A network approach to migrants’ transnational biographies

This paper reviews social network analysis (SNA) as a method to be utilized in biographical research which is a novel contribution. We argue that applying SNA in the context of biography research through standardized data collection as well as visualization of networks can open up participants’ interpretations of relations throughout their lives, and allow a creative and innovative way of data collection that is responsive to participants’ own meanings and associations while allowing the researchers to conduct systematical data analysis. The paper discusses the analytical potential of SNA in biographical research, where the efficacy of this method is critically discussed, together with its limitations, and its potential within the context of biographical research

Chicken TLR3 polymorphic sites in different breeds.

<p>¹Aa = Amino acid</p><p>² BF = Beijing Fatty chicken, BW = Beijing White 939 chicken, HL = Hy-Linevarietybrown chicken, LB = Laiwu Black chicken, LH = Luhua chicken, NN3 = Nongda No.3 chicken, RY = Royal chicken, WL = White Leghorn chicken, WS = White-Feather Silky chicken, referred sequence = Gallus gallus TLR3 NM_001011691.</p><p>³ Synonymous (n = 6) and nonsynonymous (n = 7) substitution.</p><p>Chicken TLR3 polymorphic sites in different breeds.</p

Chicken TLR7 polymorphic sites in different breeds.

<p>¹Aa = Amino acid</p><p>² BF = Beijing Fatty chicken, BW = Beijing White 939 chicken, HL = Hy-Linevarietybrown chicken, LB = Laiwu Black chicken, LH = Luhua chicken, NN3 = Nongda No.3 chicken, RY = Royal chicken, WL = White Leghorn chicken, WS = White-Feather Silky chicken, referred sequence = Gallus gallus TLR7 NM 001011688.</p><p>³ Synonymous (n = 1) and nonsynonymous (n = 4) substitution.</p><p>Chicken TLR7 polymorphic sites in different breeds.</p

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex

<div><p>Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling–dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation–induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.</p></div

Schematic model of the negative regulation of amino acid signaling by the 4F2hc/Girdin complex.

<p><b>(A)</b> In the absence of the 4F2hc/Girdin complex, various growth factor and amino acid signaling pathways converge on mTORC1 to regulate its activity and cell metabolism. <b>(B)</b> In the presence of Girdin, it interacts with 4F2hc depending on MAPK activity and amino acid stimulation, which promotes endocytosis of 4F2hc into the lysosome, leading to changes in intracellular Gln, Leu, and mTORC1 activity. AA, amino acids; Girdin, girders of actin filaments; Gln, glutamine; Leu, leucine; MAPK, mitogen-activated protein kinase; mTORC1, mechanistic target of rapamycin complex 1; TSC, tuberous sclerosis; Ub, ubiquitin; 4F2hc, 4F2 heavy chain.</p

Girdin promotes 4F2hc translocation to the lysosomes.

<p><b>(A)</b> In-house-developed 4F2hc antibody was used for IF in HeLa cells and 293FT cells; staining intensity was markedly reduced in 4F2hc knockdown cells. <b>(B)</b> In-house-developed 4F2hc antibody was used for IP in HeLa cells, and the sample was detected by commercial 4F2hc antibody to verify the quality of the antibody. <b>(C)</b> Flp-In 293 cells stably transduced with the indicated plasmids were starved for amino acids for 1 h and stimulated with amino acids for 30 min, followed by immunofluorescence staining for 4F2hc (green) and Lamp1 (red). Arrowheads indicate the localization of 4F2hc in the lysosome. <b>(D)</b> Quantification of the cells shown in <b>(C)</b> with 4F2hc localized on the lysosome (100 cells from three independent experiments). The data are presented as means ± SEs. *<i>P</i> < 0.05. The data underlying this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s002" target="_blank">S1 Data</a>. <b>(E)</b> Cell surface proteins of HeLa cells transfected with the indicated siRNAs or plasmids were biotinylated and isolated, followed by WB with the indicated antibodies. <b>(F)</b> Quantification of the bands intensity of cell surface 4F2hc from 3 independent experiments is shown. The data are presented as means ± SEs. *<i>P</i> < 0.05. The value of the surface 4F2hc in the cells transfected with empty vector or control siRNA were set as 100. The data underlying this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s002" target="_blank">S1 Data</a>. Girdin, girders of actin filaments; IgG, immunoglobulin G; <i>Mr</i>, molecular marker; N.S., not significant; siRNA; small interfering RNA; WB, western blot; WT, wild-type; 4F2hc, 4F2 heavy chain.</p

Girdin negatively regulates amino acid signaling through decreasing intracellular Gln and Leu contents.

<p><b>(A)</b> 293FT cells starved for amino acids for 1 h were stimulated with total amino acids (AA+) for 10 min. Amino acid concentrations were measured with an HPLC system. Quantification of each amino acid concentration from 3 independent experiments is shown; the data are presented as means ± SEs. *<i>P</i> < 0.05. The data underlying this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s002" target="_blank">S1 Data</a>. <b>(B, C)</b> 293FT cells transfected with indicated siRNA <b>(B)</b> or Flp-in 293 cells stably expressing empty vector, Girdin WT, Girdin AA, and 4F2hc <b>(C)</b> were starved for amino acids for 1 h and stimulated with total amino acids for 10 min. Quantification of each amino acid concentration from 3 independent experiments is shown; the data are presented as means ± SEs. *<i>P</i> < 0.05. The values in control cells (control siRNA or empty vector) starved for amino acids were set as 1. The data underlying this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s002" target="_blank">S1 Data</a>. <b>(D, E)</b> 293FT cells transduced with control or Girdin shRNA were starved for 1 h in amino acid–free medium and stimulated with Gln <b>(D)</b> or Leu <b>(E)</b> for the indicated times, followed by WB. Note that the activation of mTORC1 was observed at 60 min in Gln stimulated cells <b>(D)</b>. AA, amino acids; Arg, arginine; A.U., arbitrary unit; Gln, glutamine; Leu, leucine; Girdin, girders of actin filaments; <i>Mr</i>, molecular marker; mTORC1, mechanistic target of rapamycin complex 1; N.S., not significant; shRNA, short hairpin RNA; siRNA, small interfering RNA; WB, western blot; WT, wild-type; 4F2hc, 4F2 heavy chain.</p

Phosphorylation of Girdin is required for its interaction with 4F2hc.

<p><b>(A)</b> Amino acid sequences of Girdins from different species. Conserved serines at positions 233 (red) and 237 (green) that conform to the MAPK substrate motif are highlighted. <b>(B)</b> Endogenous interaction between MAPK1/2 and Girdin, as detected by co-IP in 293FT cells. <b>(C)</b> In vitro kinase assay showing that MAPK phosphorylates recombinant Girdin NT but not its mutants. <b>(D)</b> Lysates from 293FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. <b>(E)</b> Lysates from 293FT cells stimulated with FBS in the presence or absence of U0126 were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit. The phosphopeptides were analyzed by mass spectrometry. The mass spectrum of a tryptic fragment at m/z 843.3431 (mass error, 0.46 ppm) that matched to the peptide 219-DGLHFLPHASSSAQpSPCGpSPGMK-241 containing phosphorylated S233 and S237 is shown. This phosphopeptide was detected with a high peptide confidence (false discovery rate of less than 1%) in cells stimulated with FBS (Mascot score, 28; expectation value, 0.431) but not in cells pretreated with U0126. <b>(F)</b> 293FT cells were transfected with the indicated plasmids, followed by co-IP, showing that mutation of MAPK phosphorylation sites in Girdin disrupts 4F2hc/Girdin interaction. <b>(G)</b> 293FT cells starved for serum were pretreated with DMSO or U0126 for 30 min and stimulated with DMEM containing 10% FBS for 30 min, followed by co-IP (left panel). 293FT cells were transfected with indicated plasmids, followed by co-IP to test endogenous 4F2hc/Girdin interaction (right panel). co-IP, co-immunoprecipitation; CT, carboxyl terminal; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum; Flag-NT, Flag-tagged Girdin NT domain; GFP, green fluorescent protein; Girdin, girders of actin filaments; GST, glutathione S-transferase; IgG, immunoglobulin G; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK CA, constitutively active mutant of MAPKK; <i>Mr</i>, molecular marker; NT, amino terminal; <i>Rf</i>, relative mobility; S233, serine at position 233; S237, serine at position 237; WT, wild-type; 4F2hc, 4F2 heavy chain.</p

4F2hc/Girdin complex is modulated by ubiquitination of 4F2hc.

<p><b>(A and B)</b> 293FT cells transfected with the indicated plasmids were starved for amino acids for 1 h in amino acid–free medium supplemented with dialyzed FBS, followed by stimulation with complete growth medium for 30 min. Ubiquitinated proteins were isolated with glutathione-sepharose beads under a non-denaturing condition <b>(A)</b> or Ni-NTA agarose beads under a denaturing condition <b>(B)</b> to detect 4F2hc ubiquitination. <b>(C)</b> Primary structures of 4F2hc (short and long isoforms) and its mutants used in the study. <b>(D)</b> Lysates from 293FT cells transfected with the indicated plasmids were pulled down under denaturing conditions using Ni-NTA agarose beads, showing the ubiquitination of the transfected 4F2hc and its mutants. <b>(E)</b> 293FT cells transfected with the indicated combinations of 4F2hc, GST, and GST-NT. The lysates were pulled down with glutathione beads, followed by WB with the indicated antibodies. a.a., amino acids; FBS, fetal bovine serum; Girdin, girders of actin filaments; GST, glutathione S-transferase; His-Ub, histidine-tagged Ub; <i>Mr</i>, molecular marker; TUBE, tandem-repeated ubiquitin-binding entity; WB, western blot; WT, wild-type; 4F2hc, 4F2 heavy chain.</p

Girdin regulates amino acid stimulation–induced mTORC1 activation through interaction with 4F2hc.

<p><b>(A and B)</b> 293FT cells were transfected or transduced with the indicated siRNA or shRNA, respectively, followed by WB to monitor mTORC1 activity. In blot images for S6K1 and pS6K1, the lower bands represent S6K1 or pS6K1, as indicated by arrowhead or asterisk, respectively. Quantitative data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s003" target="_blank">S1A and S1B Fig</a>. <b>(C)</b> Basal mTORC1 activity in control Flp-In 293 cells and cells stably overexpressing Girdin WT, Girdin AA, and 4F2hc. Quantitative data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s003" target="_blank">S1C Fig</a>. <b>(D)</b> Brain sections from heterogeneous (left) and Girdin knockout (right) P14 mice were stained for pS6. The brown staining indicates pS6 signal. The regions within the black boxes are shown below at a higher magnification. <b>(E, F, G)</b> 293FT cells <b>(E)</b>, primary mouse embryonic fibroblasts isolated from WT and Girdin-deficient mice <b>(F)</b>, or Flp-In 293 cells stably transduced with the indicated constructs <b>(F)</b> were starved for amino acids for 1 h in medium supplemented with dialyzed FBS, followed by stimulation with complete medium for the indicated time. mTORC1 activity and Girdin expression were monitored by WB. Quantitative data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005090#pbio.2005090.s003" target="_blank">S1G–S1I Fig</a>. FBS, fetal bovine serum; FH, Flag-HA epitope; Girdin, girders of actin filaments; HP, hippocampus; <i>Mr</i>, molecular marker; mTORC1, mechanistic target of rapamycin complex 1; OB, olfactory bulb; RMS, rostral migratory stream; shRNA, short hairpin RNA; siRNA, small interfering RNA; S6K1, S6 kinase beta1; WB, western blot; WT, wild-type; 4F2hc, 4F2 heavy chain.</p