† These authors contribute equally to this work.Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway.

The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system1. Cyclic GMP–AMP synthase is a sensor of cytoplasmic DNA that activates the type-I interferon pathway2. Upon binding to DNA, cyclic GMP–AMP synthase is activated to catalyse the synthesis of cyclic GMP–AMP (cGAMP) from GTP and ATP3. cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING)3–9. STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines10,11. However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a β-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1.

No abstract

The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer's disease (AD), with most AD-derived mutations mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by singleparticle cryo-electron microscopy. AD-derived mutations in PS1 affect residues at two hotspots, each located at the center of a distinct four transmembrane segment (TM) bundle. TM2, and to a lesser extent TM6, exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain (ECD) following substrate recruitment. Aph-1 serves as a scaffold, anchoring the lone TM from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.A hallmark of Alzheimer's disease (AD) is accumulation of β-amyloid plaque in patient brain 1 . The intramembrane protease γ-secretase is thought to contribute to AD development by generating β-amyloid peptides, particularly those that are prone to aggregation such as Aβ42 2-5 . A mature γ-secretase consists of four components: presenilin, Pen-2, nicastrin, and Aph-1 6 . Among these components, presenilin is responsible for the Aβ-producing proteolytic activity 7,8 .