Toshiaki Sakisaka

Molecular Machinery for Insertion of Tail-Anchored Membrane Proteins into the Endoplasmic Reticulum Membrane in Mammalian Cells

Tail-anchored (TA) membrane proteins destined for the secretory pathway are posttranslationally inserted into the endoplasmic reticulum (ER) membrane, but the molecular machinery for this insertion in mammalian cells remains elusive. Here we reveal a mammalian protein complex that drives the membrane insertion. We identify calcium-modulating cyclophilin ligand (CAML) as a mammal-specific receptor for TRC40, an ATPase targeting newly synthesized TA proteins, and show that CAML mediates membrane insertion of TA proteins. We show that CAML binds to WRB, an evolutionarily conserved TRC40 receptor, through the transmembrane domains and that CAML and WRB synergistically insert TA proteins into the membrane. Mutagenesis of CAML demonstrates that binding of TRC40 to CAML is required to ensure synergistic membrane insertion. Thus, identification of CAML and WRB as components of the TRC40 receptor complex represents a crucial mechanism for driving ER membrane insertion of TA proteins in mammalian cells.

Cell adhesion molecules in the central nervous system.

Cell-cell adhesion molecules play key roles at the intercellular junctions of a wide variety of cells, including interneuronal synapses and neuron-glia contacts. u3000Functional studies suggest that adhesion molecules are implicated in many aspects of neural network formation, such as axon-guidance, synapse formation, regulation of synaptic structure, and astrocyte-synapse contacts.u3000u3000Some basic cell biological aspects of the assembly of junctional complexes of neurons and glial cells resemble those of epithelial cells. u3000However, the neuron specific junctional machineries are required to exert neuronal functions, such as synaptic transmission and plasticity. In this review, we describe the distribution and function of cell adhesion molecules at synapses and at contacts between synapses and astrocytes.

Involvement of afadin in the formation and remodeling of synapses in the hippocampus

In the hippocampus, synapses are formed between mossy fiber terminals and CA3 pyramidal cell dendrites and comprise highly developed synaptic junctions (SJs) and puncta adherentia junctions (PAJs). Dynamic remodeling of synapses in the hippocampus is implicated in learning and memory. Components of both the nectin-afadin and cadherin-catenin cell adhesion systems exclusively accumulate at PAJs. We investigated the role of afadin at synapses in mice in which the afadin gene was conditionally inactivated in hippocampal neurons. In these mutant mice, the signals for not only nectins, but also N-cadherin and beta-catenin, were hardly detected in the CA3 area, in addition to loss of the signal for afadin, resulting in disruption of PAJs. Ultrastructural analysis revealed an increase in the number of perforated synapses, suggesting the instability of SJs. These results indicate that afadin is involved not only in the assembly of nectins and cadherins at synapses, but also in synaptic remodeling.

Kif14 mutation causes severe brain malformation and hypomyelination.

We describe a novel spontaneous mouse mutant, laggard (lag), characterized by a flat head, motor impairment and growth retardation. The mutation is inherited as an autosomal recessive trait, and lag/lag mice suffer from cerebellar ataxia and die before weaning. lag/lag mice exhibit a dramatic reduction in brain size and slender optic nerves. By positional cloning, we identify a splice site mutation in Kif14. Transgenic complementation with wild-type Kif14-cDNA alleviates ataxic phenotype in lag/lag mice. To further confirm that the causative gene is Kif14, we generate Kif14 knockout mice and find that all of the phenotypes of Kif14 knockout mice are similar to those of lag/lag mice. The main morphological abnormality of lag/lag mouse is severe hypomyelination in central nervous system. The lag/lag mice express an array of myelin-related genes at significantly reduced levels. The disrupted cytoarchitecture of the cerebellar and cerebral cortices appears to result from apoptotic cell death. Thus, we conclude that Kif14 is essential for the generation and maturation of late-developing structures such as the myelin sheath, cerebellar and cerebral cortices. So far, no Kif14-deficient mice or mutation in Kif14 has ever been reported and we firstly define the biological function of Kif14 in vivo. The discovery of mammalian models, laggard, has opened up horizons for researchers to add more knowledge regarding the etiology and pathology of brain malformation.

The tail domain of tomosyn controls membrane fusion through tomosyn displacement by VAMP2

Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the tomosyn-SNARE complex is so tight that VAMP2 cannot displace tomosyn. Based on these results, the tomosyn-SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of tomosyn suggest that tomosyn positively regulates exocytosis. Therefore, it is still controversial whether tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.

Arl6IP1 has the ability to shape the mammalian ER membrane in a reticulon-like fashion.

The ER (endoplasmic reticulum) consists of the nuclear envelope and a peripheral network of membrane sheets and tubules. Two classes of the evolutionarily conserved ER membrane proteins, reticulons and REEPs (receptor expression-enhancing proteins)/DP1 (deleted in polyposis locus 1)/Yop1 (YIP 1 partner), shape high-curvature ER tubules. In mammals, four members of the reticulon family and six members of the REEP family have been identified so far. In the present paper we report that Arl6IP1(ADP-ribosylation factor-like 6 interacting protein 1), an anti-apoptotic protein specific to multicellular organisms, is a potential player in shaping the ER tubules in mammalian cells. Arl6IP1, which does not share an overall primary sequence homology with reticulons, harbours reticulon-like short hairpin transmembrane domains and binds to atlastin, a GTPase that mediates the formation of the tubular ER network. Overexpression of Arl6IP1 induced extensive tubular structures of the ER and excluded a luminal protein. Furthermore, overexpression of Arl6IP1 stabilized the ER tubules, allowing the cells to maintain the ER tubules even in the absence of microtubules. Arl6IP1 constricted liposomes into tubules. The short hairpin structures of the transmembrane domains were required for the membrane-shaping activity of Arl6IP1. The results of the present study indicate that Arl6IP1 has the ability to shape high-curvature ER tubules in a reticulon-like fashion.

Kif14 overexpression accelerates murine retinoblastoma development.

The mitotic kinesin KIF14 has an essential role in the recruitment of proteins required for the final stages of cytokinesis. Genomic gain and/or overexpression of KIF14 has been documented in retinoblastoma and a number of other cancers, such as breast, lung and ovarian carcinomas, strongly suggesting its role as an oncogene. Despite evidence of oncogenic properties in vitro and in xenografts, Kif14s role in tumor progression has not previously been studied in a transgenic cancer model. Using a novel Kif14 overexpressing, simian virus 40 large T‐antigen retinoblastoma (TAg‐RB) double transgenic mouse model, we aimed to determine Kif14s role in promoting retinal tumor formation. Tumor initiation and development in double transgenics and control TAg‐RB littermates were documented in vivo over a time course by optical coherence tomography, with subsequent ex vivo quantification of tumor burden. Kif14 overexpression led to an accelerated initiation of tumor formation in the TAg‐RB model and a significantly decreased tumor doubling time (1.8 vs. 2.9 weeks). Moreover, overall percentage tumor burden was also increased by Kif14 overexpression. These data provide the first evidence that Kif14 can promote tumor formation in susceptible cells in vivo.

Crystal structure of afadin PDZ domain–nectin‐3 complex shows the structural plasticity of the ligand‐binding site

Afadin, a scaffold protein localized in adherens junctions (AJs), links nectins to the actin cytoskeleton. Nectins are the major cell adhesion molecules of AJs. At the initial stage of cell–cell junction formation, the nectin–afadin interaction plays an indispensable role in AJ biogenesis via recruiting and tethering other components. The afadin PDZ domain (AFPDZ) is responsible for binding the cytoplasmic C‐terminus of nectins. AFPDZ is a class II PDZ domain member, which prefers ligands containing a class II PDZ‐binding motif, X‐Φ‐X‐Φ (Φ, hydrophobic residues); both nectins and other physiological AFPDZ targets contain this class II motif. Here, we report the first crystal structure of the AFPDZ in complex with the nectin‐3 C‐terminal peptide containing the class II motif. We engineered the nectin‐3 C‐terminal peptide and AFPDZ to produce an AFPDZ–nectin‐3 fusion protein and succeeded in obtaining crystals of this complex as a dimer. This novel dimer interface was created by forming an antiparallel β sheet between β2 strands. A major structural change compared with the known AFPDZ structures was observed in the α2 helix. We found an approximately 2.5 Å‐wider ligand‐binding groove, which allows the PDZ to accept bulky class II ligands. Apparently, the last three amino acids of the nectin‐3 C‐terminus were sufficient to bind AFPDZ, in which the two hydrophobic residues are important.

The emerging role of calcium-modulating cyclophilin ligand in posttranslational insertion of tail-anchored proteins into the endoplasmic reticulum membrane.

Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack the gene homologous to Get2. We recently identified calcium-modulating cyclophilin ligand (CAML) as a TRC40 receptor, indicating that CAML was equivalent to Get2 in the context of the membrane insertion. On the other hand, CAML has been well characterized as a signaling molecule that regulates various biological processes, raising the question of how the two distinct actions of CAML, the membrane insertion and the signal transduction, are assembled. In this review, we summarize recent progress of the molecular mechanism of the membrane insertion of TA proteins and discuss the possibility that CAML could sense the various signals at the ER membrane, thereby controlling TA protein biogenesis.

Cytoarchitecture of the olfactory bulb in the laggard mutant mouse.

The laggard (lag) mutant mouse, characterized by hypomyelination and cerebellar ataxia, is a spontaneously occurring mutant mouse caused by mutation in the Kif14 gene. In this mutant mouse, the laminated structures such as the cerebral and cerebellar cortices and the dentate gyrus are cytoarchitecturally abnormal. Macroscopically, the olfactory bulb of the lag mutant mouse is smaller in size and more transparent than the normal counterpart. Hematoxylin-eosin staining reveals that the mutant olfactory bulb has normal lamination in general, but detailed analysis has demonstrated that olfactory periglomerular cells and granule cells are reduced in number. In the mutant, olfactory glomeruli are cytoarchitecturally disorganized and mitral cells are arranged in multiple cell layers instead of being arranged in a single layer. The rostral migratory stream in the mutant becomes gradually thinner or obliterated during early postnatal days. Some of mitral cells and periglomerular cells are multinucleated, suggesting that Kif14 mutation leads to an abnormal cell division. In the mutant, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the subventricular zone of the lateral ventricle are increased in number, especially at perinatal age, suggesting that the decreased population of granule cells in the lag mutant mouse is caused by the increased apoptotic cell death. The olfactory input appears to be intact, as indicated by anterograde labeling of olfactory nerves with an injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the olfactory mucosa. In conclusion, the olfactory bulb of the lag mutant mouse is cytoarchitecturally affected, suggesting that the causal gene for lag mutation, i.e., Kif14, has multiple effects on the development of laminated structures in the central nervous system in addition to the myelin formation.