Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

''Cross-talk'' between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells.

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the -subunits of the stimulatory and inhibitory G-proteins of AC (G_s and G_i−2) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_s levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_s levels by more than 50% at all time-points. G_i−2 levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein -subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

Circuit for the electromanipulation of plant protoplasts

An electric circuit for plant protoplast manipulation is described. The circuit used readily available materials and was designed for use in teaching. This integrated circuit can be placed in a single small box with controls for the aligning voltage, the aligning frequency, the pulse voltage, and the pulse timing. The circuit can be supplied by any suitable source of dc power and can be easily altered for individual requirements. The circuit, as presented here, can be assembled for less than $250.     

Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro . By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis , we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.     

Should conservation strategies consider spatial generality? Farmland birds show regional not national patterns of habitat association

A key assumption underlying any management practice implemented to aid wildlife conservation is that it will have similar effects on target species across the range it is applied. However, this basic assumption is rarely tested. We show that predictors [nearly all associated with agri-environment scheme (AES) options known to affect European birds] had similar effects for 11 bird species on sites with differing farming practice (pastoral vs. mixed farming) or which differed in the density at which the species was found. However, predictors from sites in one geographical region tended to have different effects in other areas suggesting that AES options targeted at a regional scale are more likely to yield beneficial results for farmland birds than options applied uniformly in national schemes. Our study has broad implications for designing conservation strategies at an appropriate scale, which we discuss.     

Protein kinase A-mediated serine 35 phosphorylation dissociates histone H1.4 from mitotic chromosome

Global histone H1 phosphorylation correlates with cell cycle progression. However, the function of site-specific H1 variant phosphorylation remains unclear. Our mass spectrometry analysis revealed a novel N-terminal phosphorylation of the major H1 variant H1.4 at serine 35 (H1.4S35ph), which accumulates at mitosis immediately after H3 phosphorylation at serine 10. Protein kinase A (PKA) was found to be a kinase for H1.4S35. Importantly, Ser-35-phosphorylated H1.4 dissociates from mitotic chromatin. Moreover, H1.4S35A substitution mutant cannot efficiently rescue the mitotic defect following H1.4 depletion, and inhibition of PKA activity increases the mitotic chromatin compaction depending on H1.4. Our results not only indicate that PKA-mediated H1.4S35 phosphorylation dissociates H1.4 from mitotic chromatin but also suggest that this phosphorylation is necessary for specific mitotic functions.     

Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3

Summary Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of ³⁵⁹serine and ⁸³⁵serine of 14-3-3β binding sites to alanine, which slightly reduces the interaction with 14-3-3β, does not influence the PTPN3 effect. In contrast, mutation of the invariant ⁸⁴²cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3β, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3β, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression. En-Chi Hsu, Yen-Cheng Lin have equal contributions to this work.     

1alpha,25-Dihydroxyvitamin D3-induced down-regulation of the checkpoint proteins, Chk1 and Claspin, is mediated by the pocket proteins p107 and p130

A previous cDNA microarray analysis in murine MC3T3-E1 osteoblasts revealed a cluster of genes involved in cell cycle progression that was significantly down-regulated after a single treatment with 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] [L. Verlinden, G. Eelen, I. Beullens, M. Van Camp, P. Van Hummelen, K. Engelen, R. Van Hellemont, K. Marchal, B. De Moor, F. Foijer, H. Te Riele, M. Beullens, M. Bollen, C. Mathieu, R. Bouillon, A. Verstuyf, Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3, J. Biol. Chem. 280 (45) (2005) 37319-37330]. Among those genes were the DNA replication and DNA damage checkpoint proteins, Chk1 and Claspin, of which the human homologues were recently shown to be E2F-responsive. Quantitative real-time PCR experiments in 1,25(OH)(2)D(3)-treated MC3T3-E1 cells confirmed the down-regulation observed in the microarray experiment. Moreover, Chk1 and Claspin promoter activities were also reduced after incubation with 1,25(OH)(2)D(3), and this reduction was mediated through the E2F recognition motifs within their promoters because mutation of these motifs almost completely abolished the repressive effect of 1,25(OH)(2)D(3). The antiproliferative effect of 1,25(OH)(2)D(3) as well as its potential to down-regulate the expression of Chk1 and Claspin depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) lost its antiproliferative action and failed to repress these E2F-target genes in p107(-/-);p130(-/-)-cells, but not in pRb(-/-)-cells.