Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide-binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.

Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

The authors successfully performed a series of 33 living related liver transplantations (LRLT) on children (15 males and 18 females, ranging from 7 months to 15 years of age) from June 1990 to May 1992, with the informed consent of their parents and the approval of the Ethics Committee of Kyoto University. Before operation, six of the children required intensive care, another 14 were hospitalized, and 13 were homebound. Donors (12 paternal and 21 maternal) were selected solely from the parents of the recipients on the basis of ABO blood group and graft/recipient size matching determined by computed tomography scanning. Procurement of graft was performed using ultrasonic aspirator and bipolar electrocautery without blood vessel clamping and without graft manipulation. All donors subsequently had normal liver function and returned to normal life. The left lateral segment (16 cases), left lobe (16 cases), or right lobe (one case) were used as grafts. The partial liver graft was transplanted into the recipient who underwent total hepatectomy with preservation of the inferior vena cava using a vascular side clamp. Twenty-seven of 33 recipients are alive and well with the original graft and have normal liver function. The patient survival rate was 89% (24/27) in elective cases and 50% (3/6) in emergent cases. The other six recipients had functioning grafts but died of extrahepatic complications. Complications of the graft were minimal in all cases. Hepatic vein stenosis, which occurred three times in two cases, was successfully treated by balloon dilatation. In cases with sclerotic portal vein, the authors anastomosed the portal vein of the graft to the confluence of the splenic vein and the superior mesenteric vein without a vascular graft, after experiencing a case of vascular graft thrombosis. After hepatic artery thrombosis occurred in one of the initial seven recipients whose arterial anastomosis was done with surgical loupe, microsurgery was introduced for hepatic artery reconstruction. There has been no occurrence of thrombosis since then. The current results with LRLT suggested that the meticulous management of surgical factors at each stage of the LRLT procedure is crucial for successful outcome. Living related liver transplantation is a promising option for resolving the graft shortage in pediatric liver transplantation and may be regarded as an independent modality to supplement cadaver donation.

PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD) showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5'-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.

In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryo–electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.