Possible modulation of process extension by N-methyl-D-aspartate receptor expressed in osteocytic MLO-Y4 cells.

In contrast to osteoblasts, little attention has been paid to expression profiles of different glutamatergic signaling machineries in osteocytes, which are the most abundant cells with a possible role as a mechanical sensor in bone. Here, we show that N-methyl-D-aspartate receptor (NMDAR) is expressed by osteocytic cells in five-weeks-old mouse tibiae in vivo as well as by osteocytic MLO-Y4 cells in vitro. Sustained exposure to the NMDAR antagonist dizocilpine significantly increased the number of cells with processes in cultured MLO-Y4 cells. These results suggest that NMDAR would be expressed by osteocytes with an unidentified role in the process extension.

Slow diffusion on the monolayer culture enhances auto/paracrine effects of Noggin in differentiation of human iPS cells induced by BMP.

Auto/paracrine factors secreted from cells affect differentiation of human pluripotent stem cells (hPSCs). However, the molecular mechanisms underlying the role of secreted factors are not well known. We previously showed that pattern formation in hPSCs induced by BMP4 could be reproduced by a simple reaction-diffusion of BMP and Noggin, a cell-secreted BMP4 inhibitor. However, the amount of Noggin secreted is unknown. In this study, we measured the concentration of Noggin secreted during the differentiation of hPSCs induced by BMP4. The Noggin concentration in the supernatant before and after differentiation was constant at approximately 0.69 ng/mL, which is approximately 50-200 times less than expected in the model. To explain the difference between the experiment and model, we assumed that macromolecules such as heparan sulfate proteoglycan on the cell surface act as a diffusion barrier structure, where the diffusion slows down to 1/400. The model with the diffusion barrier structure reduced the Noggin concentration required to suppress differentiation in the static culture model. The model also qualitatively reproduced the pattern formation, in which only the upstream but not the downstream hPSCs were differentiated in a one-directional perfusion culture chamber, with a small change in the amount of secreted Noggin resulting in a large change in the differentiation position. These results suggest that the diffusion barrier on the cell surface might enhance the auto/paracrine effects on monolayer hPSC culture.

Glutamate preferentially suppresses osteoblastogenesis than adipogenesis through the cystine/glutamate antiporter in mesenchymal stem cells.

We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective. In cells with Runx2 adenovirus, Glu significantly decreased [14C]cystine uptake, intracellular glutathione (GSH) level, Runx2 recruitment to osteocalcin promoter and nuclear Runx2 protein level, respectively. Cystine again significantly prevented the decreases by Glu in both GSH levels and Runx2 recruitment. In mouse bone marrow stromal cells, Glu and a GSH depleter significantly decreased ALP staining without affecting Oil Red O staining. Knockdown of the cystine/Glu antiporter led to markedly decreased ALP staining and GSH levels, with concomitant prevention of the decrease by Glu, in cells with Runx2 adenovirus. These results suggest that Glu may play a role as a negative regulator at an early differentiation stage into osteoblasts than adipocytes through a mechanism relevant to nuclear translocation of Runx2 after regulation of intracellular GSH levels by the cystine/Glu antiporter expressed in mesenchymal stem cells.

Light exaggerates apical hook curvature through phytochrome actions in tomato seedlings.

Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato (Solanum lycopersicum L.) that the apical hook curvature is exaggerated by light. Experiments with several tomato cultivars and phytochrome mutants, irradiated with red and far-red light either as a brief pulse (Rp, FRp) or continuously (Rc, FRc), revealed: the hook-exaggeration response is maximal at the emergence of the hypocotyl from the seed; the effect of Rp is FRp-reversible; fluence-response curves to a single Rp or FRp show an involvement of low and very low fluence responses (LFR, VLFR); the effect of Rc is fluence-rate dependent, but that of FRc is not; the phyA mutant (phyA hp-1) failed to respond to an Rp of less than 10(-2) micromol m(-2) and to an FRp of all fluences tested as well as to FRc, thus indicating that the hook-exaggeration response involves phyA-mediated VLFR. The Rp fluence-response curve with the same mutant also confirmed the presence of an LFR mediated by phytochrome(s) other than phyA, although the phyB1 mutant (phyB1 hp-1) still showed full response probably due to other redundant phytochrome species (e.g., phyB2). Simulation experiments led to the possible significance of hook exaggeration in the field that the photoresponse may facilitate the release of seed coat when seeds germinate at some range of depth in soil. It was also observed that seed coat and/or endosperm are essential to the hook exaggeration.

Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.

Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis.

Compartmentalized microfluidic perfusion system to culture human induced pluripotent stem cell aggregates.

Microfluidic perfusion systems enable small-volume cell cultures under precisely controlled microenvironments, and are typically developed for cell-based high-throughput screening. However, most such systems are designed to manipulate dissociated single cells, not cell aggregates, and are thus unsuitable to induce differentiation in human induced pluripotent stem cells (hiPSCs), which is conventionally achieved by using cell aggregates to increase cell-cell interactions. We have now developed a compartmentalized microfluidic perfusion system with large flow channels to load, culture, and observe cell aggregates. Homogeneously sized cell aggregates to be loaded into the device were prepared by shredding flat hiPSC colonies into squares. These aggregates were then seeded into microchambers coated with fibronectin and bovine serum albumin (BSA) to establish adherent and floating cultures, respectively, both of which are frequently used to differentiate hiPSCs. However, the number of aggregates loaded in fibronectin-coated microchambers was much lower than in BSA-coated microchambers, suggesting that fibronectin traps cell aggregates before they reach the chambers. Accordingly, hiPSCs that reached the microchambers subsequently adhered. In contrast, BSA-coated microchambers did not allow cell aggregates to adhere, but were sufficiently deep to prevent cell aggregates from flowing out during perfusion of media. Immunostaining for markers of undifferentiated cells showed that cultures on both fibronectin- and BSA-coated microchambers were successfully established. Notably, we found that floating aggregates eventually adhered to surfaces coated with BSA upon differentiation, and that differentiation depends on the initial size of aggregates. Collectively, these results suggest that the microfluidic system is suitable for manipulating hiPSC aggregates in compartmentalized microchambers.

A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling.

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.

Thermodynamic characterization of viral procapsid expansion into a functional capsid shell.

The assembly of "complex" DNA viruses such as the herpesviruses and many tailed bacteriophages includes a DNA packaging step where the viral genome is inserted into a preformed procapsid shell. Packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. This transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and that the transition is fully reversible. This provides an unprecedented opportunity to evaluate the thermodynamic features of this fascinating and essential step in virus assembly. We show that urea-triggered expansion is highly cooperative and strongly temperature dependent. Thermodynamic analysis indicates that the free energy of expansion is influenced by magnesium concentration (3-13 kcal/mol in the presence of 0.2-10 mM Mg(2+)) and that significant hydrophobic surface area is exposed in the expanded shell. Conversely, Mg(2+) drives the expanded shell back to the procapsid conformation in a highly cooperative transition that is also temperature dependent and strongly influenced by urea. We demonstrate that the gpD decoration protein adds to the urea-expanded capsid, presumably at hydrophobic patches exposed at the 3-fold axes of the expanded capsid lattice. The decorated capsid is biologically active and sponsors packaging of the viral genome in vitro. The roles of divalent metal and hydrophobic interactions in controlling packaging-triggered expansion of the procapsid shell are discussed in relation to a general mechanism for DNA-triggered procapsid expansion in the complex double-stranded DNA viruses.

The transcription factor paired box-5 promotes osteoblastogenesis through direct induction of Osterix and Osteocalcin.

Although skeletal abnormalities are seen in mice deficient of particular paired box (Pax) family proteins, little attention has been paid to their role in osteoblastogenesis so far. Here, we investigated the possible involvement of several Pax family members in mechanisms underlying the regulation of differentiation and maturation of osteoblasts. Among different Pax family members tested, Pax5 was not markedly expressed in murine calvarial osteoblasts before culture, but progressively expressed by osteoblasts under differentiation toward maturation. Immunoreactive Pax5 was highly detectable in primary cultured mature osteoblasts on immunoblotting and in osteoblastic cells attached to cancellous bone in mouse tibial sections on immunohistochemistry, respectively. Knockdown by small interfering RNA (siRNA) of endogenous Pax5 led to significant inhibition of the expression of Osteocalcin, and Osterix through deterioration of gene transactivation, in addition to a1(I)Collagen expression and alkaline phosphatase (ALP) staining, without affecting runt-related transcription factor-2 (Runx2) expression and cell viability in osteoblastic MC3T3-E1 cells. The introduction of Pax5 enhanced promoter activities of Osteocalcin and Osterix in a manner dependent on the paired domain in MC3T3-E1 cells. Putative Pax5 binding sites were identified in the 5'-flanking regions of mouse Osteocalcin and Osterix, whereas chromatin immunoprecipitation assay revealed the direct binding of Pax5 to particular regions of Osteocalcin and Osterix promoters in MC3T3-E1 cells. Overexpression of Pax5 significantly increased Osteocalcin, Osterix, and a1(I)Collagen expression, ALP activity, and Ca(2+) accumulation, without affecting Runx2 expression, in MC3T3-E1 cells. In vertebrae of transgenic mice predominantly expressing Pax5 in osteoblasts, a significant increase was seen in the ratio of bone volume over tissue volume and the bone formation rate. These findings suggest that Pax5 could positively regulate osteoblastic differentiation toward maturation in vitro, in addition to promoting bone formation and remodeling in vivo, as one of the transcription factors essential for controlling osteoblastogenesis independently of Runx2.

Positive regulation of osteoclastic differentiation by growth differentiation factor 15 upregulated in osteocytic cells under hypoxia.

Osteocytes are thought to play a role as a mechanical sensor through their communication network in bone. Although osteocytes are the most abundant cells in bone, little attention has been paid to their physiological and pathological functions in skeletogenesis. Here, we have attempted to delineate the pivotal functional role of osteocytes in regulation of bone remodeling under pathological conditions. We first found markedly increased osteoclastic differentiation by conditioned media (CM) from osteocytic MLO-Y4 cells previously exposed to hypoxia in vitro. Using microarray and real-time PCR analyses, we identified growth differentiation factor 15 (GDF15) as a key candidate factor secreted from osteocytes under hypoxia. Recombinant GDF15 significantly promoted osteoclastic differentiation in a concentration-dependent manner, with concomitant facilitation of phosphorylation of both p65 and inhibitory-κB in the presence of receptor activator of nuclear factor-κB ligand. To examine the possible functional significance of GDF15 in vivo, mice were subjected to ligation of the right femoral artery as a hypoxic model. A significant increase in GDF15 expression was specifically observed in tibias of the ligated limb but not in tibias of the normally perfused limb. Under these experimental conditions, in cancellous bone of proximal tibias in the ligated limb, a significant reduction was observed in bone volume, whereas a significant increase was seen in the extent of osteoclast surface/bone surface when determined by bone histomorphometric analysis. Finally, the anti-GDF15 antibody prevented bone loss through inhibiting osteoclastic activation in tibias from mice with femoral artery ligation in vivo, in addition to suppressing osteoclastic activity enhanced by CM from osteocytes exposed to hypoxia in vitro. These findings suggest that GDF15 could play a pivotal role in the pathogenesis of bone loss relevant to hypoxia through promotion of osteoclastogenesis after secretion from adjacent osteocytes during disuse and/or ischemia in bone.

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T(m)=40.6±0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K(d,app)~50 µM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.

Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules.

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).