Lipocalin signaling controls unicellular tube development in the Caenorhabditis elegans excretory system

Unicellular tubes or capillaries composed of individual cells with a hollow lumen perform important physiological functions including fluid or gas transport and exchange. These tubes are thought to build intracellular lumina by polarized trafficking of apical membrane components, but the molecular signals that promote luminal growth and luminal connectivity between cells are poorly understood. Here we show that the lipocalin LPR-1 is required for luminal connectivity between two unicellular tubes in the Caenorhabditis elegans excretory (renal) system, the excretory duct cell and pore cell. Lipocalins are a large family of secreted proteins that transport lipophilic cargos and participate in intercellular signaling. lpr-1 is required at a time of rapid luminal growth, it is expressed by the duct, pore and surrounding cells, and it can function cell non-autonomously. These results reveal a novel signaling mechanism that controls unicellular tube formation, and provide a genetic model system for dissecting lipocalin signaling pathways.     

Molecular basis for an ancient partnership between prolyl isomerase Pin1 and phosphatase inhibitor-2

Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ～50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.     

Mitochondrial genome regulates mitotic fidelity by maintaining centrosomal homeostasis

Centrosomes direct spindle morphogenesis to assemble a bipolar mitotic apparatus to enable error-free chromosome segregation and preclude chromosomal instability (CIN). Amplified centrosomes, a hallmark of cancer cells, set the stage for CIN, which underlies malignant transformation and evolution of aggressive phenotypes. Several studies report CIN and a tumorigenic and/or aggressive transformation in mitochondrial DNA (mtDNA)-depleted cells. Although several nuclear-encoded proteins are implicated in centrosome duplication and spindle organization, the involvement of mtDNA encoded proteins in centrosome amplification (CA) remains elusive. Here we show that disruption of mitochondrial function by depletion of mtDNA induces robust CA and mitotic aberrations in osteosarcoma cells. We found that overexpression of Aurora A, Polo-like kinase 4 (PLK4), and Cyclin E was associated with emergence of amplified centrosomes. Supernumerary centrosomes in rho0 (mtDNA-depleted) cells resulted in multipolar mitoses bearing “real” centrosomes with paired centrioles at the multiple poles. This abnormal phenotype was recapitulated by inhibition of respiratory complex I in parental cells, suggesting a role for electron transport chain (ETC) in maintaining numeral centrosomal homeostasis. Furthermore, rho0 cells displayed a decreased proliferative capacity owing to a G₂/M arrest. Downregulation of nuclear-encoded p53 in rho0 cells underscores the importance of mitochondrial and nuclear genome crosstalk and may perhaps underlie the observed mitotic aberrations. By contrast, repletion of wild-type mtDNA in rho0 cells (cybrid) demonstrated a much lesser extent of CA and spindle multipolarity, suggesting partial restoration of centrosomal homeostasis. Our study provides compelling evidence to implicate the role of mitochondria in regulation of centrosome duplication, spindle architecture, and spindle pole integrity.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane

Phospholipid scramblase 1 (PLSCR1) is a Ca(2+)-binding, endofacial plasma membrane protein thought to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids that is observed upon influx of calcium into the cytosol. Expression of PLSCR1 is markedly induced by interferon and other cytokines, and PLSCR1-/- bone marrow cells exhibit defective myeloid proliferation and differentiation in response to stimulation by select growth factors, implying that PLSCR1 also functions in cytokine signaling or response pathways. PLSCR1 is multiply palmitoylated and partitions into membrane lipid raft domains. We have now identified the Cys-rich sequence (184)CCCPCC(189) in PLSCR1 as required for palmitoylation of the polypeptide. Mutation of these five cysteines abrogates PLSCR1 trafficking to the plasma membrane and results in virtually all of the expressed protein localizing to the nucleus. Consistent with this observation, cell treatment with the palmitoylation inhibitor, 2-bromo-palmitate, results in a marked redistribution of endogenous PLSCR1 from plasma membrane to nucleus. In a small percentage of untreated cells, predominantly nuclear localization of PLSCR1 is also observed. Furthermore, PLSCR1 is also found in the nucleus following its cytokine-induced expression. These data suggest that under the circumstance of rapid biosynthesis in response to gene induction by cytokines, PLSCR1 traffics into the nucleus, implying a potential nuclear function for this protein.     

Immunohistochemical localization of adenosine deaminase complexing protein in intestinal mucosa and in colorectal adenocarcinoma as a marker for tumour cell heterogeneity

Summary Adenosine deaminase complexing protein (ADCP), a dimeric glycoprotein, has been reported to be decreased or deficient in transformed or cancer-derived cell lines, indicating its potential significance as an indicator of malignant transformation. A similar deficiency was reported in total homogenates of tumours of colon, kidney, lung and liver. In previous biochemical studies we failed to confirm the consistent reduction in ADCP concentration in cancer tissues. A possible explanation for our findings was thought to be intercellular heterogeneity in ADCP expression in individual tumour cells. To study ADCP expression in individual cells we developed an immunohistochemical method which was applied to tissue sections. Paraformaldehyde-lysine-periodate (PLP) solution was found to be a suitable fixative. Fixed tissue samples were paraffin-embedded, sectioned and stained for ADCP, using an indirect peroxidase-labelled antibody procedure. The protein was localized in normal colonic mucosa, mainly in the brush border region of the luminal epithelium and in cytoplasmic granules. Intense ADCP immunoreactivity was found also in the basal part of some cells. In cancer cells, three staining patterns were observed: membranous, diffuse cytoplasmic and granular cytoplasmic. The adenocarcinomas exhibited significant intratumour and intertumour heterogeneity in their staining types.Further studies on ADCP expression in colorectal cancer in relation to clinical and histopathological characteristics are warranted in order to fully evaluate the potential significance of ADCP as a cancer associated antigen.     

Influence of zoysiagrass rhizosphere fungal isolates on growth and yield of soybean plants

Among 21 rhizosphere fungi tested, eight sterile fungi and oneTrichoderma isolate (GT2-1) from zoysiagrass rhizosphere promoted the overall growth of soybean varieties when grown in the greenhouse. Out of nine effective isolates, GS7-4, GS8-2, GS8-3, GU23-3 (all sterile fungi) and GT2-1 (Trichoderma sp.) promoted plant growth and increased yield of Toyosuzu (variety 1) significantly, while GS8-3, GS10-1, GS10-2 (sterile fungi), and GT2-1 significantly caused plant growth promotion and yield increase of Kitamusume (variety 2). Among these efficient isolates, GS8-3 and GS10-2 induced considerable and consistent increases in length, biomass and yield of plants of varieties 1 and 2, respectively. In the field, however, only GS8-3 and GU23-3 among seven selected isolates, induced consistent and significant increases in plant growth and yield of varieties 1 and 2, while the ability of other isolates decreased. The plant growth promotion by these isolates in the field followed a similar trend to that in the greenhouse, but the effect was less marked. Some isolates which were effective in the greenhouse were less effective in the field. The degree of growth promotion by different isolates depended on the variety of soybean. The nutrient condition of soils used in experiments also seemed to play a vital role, since notable growth promotion by these isolates was observed in nutrient-depleted soil.The author is grateful to Ministry of Education, Science and Culture, Japan, for financial assistance.