Its canonical function is to accelerate the intrinsic GTPase activity of heterotrimeric G-protein α subunits at the plasma membrane, thereby limiting heterotrimeric G-protein signaling. A unique, non-canonical function of RGS13 occurs following translocation to the nucleus, where it represses CREB transcriptional activity. The functional role of RGS13 in GC B cells is unknown. To create a surrogate marker for Rgs13 expression and a loss of function mutation, we inserted a GFP coding region into the Rgs13 genomic locus. Following immunization GFP expression rapidly increased in activated B cells, persisted in GC B cells, but declined in newly generated memory B and plasma cells. Intravital microscopy of the inguinal lymph node (LN) of immunized mice revealed the rapid appearance of GFP+ cells at LN interfollicular regions and along the T/B cell borders, and eventually within GCs. Analysis of WT, knock-in, and mixed chimeric mice indicated that RGS13 constrains extra-follicular plasma cell generation, GC size, and GC B cell numbers. Analysis of select cell cycle and GC specific genes disclosed an aberrant gene expression profile in the Rgs13 deficient GC B cells. These results indicate that RGS13, likely acting at cell membranes and in nuclei, helps coordinate key decision points during the expansion and differentiation of naive B cells.Germinal centers (GCs) are microanatomic structures that develop in secondary lymphoid organs in response to antigenic stimulation. Within GCs B cells clonally expand and their immunoglobulin genes undergo class switch recombination and somatic hypermutation. Transcriptional profiling has identified a number of genes that are prominently expressed in GC B cells. Among them is R The mechanisms controlling the directed migration of B cells between these GC zones and eventually out of GCs remain largely enigmatic. A model of GC B cell migration based on differential chemoattractant receptor signaling requires a rapid decline in B cell chemokine sensitivity following zonal transition to maintain discrete dark and light zones During a T cell dependent antibody response the engagement of the B cell antigen receptor by cognate antigen initiates an activation program that prepares naïve B cells to receive T cell help i subfamily of heterotrimeric G-proteins as signal transducers i subunit GDP-GTP exchange, Gαi dissociation from Gβγ, downstream effector activation, and directed migration. Since Gαi subunits possess an intrinsic GTPase activity, GTP hydrolysis facilitates re-assembly of heterotrimeric G-protein causing signaling to cease. By dramatically accelerating the intrinsic GTPase activity of Gαi subunits, RGS proteins reduce the duration that Gαi subunits remains GTP bound, thereby decreasing effector activation i, would provide a mechanism to control the sensitivity of GC B cells to chemoattractants.The sensitivity of B cells to chemokines can be rapidly modulated by two basic mechanisms: uncoupling the receptor from second messengers or by attenuating second messenger signaling RGS13 expression in a human B cell line enhanced the magnitude and duration of chemokine receptor signaling while overexpression led to the opposite phenotype Rgs13 is also expressed by mast cells and similar to the results with B cells, a mast cell line knock-down enhanced chemoattractant signaling 2+ signaling promotes the translocation of RGS13 into the nucleus where it binds phosphorylated CREB and core binding protein (CBP)/p300. This reduces CREB mediated transcription One RGS protein prominently expressed by GC B-lymphocytes and lymphomas of a GC origin is RGS13 Rgs13 expression in GC B cell chemotaxis and GC zoning, and to determine the impact of the loss of RGS13 inhibition of CREB mediated transcription in B cells, we generated mice in which the Rgs13 coding region was replaced with green fluorescent protein (GFP). Using these mice we carefully documented Rgs13 expression using GFP as a surrogate marker both in vivo using intravital microscopy and in vitro using flow cytometry. We examined the consequences of a lack of RGS13 on GC organization, GC B cell chemotaxis, and T cell dependent antibody responses. We found an expanded mucosal GC compartment and an exuberant antigen induced splenic GC response populated by B cells that exhibited a surprisingly altered gene expression profile. The implications of our results are discussed.To examine the functional importance of Rgs13GFP KI mouse was generated by replacing exon 3 of the Rgs13 gene with the coding region for enhanced GFP gene (aPep3b/BoyJ) mice were purchased from Jackson Laboratory . The mice were housed under specific pathogen-free conditions. All procedures were approved by and performed in accordance with the guidelines from the Institutional Animal Care Committee at the National Institutes of Health. When Jackson C57/BL6 mice were used as controls they were age and sex matched to the KI mice and co-housed for a minimum of two weeks. Littermate controls were used to verify germinal center formation and all studies involving Peyer's patches otherwise co-housed C57Bl/6 mice were used as controls.The GFP gene . The micSingle cells were re-suspended in PBS plus 2% FBS and stained with fluorochrome-conjugated or biotinylated antibodies against B220 (RA3-6B2), IgD (11-26c-2a), IgM (R6-60.2), CD24 (M1/69), CD3 (145-2C11), CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7), CD11c (HL3), CD11b (M1/70), CD138 (281-2), CD19 (1D3), CD38 (90), IgG1 (X56), CD93 (AA4.1), BP-1 (6C3), GL-7 , CD95 (Jo2), CD21 (4E3), CD23 (B3B4), CD43 (S7), CD184 , CXCR5 (2G8), CCR7 (4B12), CD69 (H1.2F3), CD86 (GL1), CD279 , CD45.1 (A20), CD45.2 (104) . Biotin-labeled antibodies were visualized with fluorochrome-conjugated streptavidin (eBioscience). LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit  was used in all experiments to exclude dead cells. Data acquisition was done on FACSCanto II (BD) flow cytometer and analyzed with FlowJo software . The absolute number of live cells in tissues was estimated as follows: total cell count  =  (detected live gate singlet cell number) × /(detected flow time (sec)) x dilution factor. Flow rate of FACS CANTOII is 60 µl/min at medium speed.The cell proliferation studies were performed using the cell Proliferation Dye eFluor® 450 (eBioscience) in a standard dye dilution assay. Purified B cells were stimulated with various combinations of the indicated reagents: 1 μg/ml CD40 , 1 μM terbutaline, 10 nM prostaglandin E2 (PGE2) , recombinant BAFF (100 ng/ml), IL-4 (10 ng/ml), IL-17 (20 ng/ml), and IL-21 (20 ng/ml) for 72 h. All the recombinant proteins were from R&D Systems, Minneapolis MN). Data acquisition was done on FACSCanto II (BD) flow cytometer. Proliferation indexes were calculated using FlowJo software (Tree Star), which is defined as the total number of divisions divided by the number of cells that went into division, assuming no cell death.+GL7+, CD95+CD38− and either CD45.1+ (WT) or CD45.2+ (KI) by FACSAria flow cytometer (Becton Dickinson). The CD45.2 GC B cells were greater than 90% GFP positive.Mixed chimeric mice were made by reconstituting irradiated CD45.1 mice with a 1∶1 mix of bone marrow from C57BL/6 CD45.1 mice (WT) and from KI (CD45.2). The success of each bone marrow engraftment was checked by sampling blood of the recipient mice 28 days later. No obvious bias was noted between the different sources of bone marrow cells in their ability to reconstitute the mice was noted. The mice were used 6–8 weeks after reconstitution. The mixed chimeric mice were immunized with sRBCs via intraperitoneal injection and 8–10 days later the splenocytes were isolated and immunostained with GL7, CD45.1, CD45.2, CD95, CD38, and B220 Abs. The cells were sorted for B220Murine CCL19, CXCL12, and CXCL13 were purchased from R&D Systems. Cells were purified from spleen, LN, and Peyer's patches using standard protocols and immunostained for B220, GL7, CD38 and CD95 (spleen and Peyer's patch) and with B220 and IgD (LN). Migration assays were performed using a Transwell chamber , as previously described 3-BSA or TNP34-BSA (Biosearch Technology) overnight at 4°C, washed and blocked with 5% BSA fraction V , serum titers were then added to the plates, and the plates incubated 4 h at 4°C. After washing alkaline phosphatase-labeled goat anti mouse Ig isotype specific antibodies were added for 2 h at RT . After washing, PNPP one component substrate (SouthernBiotech) was used to detect the amount of secondary antibody bound. For the ELISPOT analysis a standard protocol was used. Briefly, various dilutions of single cell suspensions prepared from bone marrow, spleen, and blood were cultured in plates previously coated with TNP-BSA or goat antibodies specific for mouse Ig. The cells were incubated for 3 h at 37°C and then washed with Tween/PBS to remove the cells. Next the plates were incubated with alkaline phosphatase-labeled goat anti-mouse isotype specific antibodies for 2 hours, washed, and nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate were used to detect the ELISPOTs. ELISPOTs were counted manually and in some instances quantitated with an ELISPOT reader .WT and KI mice were immunized with either sRBCs or TNP-KLH. For the sRBC immunizations 200 µl of 10% solution of sRBCs  was given by intraperitoneal injection. In some instances TNP-KLH  mixed with Imject® Alum  was introduced to mice (100 μg) via intraperitoneal injection or injected intradermally (50 μg). Mice were boosted with same dose of antigen at the indicated days along with Alum. Serum TNP specific Ig levels in these mice were analyzed by ELISA. Briefly, 96 well ELISA plates  were coated with TNP2 alkaline phosphatase were purchased (Jackson ImmunoResearch Laboratories). Anti-Ki67  was from Abcam, and anti-RGS13 rabbit polyclonal was previously described Freshly isolated spleens from mice were snap frozen in Tissue-Tek OCT compound . Frozen OCT splenic sections (7 μm) were acetone fixed for 2 min, and dried at room temperature. Slides were rehydrated in Tris-buffered saline (TBS) and stained in a humidified chamber in TBS/0.1% BSA/1% mouse serum overnight at 4°C or 1 h. For immunohistochemistry primary antibodies included hamster anti-mouse CD3 , rat anti-mouse IgD , rat anti-mouse CD35 , rat anti-mouse CD45R/B220  all from BD Pharmingen. Donkey anti-mouse IgG (H+L) alkaline phosphatase and goat anti-mouse IgM F(ab')Mta3, Creb1, Crebbp, EP300, Stk11, Cdkn1b, Bcl6, Smarca2, Trp53, Prdm1, Rgs2 were from Qiagen . Real-time PCR was performed using a 7500 Real-Time PCR System  following the Rotor-Gene SYBR Green PCR kit  protocol. The Rgs13 and Rgs1 primers/probes were Mn00450170_m1 & Mn00462629_m1  and used on the 7500 Real Time PCR System (Applied Biosystems). The endogenous control was Gapdh detected using a VIC/MGB Probe . The results were normalized to the expression levels of the GAPDH reference gene and the relative mRNA levels were calculated using the 2−ΔΔCt method. Quantitative PCR of mouseRNA was isolated with TRIZOL Reagent  according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg RNA with Omniscript RT Kit . Some of the real-time PCR primers used to amplify genes are listed below while those for Crtc2 Product length 71 bp: Forward primer GGCCTTCGAGGAGGTGATGTATAAGCCAGTCGCAGTTTTTGGReverse primer Ccnb2 Product length 475 bp: Forward primer GGAAGAAACTGCAGCTGGTCGCGATGAACTTGGTACGGTTReverse primer Ccna2 Product length 285 bp: Forward primer AGAGGCAGCCAGACATCACTGCTCCATTCTCAGAACCTGCReverse primer Aicda Product length 285 bp: Forward primer GATGGATGCCAACACGGTTAAACAAAGCGTCATTTCCTTGCCACGGTCReverse primer Gcet2 Product length 375 bp: Forward primer ATGGGGAACTGTTTGCAGAGATCTGTGGAAGGTAGGGGCT.Reverse primer 3VO4 with Complete and PhosStop  protease and phosphatase inhibitor cocktail tablets. The cell lysates were centrifuged at 20,000×g for 10 min at 4°C, and equivalent amounts of protein were loaded onto 10% NuPage Bis-Tris gels (Invitrogen). After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membrane was first incubated in Tris-buffered saline-Tween (TBST)  with 5% milk for 1 hour. The membrane was washed once with TBST, and the first antibody was added to the membrane in TBST containing 5% milk overnight at 4°C. The membrane was washed four times for 10 min with TBST, incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, washed six times for 10 min in TBST, and subjected to enhanced chemiluminence, which was detected with HyBlot CL film .A polyclonal antibody against RGS13 has been previously described 2HC (Molecular probes) or eFluor® 670 (eBioscience). 10–30 million labeled cells of each population in 200 ml of PBS were adoptively transferred by tail vein injection into 6∼10-week-old recipient mice. After anesthetizing the mice by intraperitoneal injection of Avertin , the skin and fatty tissue over inguinal LN were removed. The mouse was placed in a pre-warmed coverglass chamber slide . The chamber slide was then placed into the temperature control chamber on the Leica SP5 microscope. The temperature of air was monitored and maintained at 37.0±0.5°C. Inguinal LN was intravitally imaged from the capsule over a range of depths (10–220 µm). Two-photon imaging was performed with a Leica SP5 inverted 5 channel confocal microscope (Leica Microsystems) equipped with 20× multi-immersion objective, 0.7 NA (immersion medium used 80% glycerol) or 25× water dipping objective, 0.95 NA (immersion medium used distilled water). Two-photon excitation was provided by a Mai Tai Ti:Sapphire laser (Spectra Physics) with a 10 W pump, tuned to 810 or 910 nm. Emitted fluorescence was collected using a 4 channel non-descanned detector. Wavelength separation was through a dichroic mirror at 560 nm and then separated again through a dichroic mirror at 495 nm followed by 460/50 nm emission filter for second harmonics or CMF2HC; 525/50 emission filter for CMFDA (Molecular probes) and GFP; a dichroic mirror at 650 nm followed by 610/60 nm emission filter for CMTMR and phycoerythrin conjugated antibodies; and the eFluor® 670 signal was collected by 680/50 nm emission filter. Sequences of image stacks were transformed into volume-rendered four-dimensional videos using Imaris software v.7.5.0 or v.7.5.2 (Bitplane), and the spot analysis was used for semi-automated tracking of cell motility in three dimensions.Inguinal LNs were prepared for intravital microscopy as described In vitro results represent mean values of quadruplicate samples. SEM and p values were calculated with the Mann-Whitney test or unpaired t tests using Microsoft Excel 2011 or GraphPad Version 5 Prism software.In vivo results represent samples from 3 to 6 mice per experimental group. Rgs13 is expressed in mouse GCs Rgs13 expression in sorted B220+IgD+, B220+IgD−, and B220 positive cells from the spleens of mice immunized 8 days previously with sheep RBCs , Aicda (.985) and GM600 (.983). Mybl1 and GM600 are also strongly expressed in GC B cells. Despite the strong GC B expression of Rgs13, examination of the promoter regions of both the human and mouse Rgs13 genes did not identify an obvious set of transcription factor binding sites that might account for its GC B cell expression .Previous results had indicated that s sRBCs, . Using qpulation . When wepulation . Among tRgs13 for B cell function, we generated mice in which the Rgs13 coding sequence was replaced with that for green fluorescent protein (GFP). The gene targeting strategy is shown . Immunostaining Peyer's patches from WT and KI mice for RGS13 demonstrated immunoreactivity in the GC region in WT but not in KI mice . Some weaker GFP positive cells were found among the B220+CD38+ subset  already exhibited a full GC phenotype. Among these GC B cells 70% expressed GFP. The B220+CD38+GL7+ population had increased although the % of GFP+ cells in that subset had declined (+ cells had a full GC phenotype and 68% were GFP+. In the B220+CD38+GL7+ subset 28% expressed GFP (+CD38+GL7+ subset 38% were GFP+ (highCD83low) and light zone (CXCR4lowCD83high) B cells expressed similar levels of GFP  immunization with sRBCs. Prior to immunization only 0.2% of the cells in the lymphocyte gate from KI mice expressed GFP and all were B220s of GFP .Rgs13 has been reported to be expressed in murine memory B cells Prdm1) is known to repress its expression Rgs13 we checked GFP expression in switched B cells and CD138+ cells at D5, D11, and D30 post immunization. A minority of day 5 B220+CD38+IgG1+ cells expressed GFP while 50% of similar cells at day 11 did. Nearly all the B220+CD38−IgG1+ cells at D11 post immunization expressed GFP. However, by D30 the B220+CD38+IgG1 positive cells lacked GFP expression indicating that long term switched memory B cells do not express high levels of Rgs13 ; non-GC memory cell and GC precursors (B220+CD38+GL7+); GC B cells (B220+CD38−CD95+GL7+); early switched memory B cells (B220+CD38+IgG1+); early plasma cells (B220+CD138+); and mature plasma cells (B220−CD5−CD138+) in the spleens of WT and KI mice immunized with sRBCs  or both WT B and T cells (D2 and D4). The WT B and T cells helped delineated the follicle and T cell zone in the LN, respectively. Prior to immunization few if any GFP+ cells were visible near or in the LN follicle (data not shown). However even D1 post-immunization several clusters of GFP positive cells were easily discernible at the T-B cell border . However, upon sRBC immunization the GCs in the spleen sections prepared from the KI mice were noticeably larger than those observed in the sections from WT mice . GCs wer+ cells had a GC phenotype while 7% of the B220+ KI cells did so . Together these results indicate that RGS13 helps organize GC morphology and limits the size of germinal centers.While C57/BL6 mice from a clean mouse facility have relatively few spontaneous GCs in their spleens, constitutive GC formation occurs in Peyer's patches and mesenteric LNs as a result of continuous B cell stimulation by commensal bacteria. Examining the Peyer's Patches from the WT and KI mice revealed more GC B cells in the KI versus the WT . FollowiRgs13 expression affected murine GC B cell chemotaxis we prepared B cells from spleens of immunized WT and KI mice and tested the cells in standard chemotaxis assays using different concentrations of CXCL12, CXCL13, and CCL19. In contrast to our expectation we found no increased chemotaxis of the KI versus the WT splenic GC B cells . The loss of Rgs13 did not improve LN GC B or CD4 T cell responsiveness either . Nevertheless, we tested whether we could discern a difference in the in vitro proliferative potential of the WT and KI B cells using a panel of different proliferative signals. Dye loaded WT and KI B cells were cultured with different inductive signals and the amount of dye dilution monitored 4 and 6 days later. A representative example of KI and WT B cells stimulated with CD40 and IL-21 is shown , extracted RNA, and compared gene expression by quantitative RT-PCR. The results are shown as a ratio normalized to Gapdh expression. Relative to WT GC B cells the KI GC B cells expressed significantly higher levels of several GC specific and cell cycle related genes and less of Prdm1 and the cell cycle inhibitor Cdkn1b . It appears that the induction of GFP in the Rgs13GFP KI B cells serves as an early marker of B cells that have interacted with T cells.While expression of GFP in the KI mouse GC B cells mirrored that expected based on Rgs13 is not induced by antigen receptor signaling it may not impact the initial EBI2 and CCR7 dependent localization of activated B cells to the T-B border and interfollicular sites. At these sites the activated B cells form long lasting conjugates with cognate CD4 T cells, which results in extensive B cell proliferation and shape subsequent B cell fate decisions. Imaging the KI mice after immunization revealed an expanding knot of GFP positive cells that expanded over the first 3 days post-immunization. In WT mice RGS13 expression in these activated and proliferating B cells should lessen Gαi signaling. While G-protein signaling affects cell fate decisions in the central nervous system and contributes to asymmetric cell divisions in model organisms Rgs13 expression normally limits the expansion of activated B cells at the T-B cell border. In its absence precursors expand more rapidly generating additional cells that eventually enter into these different differentiation pathways.Since +CD38+GL7+ cells at the D3/4 post immunization in the KI mice at D2 only 15% of these cells co-expressed GFP and at D5 40%. Rather at the early time points post-immunization many of the GFP+ cells remained in the B220+CD38+GL7− gate. These early GFP positive CD38+GL7+ and CD38+GL7− B cells expressed an intermediate level of GFP, less than the typical germinal center B cell found later in the immunized KI mice. At D4/5 post immunization we noted a significant expansion in the numbers of B220+CD38+IgG1+ cells in both the WT and KI mice although 2–3 fold more in the KI mouse. Yet again only about 20% of these cells were GFP+. Whether some of these cells had briefly expressed GFP or never expressed GFP needs to be resolved. We are developing a new mouse strain where the Rgs13 coding region has been replaced with the Cre recombinase to address this question as well as others. Long term switched memory B cells expressed GFP at levels similar to those present in naïve B cells.Both a GC dependent and GC independent pathway can generate memory B cells following immunization. The GC independent pathway rapidly generates IgM and switched memory cells, which consistent with their non-GC origin have relatively un-mutated antigen receptors. These cells arise from activated, naive B precursors located at the T-B border that co-express CD38 and GL7 +CD38+GL7+ cells also serve as precursors for GC B cells as they return from the interfollicular region to the center of the LN follicle or splenic B cell area to establish a nascent GC. Our intravital microscopy identified KI GFP positive cells moving from the interfollicular zone into the LN follicle and then expanding within the follicle. By day 11 post immunization most of the KI GC B cells and CD38− IgG1 switched B cells expressed GFP. Although nearly 90% of centrocytes and centroblasts expressed high levels of GFP 10% of both populations lacked GFP expression. Since dark zone and light zone B cells largely remain confined to their respective regions and only occasionally move between zones, a decrease in RGS13 expression might be predicted to augment responsiveness to a CXCL12 or CXCL13 gradient facilitating cycling between the zones. In the KI mice the lack of RGS13 might perturb the cycling of cells between zones as both centroblasts and centrocytes would remain sensitive to chemokines. This is predicted to alter GC zoning in vitro evidence to support a heightened sensitivity of the Rgs13 deficient GC B cells to chemokines. This failure may be secondary to intrinsic problems in the in vitro chemotaxis assays as well as the tendency of GC B cells to undergo apoptosis ex vivo. We did note that the KI GC B cells that lacked GFP expression had a heightened response to chemokines compared to those that expressed GFP. As GC B cells express other RGS proteins there could be some compensation for the loss of RGS13. In fact the expression of Rgs1 and Rgs2, both located close to the Rgs13 locus on chromosome 1, were increased in GC B cells from the KI mice. It will be of interest to examine the GC zoning in mice with a Gnai2 G184S KI, which interferes with the binding of all RGS proteins to Gαi2B220+GFP+ and B220+GFP− cells from KI mice D8-10 following immunization, and from B220+ B cells from unimmunized WT mice, we extracted RNA, and performed microarrays. The genes known to be upregulated in GC B cells all appeared in the KI GFP positive cells. In addition, the cell cycle specific genes were upregulated only in the B220+GFP+ cells while the gene expression profile of the B220+GFP− KI B cells from immunized mice resembled the WT B220+ cells from unimmunized mice. Thus, GFP expression in the KI animals marked nearly all the proliferating B cells at D8-10 days post immunization (unpublished observations). Because of difficulties in comparing GC B cells from different WT and KI animals, we created bone marrow chimeric mice and sorted the GC B cells following immunization. Based on the quantitative PCR analysis of RNA extracted from the genetically distinct cells we found lower levels of Prdm1, which promotes B cell differentiation, and Cdkn1b, a known cell cycle inhibitor in the KI GC B cells as compared to WT GC B cells. In addition, we found higher levels of the several GC specific genes, cell cycle specific genes, and a number of CREB related and CREB target genes.Besides the early increase in GC B cell precursors in the KI mice, the numbers of splenic GC B cells increased more rapidly than they did in the WT mice. Furthermore the KI mice had more GC B cells than did WT mice in their Peyer's patches and mesenteric lymph nodes, sites of ongoing immune reactivity to commensal bacterial products. The more robust GC response in the spleen to an exogenous antigen the ongoing exaggerated GC response at mucosal sites seemed less likely to be explained by heightened heterotrimeric G-protein signaling and more likely as a consequence of RGS13's role in the cell nucleus to limit CREB signaling. To investigate the exaggerated GC response in the KI mice, we initially sorted B2202+ and cAMP signaling can promote RGS13 accumulation in the nucleus, where it forms a complex with phosphorylated CREB and CBP/p300, which suppresses CREB-mediated gene expression Mta3, Aicda, and Smarca4 are target genes of CREB/CRTC2 in human GC B cells A subset of CREB target genes have emerged as important regulators of B cells within human GCs Rgs13GFP KI mice along with other engineered mice should allow a better understanding of how RGS13 and other RGS proteins accomplish this. While we did not note any frank autoimmunity or increased incidence of lymphoma in the KI mice crossing these mice onto different genetic backgrounds with a predilection towards autoimmunity or lymphomagenesis should be of interest.Together these data indicate that RGS13 has several roles in B cells. It limits the initial expansion of recently activated B cells and the expansion of GC B cells. It likely does so in part by altering CREB signaling. In addition, it along with other RGS proteins help to coordinate the responsiveness of recently activated and GC B cells to chemoattractants. The Figure S1Construction of the Rgs13GFP KI mouse and the loss of Rgs13 expression. A. Schematics of Rgs13 genomic, targeted, and Cre deleted loci. B. Southern blot demonstrating the Rgs13 targeting and Cre mediated deletion of the Neomycin gene. C. Standard RT-PCR examining the expression of Rgs1, Rgs10, Rgs13, and Rgs19 in immunized splenic B cells or Peyer's Patch B cells from WT and KI mice.(TIF)Click here for additional data file.Figure S2Initial assessment of cell populations in Rgs13GFP KI mice. A. Flow cytometric analysis of B cell development in WT and KI bone marrow. The percentage of GFP expression in the various subsets in the KI cells is indicated. The analysis was performed on bone marrow from 4 WT versus 4 KI animals. Data is mean ± SEM and statistics from unpaired t tests. B. Coulter counter and blood smear analysis of blood from 10 WT and 10 KI animals. C. Flow cytometric analysis of B cell subsets in the spleen of WT and KI mice. The percentage of GFP expression in the various subsets in the KI cells is indicated. The analysis was performed on spleens from 4 WT versus 4 KI animals. Data is mean ± SEM and statistics from unpaired t tests. D. Flow cytometric analysis of B1 and B2 B cells in the peritoneum of WT and KI mice. The percentage of GFP expression in the various subsets in the KI cells is indicated. The analysis was performed on peritoneal cells from 4 WT versus 4 KI animals. Data is mean ± SEM and statistics from unpaired t tests.(TIF)Click here for additional data file.Figure S3Follicular helper T cells do not express high levels of GFP in the Rgs13GFP KI mice. A. Flow cytometric analysis of the number of follicular helper cells in the spleen and Peyer's patches of WT and KI animals. The % of follicular helper T cells (CD4+B220−PD-1+CXCR5+) in the CD4 gate from the analysis of cells from the spleens and Peyer's patches of sRBC immunized animals either 11 or 30 days post immunization. Analysis is from 4 WT versus 4 KI animals. Data is mean ± SEM and statistics from unpaired t tests. B. Representative flow cytometry plots examining the expression of GFP in follicular helper T cells from the D11 immunized spleen, mesenteric LN, and Peyer's patches. C. Flow cytometric analysis of the number of follicular helper T cells in mixed bone marrow chimeras derived from either WT or KI bone marrow. Cells obtained from 4 chimeric mice at 11 days post sRBC immunization. Data is mean ± SEM. No statistical difference was noted.(TIF)Click here for additional data file.Video S1Intravital microscopy of the inguinal lymph node from a Rgs13GFP KI mouse 1D post-immunization identifies GFP positive cells located at the edge of B cell follicle. Shown is the distribution and behavior of GFP expressing KI cells in the inguinal lymph node of a KI mouse. Images were taken at D1 post immunization via an intra-peritoneal injection of sRBCs. An image sequence of an 80 μm z- projection was acquired with 20× lens over 30 minutes. From shallow (top left) to deep (low right), each panel is from a 80 μm volume image stack at 10 μm intervals moving from the LN surface deeper into the lymph node cortex. The capsule of the lymph nodes was visualized by second harmonic signal (blue) from collagen. Adoptively transferred B cells labeled with CMTMR are shown in red. The GFP signals are shown in the green channel. White lines indicate edge of the B-cell follicle, which was based on the distribution of adoptively transferred WT B-cells. The scale bar is 200 μm. Time counter is h:min:sec.(MOV)Click here for additional data file.Video S2Intravital microscopy of the inguinal lymph node from a Rgs13GFP KI mouse 2D post immunization identifies an expanding population of GFP positive cells located at the edge of B cell follicle. Shown is the distribution and behavior of GFP expressing KI cells in the inguinal lymph node of a KI mouse. Images were taken at D2 post immunization via intra-peritoneal injection of sRBCs. An image sequence of an 80 μm z- projection was acquired with 20× lens over 30 minutes. From shallow (top left) to deep (low right), each panel is from a 80 μm volume image stack at 10 μm intervals moving from the LN surface deeper into the lymph node cortex. The capsule of the lymph nodes was visualized by second harmonic signal (blue) from collagen. Adoptively transferred B cells labeled with eFluor® 670 are shown in white. The follicular dendritic cell (FDC) network (red) was visualized by means of subcutaneous injection of anti-CD21/35 PE conjugated antibody (3 μg) prior to imaging. GFP signals are shown in the green channel. White lines indicate edge of the B-cell follicle based on distribution of WT B cells adoptively transferred 1D prior to imaging. The scale bar is 150 μm. Time counter is h:min:sec.(MOV)Click here for additional data file.Video S3Intravital microscopy of the inguinal lymph node from a Rgs13GFP KI mouse 8D post immunization identifies a large population of GFP positive cells residing in a germinal center located in the LN follicle. Shown is the distribution and behavior of GFP expressing KI cells in the LN follicle of the inguinal lymph node of a KI mouse. Images were taken D8 post immunization via intra-peritoneal injection of sRBCs. An image sequence of an 80 μm z- projection was acquired with 25x lens over 30 minutes. From shallow (top left) to deep (low right), each panel is from the 80 μm volume image stack at 10 μm intervals moving from the LN surface deeper into the lymph node cortex. The capsule of the lymph nodes was visualized by second harmonic signal (blue) from collagen. Adoptively transferred B cells labeled with CMF2HC are shown in blue. The follicular dendritic cell (FDC) network (red) was visualized by means of subcutaneous injection of anti-CD21/35 PE conjugated antibody (3 μg) prior to imaging. GFP signals are in the green channel. White lines indicate edge of the B-cell follicle, and were drawn based on distribution of WT B cells adoptively transferred 1D prior to imaging. The scale bar is 200 μm. Time counter is h:min:sec.(MOV)Click here for additional data file.


S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation. Protein S-acylation  is a common post-translational modification covalently linking long-chain fatty acids  to cysteine thiols in proteins via a labile thioester bond, which can be highly dynamic et al. identified and validated 35 proteins out of 58 novel candidate yeast palmitoylated proteins et al. extended this method to study whole rat brain, embryonic rat neurons or rat synaptosomes, confirming 21 novel palmitoylated proteins out of >200 candidates The recent development of palmitoyl-protein enrichment methods has significantly advanced the study of palmitoylated proteins. Acyl-biotin exchange (ABE) in vitro with a tag such as biotin A second method utilizes the metabolic incorporation of a bioorthogonal palmitate analog into endogenous sites of palmitoylation, which may be subsequently conjugated Given the diversity of palmitoylated proteins identified in each of these experiments, and the growing understanding of protein palmitoylation in immune regulation, we sought to identify novel palmitoylated proteins in B lymphocytes. Palmitoylated proteins from immortalized B lymphoid cells were enriched using ABE chemistry, and samples prepared with or without hydroxylamine treatment were compared by a semi-quantitative differential proteomic analysis based on spectral counting In order to identify potentially novel palmitoylated proteins from human B lymphoid cells, ABE experiments were performed following the protocol described by Drisdel and Green Putative palmitoylated proteins were partitioned into two groups: proteins exclusively identified in specific (HA+) samples, and proteins common to both control (HA−) and HA+ samples. Data from the first group were filtered to proteins with at least 5 spectral counts, which led to the identification of 57 proteins. This group includes 16 known palmitoylated proteins, 30 proteins annotated as candidates in previous proteomic studies Altogether, we identified 95 putative palmitoylated proteins, out of which 46 are MHC molecules and 38 have been annotated as candidates in previous palmitoyl-proteome studies The main goal of this study was to identify B cell novel palmitoylated proteins. Among the proteins specifically enriched in the HA+ samples, we focused on two immune regulators, CD20 and CD23. CD20 was identified by 5 peptides (39 MS2 spectra), corresponding to 4 different sequences covering 11.3% of the protein sequence . CD20 wa81, Cys111 and Cys220) are candidate sites of CD20 palmitoylation. Cys81 is predicted to be embedded within the first or the second transmembrane domain, Cys111 reside between the second and the third transmembrane domains in the cytoplasmic loop and Cys220 is in the C-terminal intracellular tail CD20 is a non-glycosylated four-pass transmembrane protein with a large extracellular loop located between the third and fourth transmembrane segments 17 and Cys18), which are likely sites of palmitoylation. These cysteines are juxtaposed to the predicted cytoplasmic/transmembrane domain boundary and surrounded by clusters of hydrophobic and positively charged amino-acids, characteristics that are often found in transmembrane palmitoylated proteins CD23 is a single-pass type II integral membrane protein  which is6 epitope. Additionally, in order to identify the palmitoylated cysteines in CD20 and CD23, we generated single point mutations in CD20  and CD23 (C17A and C18A). As aberrant palmitoylation frequently occurs after mutating a cysteine in a context of neighbouring cysteines In order to validate the palmitoylation of CD20 and CD23, we developed a heterologous expression system, where palmitoylation of these specific candidates was assessed by transient transfection of plasmids encoding CD20 and CD23 tagged with a C-terminal (His)6-tagged species  of CD23 did not reduce the incorporation of 17-ODYA, but the double mutant (C17A/C18A) completely abrogated the observed labeling. Therefore, CD23 is palmitoylated on either Cys17 and/or Cys18.Mutational analysis also demonstrated that the C81A and C111A mutants of CD20, but not the C220A mutant, incorporated equivalent amounts of 17-ODYA, relative to their expression levels. The double mutant C111A/C220A completely eliminated 17-ODYA incorporation. Accordingly, CD20 is palmitoylated on its intracytoplasmic cysteines CysIn order to identify novel palmitoylated proteins in human B lymphocytes, we used the ABE method Given the overlap with previous efforts to profile palmitoylated proteins in immune cells, we focused our interest on two proteins involved in the immune response, CD20 and CD23, which were both identified unambiguously as candidate palmitoylated proteins in the present study. Interestingly, CD20 and CD23 are both effective or promising targets for immunotherapy of haematological malignancies, autoimmune diseases or allergy disorders 111 belongs to the intracellular loop between the second and the third transmembrane domains, while Cys220 is located in the C-terminal tail. Despite their distance in the primary sequence, they could nevertheless be in close proximity in the folded protein and behave like vicinal cysteines in these palmitoylation experiments. Our observation that the C220A mutation was more effective in reducing CD20 palmitoylation than did the C111A one, indicative of Cys220 modification, was corroborated by previously published data. Indeed, the presence of post-translational modifications in the C-terminal region, such as phosphorylation or acylation, had been proposed, as the observed molecular weight of a CD20 C-terminal fragment resulting from protease digestion of intact cells was found to significantly exceed its expected size , protein stability, protein-protein interactions or cellular signaling Protein palmitoylation is a key regulatory mechanism for numerous cellular processes that include membrane targeting, segregation to membrane microdomains  was obtained from Cayman. The Colloidal Blue solution (Biosafe) and the BCA assay were from BioRad. The modified trypsin (sequencing grade) was purchased from Promega. Formic acid was obtained from Aristar.RPMI-GlutamaxI, penicillin-steptomycin-glutamine, and Dynabeads M-280 streptavidin were obtained from Invitrogen. FBS was from Invitrogen or Gemini. DMEM (High Glucose) and D-PBS were purchased from Mediatech.A human Epstein-Barr virus-transformed B lymphoid cell line was established in the lab from a donor whose parents had given written informed consent for the establishment and storage of the cell line, according to the protocols approved by the ethics review board of the Toulouse Hospital. Research was performed in compliance with the Declaration of Helsinki. Cells were grown in RPMI-GlutaMaxI containing 10% FBS.6 B cells according to Wan et al.6 cells/mL , and finally broken by 4 cycles of freezing/thawing before sonication. Total membranes were recovered by high-speed centrifugation  and resolubilized in 50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1.7% Triton X-100, 10 mM N-ethylmaleimide (NEM), protease inhibitors. Then, proteins were extracted by methanol-chloroform precipitation. Resolubilized proteins were incubated successively with NEM for blockade of free thiols, and with hydroxylamine (HA+ sample) to release the thioester-linked palmitate and restore the modified cysteines to thiols. Formerly thioester-containing  proteins were biotinylated using a thiol-reactive biotinylation reagent (EZ-Link biotin-HPDP) and finally captured on streptavidin-coated magnetic beads (Dynabeads M-280 streptavidin). Specifically bound proteins were eluted by β-mercaptoethanol reduction of the protein-biotin disulfide linkage. An essential control of non-specific biotinylation and binding to the streptavidin matrix consisted in replacing hydroxylamine by 50 mM Hepes, pH 7.4, in half of the NEM-treated sample (HA− sample).Palmitoylated proteins were purified from 200×10HEK293T cells were cultured in DMEM (High Glucose) supplemented with 10% FBS and 1% penicillin-steptomycin-glutamine. Cells were transiently transfected using purified plasmid DNA and Fugene HD transfection reagent. Twelve hours after transfection, cells were labeled with 20 µM 17-ODYA for 8 hours, washed twice with D-PBS, and then harvested and frozen at −80°C. Cell pellets were later sonicated in D-PBS and separated into soluble and insoluble fractions by ultracentrifugation for 45 minutes at 100 000× g. The insoluble pellet was resuspended by sonication in D-PBS, and then quantified using the BCA protein assay using a microplate reader. Cell lysates were normalized to 1 mg/mL. To 100 µL of each lysate, click chemistry reagents were added at room temperature to yield a final concentration of 20 µM rhodamine-azide, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), 100 µM Tris[methyl]amine (TBTA), and 1 mM CuSO4 in PBS. After 1 hour, half of the sample (50 µL) was transferred to a fresh tube and mixed with 1 M hydroxylamine and boiled for 5 minutes. All samples were mixed with 4× SDS sample loading buffer and 2/3 (33 µg) of the sample was loaded on a 10% SDS-PAGE gel, separated over 850 volt-hours, and analyzed using a Hitachi FMBIO-II flatbed fluorescence scanner. Gels were then transferred to nitrocellulose (100 volt-hours), blocked with 5% non-fat dry milk in Tris-buffered saline Tween-20 buffer (TBST), incubated overnight in 2% bovine serum albumin with 1∶2000 mouse anti-His (C-term) (Invitrogen) in TBST, washed, and incubated with 1∶10 000 goat anti-mouse IRDye 800CW (LiCOR) for 1 hour. After washing, the membrane was imaged using an Odyssey Imager (LiCOR).ABE purified proteins were separated on SDS-polyacrylamide gels . They were revealed either by silver staining for analytical purpose or by colloidal blue staining for mass spectrometry analysis.(6) sequence were purchased as pDEST51-based plasmids . The CD20 cDNA was amplified by PCR from human B cells cDNA, with the following primers: 5′-ATTGGATCCGCCGCCATGACAACACCCAGAAATTC-3′ and 5′-GCGCTCGAGAGGAGAGCTGTCATTTTC-3′, and cloned into pcDNA3.1/myc-His (Invitrogen) at the BamHI and XhoI sites to generate pcDNA3.1-CD20-myc-His.cDNAs encoding CD23a and flotillin-1 tagged with a C-terminal V5-HisNdeI and XbaI sites at both steps. The CD23 insert was transferred by use of the Gateway technology (Invitrogen) into a pENTR221 vector, subjected to directed mutagenesis and returned to the pDEST51 Gateway vector. The primers used for generating the mutations were the following: 5′-GGATCTATGCACCCATCGCTGTGACTGTGTGGTACC-3′ and 5′-GGTACCACACAGTCACAGCGATGGGTGCATAGATCC-3′ for CD20 C81A; 5′-CGGAGAAAAACTCCAGGAAGGCTTTGGTCAAAGGAAAAATG-3′ and 5′-CATTTTTCCTTTGACCAAAGCCTTCCTGGAGTTTTTCTCCG-3′ for CD20 C111A; 5′-GAGAATGAATGGAAAAGAACGGCCTCCAGACCCAAATCTAAC-3′ and 5′-GTTAGATTTGGGTCTGGAGGCCGTTCTTTTCCATTCATTCTC-3′ for CD20 C220A; 5′-TCCCAGGAGGCGGGCTTGCAGGCGTGGG-3′ and 5′-CCCACGCCTGCAAGCCCGCCTCCTGGGA-3′ for CD23 C17A; 5′-CCAGGAGGCGGTGTGCCAGGCGTGGGACT-3′ and 5′-AGTCCCACGCCTGGCACACCGCCTCCTGG-3′ for CD23 C18A; 5′-GCTTCCCAGGAGGCGGGCTGCCAGGCGTGGGACT C-3′ and 5′-GAGTCCCACGCCTGGCAGCCCGCCTCCTGGGAAGC-3′ for CD23 C17A/C18A.Cys-to-Ala point mutations were generated by site-directed mutagenesis , according to the manufacturer instructions. The CD20 insert was first transferred into the pUC18 vector for directed mutagenesis, and then the mutated insert was returned back to the pcDNA3.1/myc-His vector, using All constructs were checked by DNA sequencing (Cogenics).4HCO3 (solution A) for 15 min followed by 50% (v/v) acetonitrile containing 25 mM NH4HCO3 (solution B) for 15 min. Gel pieces were dehydrated with 100% acetonitrile and then incubated with 7% H2O2 for 15 min before being washed again with solutions A and B as previously. 0.15 µg of modified trypsin in solution A was added to the dehydrated gel bands for an overnight incubation at 37°C. Peptides were then extracted from gel pieces in three 15 min sequential extraction steps in 30 µL of 50% acetonitrile, 30 µL of 5% formic acid and finally 30 µL of 100% acetonitrile. The pooled supernatants were finally dried under vacuum.Protein bands covering the entire migration zone of the SDS-polyacrylamide gel were systematically and manually excised and washed several times by incubation in 25 mM NHThe dried extracted peptides were resuspended in 4% acetonitrile and 0.5% trifluoroacetic acid and analyzed by online nanoLC-MS/MS . The nanoLC method consisted in a 40 min gradient ranging from 5% to 55% acetronitrile in 0.1% formic acid at a flow rate of 300 nL/min. Peptides were sampled on a 300 µm×5 mm PepMap C18 precolumn and separated on a 75 µm×150 mm C18 column . MS and MS/MS data were acquired using Xcalibur (Thermo Fischer Scientific) and processed automatically using Mascot Daemon software .Homo sapiens taxonomy, 92 013 entries), the corresponding reverse sequences and classical contaminants . ESI-TRAP was chosen as the instrument, trypsin as the enzyme and 2 missed cleavages allowed. Precursor and fragment mass error tolerance were set respectively at 10 ppm and 0.6 Da. Peptide modifications allowed during the search were: acetyl (N-ter), dioxidation (M), oxidation (M) and trioxidation (C). Proteins identified with a minimum of 1 peptide and with a score higher than the identity threshold  were automatically validated using IRMa http://www.ebi.ac.uk/pride) under accession number 20197 Consecutive searches against a homemade polypeptide sequence database were performed for each sample using Mascot 2.3. The search database consisted in the concatenation of the SwissProt-Trembl database  or identified as candidates in previous proteomic screens (“Proteomics”) are indicated along with the corresponding reference. The list of references is below the table. The spectral index has been calculated as described in Fu et al. :845–854). NA (not applicable): a spectral index is calculated only for proteins that are present in both HA+ and HA− samples. For the proteins that are not identified in one category of samples (HA+ or HA−), the averaged normalized spectral count of this sample is considered as equal to 0. Sc: spectral count; nsc: normalized spectral count . Exp1 to 3 correspond to the three biological replicates.(XLS)Click here for additional data file.Table S2Detail of the distribution of the proteins meeting selection criteria. HA+ only : proteins identified in HA+ samples exclusively. Common proteins: proteins identified in both HA+ and HA− samples. P-value: p-value related to the spectral index analysis.(XLS)Click here for additional data file.Table S3Candidate palmitoylated proteins. Proteins meeting our criteria of potential palmitoylation are listed. They include well-established palmitoylated proteins («Established»), proteins previously identified as candidates in proteomic screens («Proteomics»), and novel candidates («Novel»). They are classified according to their associated biological process as indicated in the Human Protein Reference Database and manually reviewed. NA (not applicable): no SpI was calculated for proteins identified exclusively in HA+ samples.(XLS)Click here for additional data file.


The marine annelid Platynereis dumerilii has become a model system for evo-devo, neurobiology and marine biology. The functional assessment of its cell types, however, has so far been very limited. Here we report on the establishment of a generally applicable, cell type specific ablation technique to overcome this restriction. Using a transgenic strain expressing the bacterial enzyme nitroreductase (ntr) under the control of the worm’s r-opsin1 locus, we show that the demarcated photoreceptor cells can be specifically ablated by the addition of the prodrug metronidazole (mtz). TUNEL staining indicates that ntr expressing cells undergo apoptotic cell death. As we used a transgenic strain co-expressing ntr with enhanced green fluorescent protein (egfp) coding sequence, we were able to validate the ablation of photoreceptors not only in fixed tissue, using r-opsin1 riboprobes, but also by monitoring eGFP+ cells in live animals. The specificity of the ablation was demonstrated by the normal presence of the eye pigment cells, as well as of neuronal markers expressed in other cells of the brain, such as phc2, tyrosine hydroxylase and brn1/2/4. Additional analyses of the position of DAPI stained nuclei, the brain’s overall neuronal scaffold, as well as the positions and projections of serotonergic neurons further confirmed that mtz treatment did not induce general abnormalities in the worm’s brain. As the prodrug is administered by adding it to the water, targeted ablation of specific cell types can be achieved throughout the life of the animal. We show that ablation conditions need to be adjusted to the size of the worms, likely due to differences in the penetration of the prodrug, and establish ablation conditions for worms containing 10 to 55 segments. Our results establish mtz/ntr mediated conditional cell ablation as a powerful functional tool in Platynereis. Platynereisdumerilii has become a reference system in the fields of evolution and development, as well as in comparative neuroscience. It has retained a more ancestral-type gene repertoire than conventional invertebrate molecular model systems [Platynereis cells can be specifically ablated by cold laser nanosurgery [The marine bristle worm  systems , a featu systems , the hyp systems  and eye  systems  reveal t systems ,6. The fosurgery , this tentr expressing cells, whereas the adjacent bystander cells are unaffected [Nitroreductase (ntr)-mediated, spatially and temporally controlled cell ablation, has been pioneered in zebrafish, and since used as a functional tool for the analyses of various tissues, such as pancreatic, heart and germ cells, as well as neuron types -10. For affected .Platynereisdumerilii. For establishment, we used a transgenic strain that expresses both egfp and ntr, under the control of the recently described r-opsin1 enhancer specifically in the worm’s r-opsin1+ photoreceptor cells (PRCs) [r-opsin1+ photoreceptors, but does not affect directly adjacent cell types, such as the pigment cells of the Platynereis adult eyes or other neuronal cell types in the worms’ brains, including their projections. Using this tool, we were able to ablate the r-opsin1+ photoreceptors from a broad range of stages. Our work establishes the ntr/mtz system as a powerful technique to determine the functional requirement of specific cell types throughout the life of the bristle worm.Here we report on the establishment of space and time controlled Ntr-mediated cell ablation in the marine bristle worm s (PRCs) . AdminisAll animal work was conducted according to Austrian and European guidelines for animal research.Platynereisdumerilii were raised and bred in MFPL marine facility according to established protocols [rotocols .nitroreductase was PCR amplified from pNTR-EGFP (gift of Lazaro Centanin from Jochen Wittbrodt’s lab) adding the ribosomal skip site F2A, a stop codon, a BamHI restriction site at 5’ and a XhoI restriction site at 3’ using the following primers:The bacterial GGATCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCAAGCTTATGGATATCATTTCTGTCGCCTT; NTR-Stop-XhoI-rev: GTCACTCGAGGAGCTCCACCGCGGTGACTAGTAGTATCGATACGTCGACTTACACTTCGGTTAAGGTGATGTTTTGNTR-F2A-BamHI-fwd: The fragment was cloned into the p3E-EGFPpA vector  at the BGAAAGGTCAGCCTTCTGTCGCCTACAACCACCAGCTTACCATGTCTCGGTCAGAGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGr-opsin1_upperGFPfusion: GGAAATTTTAAGGTTACATTTCAGCAAATAAGTGGGAAGAAACAAAGCGAAAAGACTTACGGAAAAAACCTCCCACACCTCCCCr-opsin1_lowerE2N: r-opsin1 locus [frkt318 as described in reference [and recombined to the first exon of a previously identified BAC containing the n1 locus . An 8kbpeference   in a 1:1000 dilution in 2.5% sheep serum/1xPTW for overnight at 4°C. Subsequently, specimens were washed in 1xPTW for 4x15min and equilibrated in 0.1MTris/Hcl (pH 8.2) for 15min. Fast red staining reactions were performed using Fast Red tablets (Roche) dissolved in 0.1M TrisHCl (pH 8.2) according to manufacturer’s recommendations. After staining, specimens were washed twice in 1xPTW to stop staining and incubated in 0.1M glycine/HCl (pH2.2)/0.1% Tween 20 for 30min to inactivate the alkaline phosphatase. After washing 4x in 1xPTW, specimens were blocked for 1h in 5% sheep serum /1xPTW followed by incubation with anti-DIG-AP Fab fragment (Roche) in a 1:2000 dilution in 2.5% sheep serum /1xPTW for overnight at 4°C. Specimens were washed 5x10min in 1xPTW and NBT-BCIP staining was performed.Co-localization and levels of protocol  with min2-EDTA and 20 mg FeCl3 in 100 ml of distilled water. Stock solution 2: 200 mg H3BO4 and 20 mg Na 2MoO 4 in 50 ml of distilled water are added to 50 ml containing 230 mg ZnSO 4, 65 mg MnSO4, 0.6 mg CoSO4, 0.1 mg CuSO 4. Stock solution 3: 2.2 g KBr, 2 mg KI, 0.6 mg LiCl, 6 mg RbCl, 380 mg SrCl2, 3 mg AlCl3 consecutively dissolved in 100 ml of distilled water. Stock solution 4: 10 g NaNO3 and 2 g Na2HPO4 in 100 ml of distilled water. Stock solution 5: 2 g Sodiumsilicate in 100 ml of distilled waterMetronidazole  was dissolved in 0.2% DMSO in artificial seawater with vigorous shaking and protected from ambient light. Artificial seawater (ASW) was prepared in accordance with ref . : 10l AWe tested concentrations between 7-25mM mtz. Worms containing 10-20, 25-35 and 45-55 segments were treated with freshly prepared mtz or DMSO control dilutions, respectively, at 18±1°C in the dark for the time indicated. After treatment, the mtz containing or DMSO control medium was replaced by several washes with artificial seawater. Worms were subsequently maintained under normal culture conditions  .Platynereis worms (45-55 segmented) were anesthetized in 50:50 7.5M MgCl2: Natural Sea Water (NSW) and subsequently decapitated. The heads were fixed with 4% PFA/PTW for 20min, subsequently incubated in 15% sucrose/phosphate-buffered saline (PBS) for two hours and in 30% sucrose/PBS overnight at 4°C. We used Tissue-Tek (O.C.T. Compound) to embed the specimens in molds and froze them in dry ice. A MicromHM500 OM Cryostat was used for obtaining 10µm thick sections, which were collected on cryosection-optimized coated glass slides . The sections were stored immediately at -20°C.Ablated and control adult Tissue sections were re-hydrated for 2x5 min in 1xPTW and immediately blocked in 5% (vol/vol) sheep serum in 1xPTW for 30 min at room temperature. Polyclonal rabbit anti-5-HT serotonin antiserum  and monoclonal mouse anti-acetylated tubulin antibody  were diluted in PBS/ 5% sheep serum, and incubated with the sections overnight at 4°C in a wet box covered with parafilm strips. Slides were washed 4x5mins in 1xPTW and further incubated with Cy3-coupled goat anti-rabbit antiserum and Alexa fluor 488-coupled goat anti-mouse antiserum  overnight at 4°C. Nuclei were stained by DAPI . Slides were washed with 1xPTW for 4x5mins and mounted in Prolong Gold antifade reagent (Invitrogen Cat No. P36934).Platynereis (45-55 segmented) worms were anesthetized and decapitated after ~28 hours treatment. The heads were fixed and mounted in Tissue Tek compound as described in the cryosectioning protocol. The TUNEL apoptotic cell death assay was performed by using the In Situ Cell death Detection Kit, TMR Red (Roche Cat No. 12156792910) immediately after sectioning without storage. The sections were fixed with 4% PFA/PBS for 20 min and equilibrated in 1xPBS for 30 min. Slides were incubated in freshly prepared permeabilization solution (0.1% Triton X-100 in 0.1% Sodium citrate) for 2 mins on ice. Slides were washed immediately with 1xPBS twice and re-suspended in TUNEL reaction mixture for 60mins at 37°C in a dark, humidified chamber. After reaction, the slides were washed in 1xPBS for 3x5 min and mounted in Prolong Gold antifade reagent (Invitrogen Cat No. P36934).Mtz- and DMSO-treated adult 2: natural seawater (NSW) and mounted on glass slides. Worms were covered with the same MgCl2:NSW mixture. Several layers of sticky tape (Tesa) served as spacer to avoid squashing. The eGFP expression in live worms was captured with a Zeiss Axioplan2 microscope.For live imaging, premature adult worms of the desired genotype were anesthetized in 50:50 mixture of 7.5M MgClhttp://imagej.nih.gov/ij/), to generate maximum intensity z-projections of scans.Head images were taken with a 20x air objective, the tail images with a 40x oil immersion objective. Confocal images for sections were taken using a Zeiss LSM 710 confocal microscope with 405, 488 and 561-nm excitation wavelength. Sections were mounted in Prolong Gold antifade reagent. Images were processed using the ImageJ software package  using antisense riboprobes against ntr and egfp. As expected, the spatial expression of ntr in transgenic worms was identical to egfp expression in the r-opsin1+ photoreceptors of the worm . After 24hrs, 48hrs and 72hrs, we transferred the worms to plain seawater and checked for the pattern and cellular morphology of eGFP+ cells. DMSO-treated control worms never showed any effects on the number and morphology of eGFP- fluorescent cells . In accordance with the determined optimal concentrations of mtz, we treated worms with 25mM mtz. When analyzed after 28hrs, a well-detectable TUNEL staining was detectable in the areas of eGFP expression, such as the photoreceptors of the adult eyes  staining. We therefore established TUNEL detection in premature adult ult eyes . Untreatult eyes . These dPlatynereis ropsin1 endogenously expressed in the transgenically labeled cells [r-opsin1 expression in the adult eyes. No r-opsin1 mRNA can be detected in the adult eyes after mtz treatment (17mM mtz based on segment number) of r-opsin1::egfp-f2a-ntr animals  using a riboprobe against ed cells . Worms wr-opsin1 mRNA expression – along with the presence of a TUNEL signal, provide strong evidence that mtz incubation leads to apoptosis of nitroreductase expressing cells in Platynereis.Taken together, the analyses of two independent markers – eGFP fluorescence and Platynereis, we next investigated if cell ablation was restricted to the ntr expressing cells, or caused more general defects. We tested this by three independent approaches.In order to assess the specificity of cell ablation in Platynereis. In these eyes, r-opsin1+ photoreceptor cells are directly adjacent to pigment cells [First, we focused again on the large adult eyes of nt cells . If the ropsin1+ photoreceptors. We visualized the cells expressing Pdu-prohormone convertase 2 (phc2) a prohormone processing enzyme, demarcating neurosecretory cells [Pdu-brn1/2/4 a pou-class transcription factor [Pdu- tyrosine hydroxylase (th) [r-opsin1 mRNA in the same animals with the red Fast Red substrate . In contrast, phc2, brn1/2/4 and th staining was indistinguishable in mtz treated vs. untreated or DMSO treated animals  showed no such difference in any of the treatments  versus GFP- (and hence NTR-) cells. Such quantification is difficult to perform with the pigment cells of the adult eyes, as they are very densely packed and thus difficult to count reliably. Therefore, we performed WMISH with three different genes, whose mRNA is present in cells located in different areas in the head, but not in the ry cells , Pdu-brnase (th)  with the blue in ; S1A-C.  blue in ; S1D. ByThird, we assessed, if the axonal and dendritic scaffold of the worms is affected by the mtz treatment. Neurite connections are highly stereotypic and have thus been used as alignment grid for the comparison of expression patterns -4. If mtntr, and can therefore be used as tool to study specific cell functions in Platynereisdumerilii.Taken together, these data strongly suggest that NTR/mtz-mediated cell ablations are confined to the cells expressing Platynereisdumerilii. This technique now provides a tool to test the function of cell types that were previously not accessible. As the system is inducible, any given cell type can first function normally during the development of the animals, excluding indirect developmental defects prior to the time of assay. Currently, the major limitation for its use in Platynereisdumerilii is the availability of cell type specific enhancer constructs.Here we report on the establishment of a technique to specifically ablate cell types in the marine bristle worm Platynereisdumerilii, we noted two main points that are likely of general relevance for the usage of this technique in other animal systems. First, we noted a dependence of the effectiveness of the treatment on the age (size) of the animals, and likely also on the expression levels of nitroreductase within the targeted cells. This suggests that the exact concentrations and incubation time with mtz should be cross-checked for different animal stages and new expression constructs. Secondly, it has recently been suggested that mtz can induce general DNA damage in Drosophila cells [ntr expressing cell population. This makes mtz induced ablation of transgenically ntr expressing cells a useful tool to obtain a functional understanding of Platynereisdumerilii cell types.Whereas mtz- mediated cell ablation has been used in well-established molecular model systems, most prominently in zebrafish, with great success, its use in less conventional molecular animal systems – common in the field of evolutionary development – has so far not been explored. We therefore anticipate that this study will also be useful for the establishment of mtz-mediated cell ablation in other model systems. From our establishment of the system in a cells . The resFigure S1brn1/2/4 in control vs. mtz-treated animals.Expression of Related to brn1/2/4 (blue), counterstained with r-opsin1 (red), white dotted circle indicates major expression domain of brn1/2/4, used for quantification. (A’-C’) Fluorescent visualization of the Fast Red precipitate, used for r-opsin1 expression detection. Dorsal views, anterior down(A-C) WMISH of brn1/2/4 cells in fixed animals (can only be counted once).Blue staining at the very anterior of the head (out of focus in A) is unspecific background staining caused by probe trapping in gland cells. (D) Quantification of cell numbers in mtz- treated vs. control worms. Green bars: number of GFP+ cells in live animals . Black bars: number of p<0.0001; ns. - no statistically significant difference. The two-tailed paired Student t-test was used for statistical analyses. Scale bar: 50µm.Details of treatment conditions and quantification as described for (TIF)Click here for additional data file.


Antimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane.Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles.We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity. Ribosomally-made cationic antimicrobial peptides (AMPs) are the first line of innate defense of almost all living organisms against microbial pathogens ++) through salt bridges neutralizing the repulsive forces of adjacent LPS molecules. Translocation of AMPs across the OM has been proposed to occur by a ‘self-promoted’ uptake mechanism involving displacement of Mg++ cations by the cationic AMPs and the formation of ionic interactions with the negatively charged LPS layer The membrane-AMP interactions are initiated by an ionic bonding between the positively charged residues of AMPs and the anionic head groups of the microbial membrane phospholipids .This initial binding event may lead to an insertion of the hydrophobic residues of AMPs into the non-polar core of membranes Escherichia coli O111:B4 induces self-associations of TA  Temporins are a group of short AMPs (8 to 14 amino acid long) found in skin secretion of frogs E. coli 0111:B4 LPS, used here, was estimated to be ∼ 1.3 to 1.6 µM 2 proton resonances of residue P3 are non-degenerate and show NOEs with the NH resonance of residue L2  e.g. CαH/CαH of G6/G11, CαH/CαH P3/G11, in the anti-parallel dimeric structures, however, corresponding NOE cross-peaks involving these protons were not detectable in tr-NOESY spectra. Thus, it is likely that TA may form an open helical dimer in LPS micelles employing residues from the N- and C-termini  towards human cells make them as attractive templates for the generation of new antibiotics NMR structures of TL and TA have been previously determined in SDS and DPC micelle solutions which are considered to be good models of anionic  and zwitterionic (eukaryotic) membranes, respectively TM . E. coli 0111:B4 LPS, trichloroacetic acid extracted, was purchased from Sigma. The molecular mass of LPS was considered to be 10 KD TA was commercially synthesized by GLBiochem2O at pH 4.5 with peptide concentration of 0.5 mM. A series of 1-D 1H NMR spectra of TA were recorded with various concentrations of LPS, ranging from 2-10 µM, 2-D tr-NOESY experiments were performed, at 0.5 mM of peptide concentration, in the presence of 5 µM LPS, with four different mixing time: 50, 100, 150, and 200 ms. Spin systems of the peptide were assigned by 2-D TOCSY spectra (mixing time: 80 ms).All NMR experiments were performed on a BRUKER DRX 600 spectrometer equipped with a cryo-probe and pulse field gradients. NMR data were collected at 298 K using DSS  as an internal standard (0.0 ppm). NMR spectra were processed with Topspin software (BRUKER) and analyzed using SPARKY  running on a Linux workstation. Two-dimensional NOESY spectra (mixing time:150 ms) of free TA were acquired in an aqueous solution containing 10% D2O and pH was adjusted to 4.5. STD experiments were performed at 298 K in the presence of 5.4 µM LPS using standard STD pulse sequences 1 and 80 transients using a MLEV-17 spin lock field of 80 ms. The relaxation delay was fixed to 2.1 s. 40 selective Gaussian 270 pulses with duration of 50 ms were applied to achieve saturation transfer.For Saturation transfer difference (STD) experiments, peptide TA (0.5 mM) and LPS samples, were prepared in DAn ensemble of monomeric helical structures of TA was determined using the CYANA program Figure S1Conformations of TA in free solution. A section of 2-D NOESY spectrum of TA in aqueous solution, pH 4.5, 298 K.(TIF)Click here for additional data file.Figure S2Interactions of TA with LPS micelles. Low field region of 1-D NMR spectra of TA in free solution (lower panel), in 3 µM LPS (middle panel) and in 5 µM LPS (top panel) in aqueous solution, pH 4.5, 298 K. The amide proton resonances (7.9–8.4) and aromatic ring proton resonances of F1 are marked. Other resonances in 7.4–6.9 ppm are arising from sidechain of Arg7 and amide groups of the C-terminus.(TIF)Click here for additional data file.Figure S3Interactions of TA with LPS micelles by STD. A section of 2-D STD-TOCSY spectrum of TA in LPS micelles showing STD effect for ring protons of the F1 residue.(TIF)Click here for additional data file.