Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system.

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

Horizontal Boundary Cells, a Special Group of Somitic Cells, Play Crucial Roles in the Formation of Dorsoventral Compartments in Teleost Somite.

Establishment of robust gene expression boundary is crucial for creating elaborate morphology during development. However, mechanisms underlying boundary formation have been extensively studied only in a few model systems. We examined the establishment of zic1/zic4-expression boundary demarcating dorsoventral boundary of the entire trunk of medaka fish (Oryzias latipes) and identified a subgroup of dermomyotomal cells called horizontal boundary cells (HBCs) as crucial players for the boundary formation. Embryological and genetic analyses demonstrated that HBCs play crucial roles in the two major events of the process, i.e., refinement and maintenance. In the refinement, HBCs could serve as a chemical barrier against Wnts from the neural tube by expressing Hhip. At later stages, HBCs participate in the maintenance of the boundary by differentiating into the horizontal myoseptum physically inhibiting cell mixing across the boundary. These findings reveal the mechanisms underlying the dorsoventral boundary in the teleost trunk by specialized boundary cells.

Synaptic activity prompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation.

Alzheimer's disease is an age-dependent neurodegenerative disorder that is characterized by a progressive decline in cognitive function. gamma-secretase dysfunction is evident in many cases of early onset familial Alzheimer's disease. However, the mechanism by which gamma-secretase dysfunction results in memory loss and neurodegeneration is not fully understood. Here, we demonstrate that gamma-secretase is localized at synapses and regulates spine formation. We identify EphA4, one of the Ephrin receptor family members, as a substrate of gamma-secretase, and find that EphA4 processing is enhanced by synaptic activity. Moreover, overexpression of EphA4 intracellular domain increases the number of dendritic spines by activating the Rac signaling pathway. These findings reveal a function for EphA4-mediated intracellular signaling in the morphogenesis of dendritic spines and suggest that the processing of EphA4 by gamma-secretase affects the pathogenesis of Alzheimer's disease.

Hyaluronan-CD44 pathway regulates orientation of mitotic spindle in normal epithelial cells.

Orientation of mitotic spindle and cell division axis can impact normal physiological processes, including epithelial tissue branching and neuron generation by asymmetric cell division. Microtubule dynamics and its interaction with cortical proteins regulate the orientation of mitotic spindle axis. However, the nature of extracellular signals that control proper orientation of mitotic spindle axis is largely unclear. Here, we show that signals from two distinct surface contact, "bi-surface-contact," sites are required for the orientation of mitotic spindle axis in normal epithelial cells. We identified apical and basal surface-membrane as required bi-surface-contact sites. We showed that high molecular weight (HMW) hyaluronan (HA)-CD44 signaling from the apical surface-membrane regulated the orientation of mitotic spindle axis to align parallel to the basal extracellular matrix (ECM). The same effect was achieved by fibronectin-integrin alphavbeta6 signaling from the basal surface-membrane or by inhibition of ROCK activity. On the contrary, HMW HA-CD44 signaling from the basal surface-membrane regulated the orientation of mitotic spindle axis to align oblique-perpendicular to the basal ECM. We also found that microtubule dynamics is required for HMW HA-CD44 mediated regulation of mitotic spindle orientation. Our findings thus provide a novel mechanism for the regulation of mitotic spindle orientation.

ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation.

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation.

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.