Ultrasmall compact CMOS imaging system for bioluminescence reporter-based live gene expression analysis.

SIGNIFICANCE: Gene expression analysis is an important fundamental area of biomedical research. However, live gene expression imaging has proven challenging due to constraints in conventional optical devices and fluorescent reporters. AIM: Our aim is to develop smaller, more cost-effective, and versatile imaging capabilities compared with conventional devices. Bioluminescence reporter-based gene expression analysis was targeted due to its advantages over fluorescence-based imaging. APPROACH: We created a small compact imaging system using micro-CMOS image sensors (µCIS). The µCIS model had an improved pixel design and a patterned absorption filter array to detect the low light intensity of bioluminescence. RESULTS: The device demonstrated lower dark current, lower temporal noise, and higher sensitivity compared with previous designs. The filter array enabled us to subtract dark current drift and attain a clearer light signal. These improvements allowed us to measure bioluminescence reporter-based gene expression in living mammalian cells. CONCLUSION: Using our µCIS system for bioluminescence imaging in the future, the device can be implanted in vivo for simultaneous gene expression imaging, behavioral analysis, and optogenetic modulation.

BNIP-2 binds phosphatidylserine, localizes to vesicles, and is transported by kinesin-1.

BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.

Aberrant gene expression and sexually incompatible genomic imprinting in oocytes derived from XY mouse embryonic stem cells in vitro.

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

A photo-triggerable drug carrier based on cleavage of PEG lipids by photosensitiser-generated reactive singlet oxygen.

To circumvent the limitations of polyethylene glycol (PEG) modified carriers, a photo-triggerable liposome was prepared which was modified by cholesterol derivatives via a cleavable vinyl ether linkage so that the PEGylated coating can be efficiently removed by a photoactivated fullerene. After the photocleavage of the PEG moiety, the intracellular uptake of the photo-triggerable liposome improved.

Cyclodextrin complexed [60]fullerene derivatives with high levels of photodynamic activity by long wavelength excitation.

We have evaluated the photodynamic activities of C60 derivative·Î³-cyclodextrin (γ-CDx) complexes and demonstrated that they were significantly higher than those of the pristine C60 and C70·Î³-CDx complexes under photoirradiation at long wavelengths (610-720 nm), which represent the optimal wavelengths for photodynamic therapy (PDT). In particular, the cationic C60 derivative·Î³-CDx complex had the highest photodynamic ability because the complex possessed the ability to generate high levels of (1)O2 and provided a higher level of intracellular uptake. The photodynamic activity of this complex was greater than that of photofrin, which is the most widely used of the known clinical photosensitizers. These findings therefore provide a significant level of information toward the optimization of molecular design strategies for the synthesis of fullerene derivatives for PDT.

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12).

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.

Abl-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion.

Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 (Abl interactor 1) promotes phosphorylation of Mena and BCAP (B-cell adaptor for phosphoinositide 3-kinase) by bridging the interaction between c-Abl and the substrate. In the present study we have identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is co-expressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukaemic cells in an Abi-1-dependent manner. Co-expression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less accumulation of VASP at focal adhesions. VASP Y39D had a reduced affinity to the proline-rich region of zyxin. Interestingly, overexpression of both phosphomimetic and unphosphorylated forms of VASP, but not wild-type VASP, impaired adhesion of K562 cells to fibronectin. These results suggest that the phosphorylation and dephosphorylation cycle of VASP by the Abi-1-bridged mechanism regulates association of VASP with focal adhesions, which may regulate adhesion of Bcr-Abl-transformed leukaemic cells.

The l-Ser analog #290 promotes bone recovery in OP and RA mice.

We previously characterized the l-Ser analog #290, H(tBut)-l-Ser-O-Methyl·HCl, as a novel inhibitor of osteoclastogenesis which functions in both mouse and human cells. Here, we assessed the activity of #290 in animal models of osteoporosis and rheumatoid arthritis. Treatment of animals with #290 both prevented bone loss and led to the recovery of lost bone in osteoporotic mice. When inflammatory arthritis was induced in SKG mice, #290 treatment suppressed arthritis scores and significantly prevented the destruction of calcaneous bones. Additionally, #290 reciprocally modulated the mammalian target of rapamycin (mTOR) pathway in osteoclasts and osteoblasts in vitro, suggesting a dual effect on bone homeostasis. Our results demonstrate that #290 is a potential novel therapeutic tool for the treatment and/or study of diseases associated with bone destruction.

Identification and functional analysis of a new phosphorylation site (Y398) in the SH3 domain of Abi-1.

Abi-1 is an adaptor protein for Abelson kinase (c-Abl), and Abi-1 promotes the Abl-mediated phosphorylation of Mammalian Enabled (Mena) by binding both c-Abl and Mena. Here, we identified a new phosphorylation site (Y398) in the SH3 domain of Abi-1, and disruption of Y398, combined with the previously identified phosphorylation site Y213, significantly weakens the binding of Abi-1 to c-Abl. The SH3 domain of Abi-1 and the proline-rich domain of c-Abl are involved in this interaction. Abi-1 phosphorylation at both sites stimulates the phosphorylation of Mena through the activation of c-Abl kinase. The phosphorylation of Abi-1 also plays a role in enhancing the adhesion of Bcr-Abl-transformed leukemic cells.

Acid sphingomyelinase regulates osteoclastogenesis by modulating sphingosine kinases downstream of RANKL signaling.

Acid sphingomyelinase (ASM) was identified as a gene induced by NFAT2 activation in osteoclasts. Suppression of ASM expression in bone marrow macrophages by knockdown enhanced c-Fos/NFAT2 expression, increasing the number of TRAP-positive multinucleated cells in vitro. SphK1 was upregulated during the late stage of osteoclastogenesis, while SphK2 expression remained constant. SphK1 was downregulated following ASM knockdown, while SphK2 levels were unchanged. Experiments using shRNA and catalytically-inactive form demonstrated inhibitory and stimulatory activities on osteoclast formation of SphK1 and SphK2, respectively. These results suggest that ASM regulates osteoclastogenesis by modulating the balance between SphK1 and SphK2 downstream of RANKL signaling.

Potential involvement of Twist2 and Erk in the regulation of osteoblastogenesis by HB-EGF-EGFR signaling.

Epidermal growth factor (EGF) family members play important roles in the skeletal system. In this study, we examined the role of EGF receptor (EGFR) signaling in osteoblastogenesis in vitro. The expression of HB-EGF and epiregulin (EPR) was transiently induced within 24 h after osteogenic stimulation, but when preosteoblastic MC3T3-E1 cells were incubated with HB-EGF or EPR, osteoblast differentiation was inhibited. These effects were Ras-dependent, and ERK modulated Runx2 activity through the localization of Smad1 and the induction of Twist2. PI3-kinase was also required for the induction of Twist2. However, the inhibition of individual signaling pathways was not sufficient to overcome HB-EGF-mediated inhibition of osteoblast differentiation. Additionally, HB-EGF treatment promoted the proliferation of preosteoblasts, and this was associated with the downregulation of p27 at the protein level. These results suggest that HB-EGF-EGFR signaling inhibits the differentiation of osteoblasts by suppression of Runx2 transcriptional activity and enhances proliferation of preosteoblasts by downregulation of expression of p27.

Identification of a novel L-serine analog that suppresses osteoclastogenesis in vitro and bone turnover in vivo.

Osteoclasts are multinucleated giant cells with bone resorbing activity. We previously reported that the expression of the transcription factor NFAT2 (NFATc1) induced by receptor activator of NF-kappaB ligand (RANKL) is essential for the formation of multinucleated cells. We subsequently identified L-Ser in the differentiation medium as necessary for the expression of NFAT2. Here we searched for serine analogs that antagonize the function of L-Ser and suppress the formation of osteoclasts in bone marrow as well as RAW264 cells. An analog thus identified, H-Ser(tBu)-OMe x HCl, appeared to suppress the production of 3-ketodihydrosphingosine by serine palmitoyltransferase, and the expression and localization of RANK, a cognate receptor of RANKL, in membrane lipid rafts was down-regulated in the analog-treated cells. The addition of lactosylceramide, however, rescued the osteoclastic formation. When administered in vivo, the analog significantly increased bone density in mice and prevented high bone turnover induced by treatment with soluble RANKL. These results demonstrate a close connection between the metabolism of L-Ser and bone remodeling and also the potential of the analog as a novel therapeutic tool for bone destruction.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells.

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

A novel role of L-serine (L-Ser) for the expression of nuclear factor of activated T cells (NFAT)2 in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis in vitro.

Multinucleated cell formation is crucial for osteoclastogenesis, and the expression of nuclear factor of activated T cells (NFAT)2 (NFATc1) is essential for this process. We previously found, using mouse RAW264 cells, that culture at high cell density blocked progression to the multinucleated cell stage induced by stimulation with receptor activator of nuclear factor kappaB ligand (RANKL). Here, we have confirmed this finding in a bone marrow cell system and extended the analysis further. A high cell density appeared to cause a change in the composition of the culture medium accompanying downregulation of NFAT2 expression, and we identified L-serine (LSer) as essential for the expression of NFAT2 induced by RANKL. Namely, culture at high cell density caused a depletion of LSer in the medium. Consequently, L-Ser appeared to exert its effect at an early stage under the regular conditions used for inducing the expression of c-Fos, an upstream regulator of NFAT2. D-Ser, an enantiomer of L-Ser, showed no NFAT2-inducing activity. The expression of NFAT2, using a retrovirus vector, could compensate for the depletion of L-Ser and resume the progression to the multinucleated cell stage. These results demonstrate a novel role for L-Ser in RANKL-induced osteoclastogenesis in vitro.

Src homology 2 (SH2)-containing 5'-inositol phosphatase localizes to podosomes, and the SH2 domain is implicated in the attenuation of bone resorption in osteoclasts.

c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship(-/-) osteoclasts that Src homology 2 (SH2)-containing 5'-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization. Although SHIP lacking a functional SH2 domain was still found in podosomes, it could not rescue the hyper-bone resorbing activity and hypersensitivity to receptor activator of nuclear factor-kappaB ligand in ship(-/-) osteoclasts, suggesting that the localization of SHIP to podosomes per se was not sufficient and the SH2 domain was indispensable for its function. Cas and c-Cbl, known to function in podosomes of osteoclasts, were identified as novel proteins binding to the SHIP SH2 domain by mass spectrometric analysis, and this interaction appeared to be dependent on the Src kinase activity. These results demonstrate that c-Src enhances the translocation of SHIP to podosomes and regulates its function there through the SH2 domain, leading to an attenuation of bone resorption.

PKA implicated in the phosphorylation of Cx43 induced by stimulation with FSH in rat granulosa cells.

Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that follicle-stimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSH-induced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca2+-dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant C-terminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.