytosis of pMHC–TCR conjugates is yet to be established.Synaptic ectosomes are important for delivery of T cell help through CD40 ligand and additional signals (35). TCR endocytosis is required for postendocytic signaling and regulation of pMHC availability (36, 37). Therefore, it is critical to understand the balance between ectocytosis and endocytosis of the TCR. Here, we show that clathrin is pivotal during T cell activation through its essential role in two sequential processes. First, clathrin is essential for ESCRT-mediated release of TCR loaded synaptic ectosomes at the cSMAC. As the IS matures, there is a temporal switch from the clathrin-associated ESCRT components HRS and STAM2 to the endocytic clathrin adaptor EPN1. Remaining antigen-ligated TCRs are then internalized by clathrin-mediated trans-endocytosis.     

利用苯乙烯染料揭示突触囊泡循环过程的研究进展

苯乙烯类染料 (Styryldyes) ,如FM 1 4 3和FM 2 10等是近年来发展和广泛应用于突触囊泡循环及突触传递研究领域的一类工具药。这类化合物特异性地标记突触囊泡 ,发出荧光 ,而又不影响囊泡的活性和运转。因此在突触事件中依照荧光强度的变化就可以推断相应的神经胞吞及胞吐的程度 ,跟踪囊泡循环过程。一些突触事件的具体环节 ,在应用了苯乙烯类染料之后 ,得到了进一步的阐明。本文简要介绍苯乙烯类染料的性质和近年来在突触事件研究中的一些主要应用及进展。     

网格蛋白介导的突触囊泡内吞机制

突触囊泡在神经末梢进行着精确而快速地胞吐、内吞循环。近年来进行的研究发现网格蛋白介导的内吞是囊泡蛋白回收的主要途径。本文介绍了这些在内吞早期阶段发生的事件和参与分子的研究进展。     

The binding of antibodies from Plasmodium berghei-infected rats to isoantigenic and parasite-specific antigenic sites on the surfaces of infected reticulocytes

Summary Ferritin-labelling techniques at the ultrastructural level have shown that antiserum from August rats immune to P. berghei infection contains antibodies which bind to the surfaces of parasitized reticulocytes but not to uninfected cells. Two antibody specificities have been demonstrated by comparing antisera i absorbed with infected reticulocytes, ii absorbed with uninfected reticulocytes, and iii unabsorbed. Ferritin labelling was much increased with antiserum preabsorbed with uninfected reticulocytes, and also with heat-inactivated serum, indicating a blocking effect on parasite-specific antibody binding by cold-reacting anti-erythrocyte isoantibodies known to be present. Energy-dependent aggregation, shedding and endocytosis of labelled material was observed at the surfaces of unfixed infected reticulocytes.