Despite being among the most celebrated taxa from Cambrian biotas, anomalocaridids (order Radiodonta) have provoked intense debate about their affinities within the moulting-animal clade that includes Arthropoda. Current alternatives identify anomalocaridids as either stem-group euarthropods1, 2, 3, crown-group euarthropods near the ancestry of chelicerates4, or a segmented ecdysozoan lineage with convergent similarity to arthropods in appendage construction5. Determining unambiguous affinities has been impeded by uncertainties about the segmental affiliation of anomalocaridid frontal appendages. These structures are variably homologized with jointed appendages of the second (deutocerebral) head segment, including antennae and ‘great appendages’ of Cambrian arthropods, or with the paired antenniform frontal appendages of living Onychophora and some Cambrian lobopodians. Here we describe Lyrarapax unguispinus, a new anomalocaridid from the early Cambrian Chengjiang biota, southwest China, nearly complete specimens of which preserve traces of muscles, digestive tract and brain. The traces of brain provide the first direct evidence for the segmental composition of the anomalocaridid head and its appendicular organization. Carbon-rich areas in the head resolve paired pre-protocerebral ganglia at the origin of paired frontal appendages. The ganglia connect to areas indicative of a bilateral pre-oral brain that receives projections from the eyestalk neuropils and compound retina. The dorsal, segmented brain of L. unguispinus reinforces an alliance between anomalocaridids and arthropods rather than cycloneuralians. Correspondences in brain organization between anomalocaridids and Onychophora resolve pre-protocerebral ganglia, associated with pre-ocular frontal appendages, as characters of the last common ancestor of euarthropods and onychophorans. A position of Radiodonta on the euarthropod stem-lineage implies the transformation of frontal appendages to another structure in crown-group euarthropods, with gene expression and neuroanatomy providing strong evidence that the paired, pre-oral labrum is the remnant of paired frontal appendages1.
Figure 1:L. unguispinus from the Chengjiang Lagersttte.
a, b, Ventral view of YKLP 13304b (counterpart) showing mouth cone (arrowed mo), eye stalks (eys), retinal pigmentation (rep), four neck segments (ne), traces of putative vascular system (vs) and its branches (open arrows) leading to metameric muscle blocks (m) aligned with lateral flaps, including oar-like first pair (fl, between arrowheads), and partly overlapping blades of tail fan (tf). c, d, Ventral view of YKLP 13304a (part) showing one frontal appendage (fa), part of the dorsal cowl (cw) and pigmented head shield anterior rim (hs). e, Frontal appendage: large proximal podomere (pp) equipped with serial spines allowing apposition against inner teeth of more distal podomeres. f, Enlarged oral cone showing concentric ridges and triangular areas (stars) suggestive of denticles. g, Pigmented rim of head shield. Scale bars: a, also for b–d, 5 mm; e, f, 1 mm; g, 3 mm.
Figure 2:L. unguispinus.
a, b, Dorsal view of YKLP13305 (left side slightly tilted downwards) resolving straight midgut (mg) and sinusoidal alimentary tract (alt). Four neck and eleven trunk segments, the first providing paired oar-like flaps (fl between arrowheads), the last providing the tail fan (tf). Dark areas in the head indicate paired frontal appendage ganglia (frg), optic tract (opt) linking retinas (re) in eyes (ey) to flattened lateral protocerebral lobes (lpr in h) flanking an approximately bilaterally symmetric protocerebrum (pr). Metameric striate areas indicate muscle (m). c–e, Raised and indented grooves of muscle blocks (enlargements of boxed areas in b). f–h, Neural traces: blue digital filter (f) cancels colours in fossil except dark neural regions (for example, medial protocerebrum, mpr) that are resolved by scanning electron microscopy and energy-dispersive X-ray spectroscopy (g), as carbon-rich domains, and shown by oblique illumination relative to eye and head margins (h); bm, basement membrane and first optic neuropil. Raised neck segments gradually obscure caudally directed descending tracts (dt). Scale bars: a, b, 1 cm; c–e, 0.5 mm; f (also for g) and h, 5 mm.
Figure 3:Comparison of onychophoran and L. unguispinus brain.
a, Horizontal section of hemibrain of E. rowelli stained with osmium–ethyl gallate, showing frontal appendage ganglion (frg) anterior to optic tract (opt) and second optic neuropil (on2) connected to medial protocerebral neuropil (mpr; lateral protocerebrum, lpr). b, c, Both sides of brain of L. unguispinus YKLP 13305 aligned to match orientation of a. Corresponding areas (and retinas, re) indicated, as is one root of descending tracts (dt). d, e, Comparison of E. rowelli and L. unguispinus brains. Nervous extensions into the frontal appendages (fa; on subsequent sections of E. rowelli; Extended Data Fig. 2e), not visible in the fossil, are added (paler blue). Incomplete distal part of frontal appendage of L. unguispinus reconstructed (paler grey). Eye, ey. Scale bars: a, 100 m; c (also for b), 2 mm.
Figure 4:Evolutionary shift of frontal appendage and its ganglia.
a–c, Proposed transformation of frontal appendages (fa, orange) and cogn
E.S.F., N.H.T., J.L.J. and W.C.F. initiated the project. E.S.F. and K.B. conducted the protein purification and crystallization. G.M.L. provided recombinant CSN, and S.C. pre-screened protein complexes by electron microscopy. E.S.F. collected data and processed and refined X-ray data. E.S.F. and N.H.T. analysed the structures. E.S.F. performed in vitro experiments and, with the help of U.H., developed and performed TR-FRET and fluorescence polarization assays. E.S.F. performed protein array experiments. M.B.S. and E.S.F. analysed the data. E.S.F., K.B., J.R.L., H.Y., M.H., J.W.H. and N.H.T. conceived and performed the cell-biological characterization. R.B.T. and R.E.J.B. conceived and conducted the chemical syntheses. J.N. and M. Schirle performed proteomics. V.A. and J.O. carried out the differential scanning fluorimetry experiments. F.S. and M. Schebesta carried out the zebrafish experiments. E.S.F. and N.H.T. wrote the manuscript. All authors assisted in editing the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Structural coordinates for human DDB1–G. gallus CRBN–thalidomide, human DDB1–G. gallus CRBN–lenalidomide and human DDB1–G. gallus CRBN–pomalidomide have been deposited in the Protein Data Bank under accession numbers 4CI1, 4CI2 and 4CI3. Human protein microarray data sets for this study have been deposited in the Gene Expression Omnibus database under accession number GSE57554.
Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA
Jae Myoung Suh,
Ruth T. Yu,
Annette R. Atkins,
Michael Downes &
Ronald M. Evans
Center for Liver, Digestive and Metabolic Diseases, Departments of Pediatrics and Laboratory Medicine, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Johan W. Jonker,
Theo H. van Dijk &
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, USA
Zhifeng Huang &
Department of Medicine, Division of Endocrinology and Metabolism, University of California at San Diego, La Jolla, California 92093, USA
Olivia Osborn &
Jerrold M. Olefsky
The Storr Liver Unit, Westmead Millennium Institute and University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia
Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California 92037, USA
Ronald M. Evans
Present address: School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China.
J.M.S., J.W.J. M.D. and R.M.E. designed and supervised the research. J.M.S., J.W.J., M.A., R.G., D.L., O.O., Z.H., W.L., E.Y., T.H.D., R.H., W.F., Y.-Q.Y. and A.R.A. performed research. J.M.S., J.W.J., M.A., R.T.Y., C.L., A.R.A., J.M.O., M.M., M.D. and R.M.E. analysed data. J.M.S., J.W.J., M.A., R.G., A.R.A., M.D. and R.M.E. wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol1, 2, 3, 4, 5, 6. These proteins have a variety of roles that are essential to virulence or parasite growth7. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins8, 9. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX)10. Although PTEX has been linked to the export of one class of exported proteins10, 11, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D)1, 2 and PEXEL-negative exported proteins (PNEPs)6. Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite’s capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.
Figure 1:Inducible knockdown of P. berghei HSP101 (i101 KD).
a, Diagram of a parasite-infected erythrocyte (RBC), the location of PTEX and proteins investigated in this study. ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. b, c, PCR (b) and Southern blot (c) of Pbi101 KD (I) and PbANKA wild-type (WT) parasites. kb, kilobases; INT, integration. d, Representative experiments (n = 3) showing that growth of Pbi101 KD in vivo is affected by ATc. Error bars show s.e.m. for three mice per condition, performed in parallel. e, Giemsa-stained blood smears from representative experiments (n = 3), showing parasites treated with ATc for the indicated period fail to recover in vitro. For d and e, grey bars and asterisks indicate when export was analysed. f, Downregulation of hsp101 transcript in Pbi101 KD parasites exposed to ATc. gDNA, genomic DNA. g, Western blot analysis showing a more than 85% decrease in HSP101 expression in parasites exposed to ATc (n = 2).
Figure 2:Knockdown of P. berghei HSP101 blocks export of PEXEL and PNEP proteins.
a, Surface labelling of parasite antigens on Pbi101 KD parasites harvested between days 1 and 2 post infection from mice pretreated with ATc was substantially decreased compared with infected erythrocytes not exposed to ATc as measured by FACS (n = 8; error bars represent s.e.m.; ***P < 0.001, using unpaired t-test). Boxes and whiskers delineate all data points, with whiskers indicating minimum and maximum values. b, Surface labelling of parasite antigens on asynchronous Pbi101 KD parasites grown to high parasitaemia and then treated with ATc for either 12 or 24 h (n = 8; error bars represent s.e.m.). c, Representative IFA of 100 Pbi101 KD intraerythrocytic stages, showing that exposure to ATc blocks export of PEXEL (top and middle panels) and PNEP (bottom panel) proteins. Yellow bar in all diagrams, signal sequence; red bar, transmembrane domain; black bar, glycosylphosphatidylinositol anchor. DIC, differential interference contrast. d, Expression of MSP8 is not affected by ATc. Scale bars, 5 m.
Figure 3:Generation of a PTEX150 knockdown line in P. falciparum.
a, PTEX150-HAglmS parasites (left), but not the control PTEX150-HA parasites (right), fail to proliferate when treated with glucosamine (GlcN) at a concentration of 0.6 mM or higher in the previous cycle (n = 2). b, Giemsa-stained P. falciparum cells, showing arrest of growth in the pigmented trophozoite stage (21–33 h post invasion (hpi)) in 2.5 mM GlcN added to the previous cycle. c, Western blot analysis: similarly treated PTEX150-HAglmS parasites, but not the control PTEX150-HA parasites, show an up to 80% decrease in PTEX150 protein levels (n = 2). HA, haemagglutinin epitope tag.
Figure 4:PTEX150 knockdown blocks protein export in P. falciparum.
a–e, IFAs (right) and graphs (left) showing a decrease in the export of RESA (a) and KAHRP (b) (mean fluorescence intensity, MFI) and of SBP1 (c) and Hyp8 (d) (Maurer’s clefts, MCs) (n = 12–47 cells for each antibody or GlcN concentration) but similar levels of MSP8 (e) (n = 15–35) after treatment with GlcN. Boxes and whiskers delineate 25th–75th and 10th–90th centiles, respectively. Colours of bars in diagrams as in Fig. 2. Scale bars, 5 m. f, Flow cytometry analysis showing decreased export of VAR2CSA onto the erythrocyte surface after treatment with GlcN (n = 3), at 24–28 h after invasion (top) and at 32–36 h after invasion (bottom). g, Cytoadherence of PTEX150-HAglms to chondroitin sulphate A (n = 2). Bars represent means ± s.d. *P
a–d, f, j, k, SDS–PAGE analysis of CSN variants used in the study. g, The N8CRL4ADDB2 substrate used for enzymatic measurements. a, The CSN5 E76A mutant catalytically inactivates CSN: activity of CSN (full-length) and CSN (CSN5(E76A)), a holocomplex carrying the active site mutant, was determined by fluorescence polarization measurements using a substrate with a PT22-labelled N8CRL4ADDB2. The decrease in signal for CSN (CSN5(E76A)) and in the buffer control is due to fluorophore photobleaching. Determination of steady-state kinetics using N8CRL4ADDB2 as substrate: initial rates observed following incubation of CSN or mutants thereof, with increasing concentrations of PT22-labelled N8CRL4ADDB2 substrate, as indicated on the abscissa. The fit of the observed initial velocities to the Michaelis–Menten equation is shown as a red line in b, endogenous CSN purified from HEK293T cells (CSN (HEK293T)); c, recombinant full-length CSN (CSN (full-length)); d, CSN with the boundaries used for crystallization (CSN); e, CSN (CSN6MPN), lacking the CSN6 MPN domain (comprising residues 192–327); and f, CSN (CSN6loop), lacking residues 174–179 of the CSN6 Ins-2 loop. a–f, Data are the average of three technical replicates. c, e, f, Error bars show standard error of the mean (s.e.m.). l, Table of steady-state kinetic parameters (errors show ± s.e.m.). A previous study46 described a CSN variant where the mouse CSN6 MPN domain was deleted (retaining CSN6 residues 171–324). This construct when immunoprecipitated from cells with the remainder of CSN was found to be active. For human CSN (CSN6MPN), detailed quantitative kinetic analysis revealed a ~100-fold reduction in kcat compared with CSN (full-length). Interestingly, mouse CSN6 171–324 retains the residues involved in the CSN4–CSN6 interface including the Ins-2 loop. Determination of steady-state kinetics using ubiquitin-rhodamine 110 as substrate: CSN variants were assayed for proteolytic release of the rhodamine 110 fluorescent group from the C-terminal glycine of ubiquitin (ubiquitin-rhodamine), using fluorescence quenching as readout. h, Wild-type CSN was not sufficiently active on ubiquitin-rhodamine to determine Michaelis–Menten parameters. To benchmark wild-type CSN against CSN (CSN5(E104A)), we assessed the relative rates at a fixed concentration of 0.5 µM ubiquitin-rhodamine substrate and 1 nM CSN (CSN5(E76A)), CSN (full-length) (23.0 ± 2.9 fmol s1) and CSN (CSN5(E104A)) (137.8 ± 2.2 fmol s1), using the CSN (CSN5(E76A)) active site mutant as a control. i, j, k, Increasing concentrations of ubiquitin-rhodamine with CSN (CSN6loop) (i), CSN (CSN5(E104A)) (j) and CSN (CSN6loop, CSN5(E104A)) (k) double mutant were assayed. Fit of the initial velocities to the Michaelis–Menten equation is shown as a red line. h–k, Data are the average of three technical replicates. m, Table summarizing the activity of CSN variants on ubiquitin-rhodamine (errors show ± s.e.m.). Assayed protein concentrations and Vmax values are indicated.
To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte1. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive2, 3, 4. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.
Figure 1:HSP101 is essential for development of asexual and sexual blood stages.
a, Auto-inhibition strategy for HSP101DDD. TMP, trimethoprim; HA, haemagglutinin tag; DDD, DHFR destabilization domain. b, Growth analysis of asynchronous cultures of the two independent clones 13F10 and 14G11 with or without TMP. Error bars represent s.d. of three technical replicates. Data are representative of three independent experiments. c, Giemsa-stained smears of cultures following 48 h with or without TMP. Accumulation of late ring-stage parasites is observed in the absence of TMP (arrows). Images are representative of three independent experiments. d, Growth analysis of synchronous 13F10 parasites. TMP was removed at the early trophozoite stage and added back to cultures after 24, 48 or 72 h. Equivalent parasitaemia in all samples at 24 h shows that development through trophozoite and schizont stages, egress and reinvasion were not affected by TMP removal. Error bars as in b. Data are representative of three independent experiments. e, Analysis of gametocyte formation by 13F10 parasites. TMP was removed from late schizonts following gametocyte induction (0 h) or at subsequent 24 h intervals. In one sample, TMP was removed at 0 h and restored after 24 h (/+ TMP 24 h). Gametocytaemia of various stages on day nine post-induction is shown. Error bars as in b. Data are representative of four independent experiments. f, Giemsa-stained smears of gametocyte cultures 9 days post induction. Images are representative of four independent experiments. d, f, Original magnification 1,000.
Figure 2:HSP101 is required for export of PEXEL and PNEP proteins.
a, c, Immunofluorescence assay (IFA) of ring-stage 13F10 parasites with or without TMP. TMP was removed in late schizont stage and parasites were allowed to reinvade and grow 18–24 h before fixation with paraformaldehyde (a) or acetone (c). a, IFA of the exported PEXEL-containing protein HRP2. SERP is a marker for the PV. Export was scored as complete (no HRP2 signal enrichment around the parasite as shown in the +TMP IFA), partial (HRP2 signal within the host cell but also enriched around the parasite) or no export (HRP2 signal only seen around the parasite and not in the host cell, as shown in the –TMP IFA). Error bars represent s.d. of three technical replicates. Data are representative of five independent experiments. DIC, differential interference contrast. b, Sequential fractionation of infected ring-stage parasites with or without TMP analysed by western blot. The host cytosol was released with tetanolysin (TTL) and subsequently the PV contents were released with saponin (SAP). Blocked HRP2 is found in the PV fraction. Haemoglobin (Hb) was detected by Coomassie staining and serves as a control for host cytosol release. SERP serves as a control for PV release. BiP serves as a parasite integrity control. Data are representative of two independent experiments. c, IFAs of the PNEP REX1, which colocalizes with HSP101DDD at the PVM in the absence of TMP. d, e, IFA of trophozoite-stage 13F10 parasites with or without TMP. TMP was removed in late ring stage and parasites were allowed to develop 12–24 h before fixation with acetone. f, Live fluorescence imaging of 13F10 parasites expressing a PFA660–GFP fusion and labelled with Bodipy TR Ceramide to demarcate the PVM (other membranes are also labelled). TMP treatment as in d, e. All scale bars, 5 µm. Images in c–f are representative of two independent experiments.
Figure 3:HSP101 is required for activation of PSAC but not trafficking of CLAG3 to the RBC periphery.
a, b, Osmotic lysis assay on 13F10 parasites. Sorbitol-sensitivity of arrested, late ring stage (TMP) and control (+TMP) parasites at 25% parasitaemia (a) or later stages with or without TMP magnet-purified to >95% parasitaemia (b) is shown. Error bars represent s.d. of three technical replicates. Results are representative of two independent experiments. c, IFA of ring-stage 13F10 parasites showing CLAG3 localization to the RBC membrane with or without TMP. TMP was removed in the late schizont stage and parasites were allowed to develop 18 h before fixation with 90% acetone/10% methanol. Scale bar, 5 µm. Images are representative of two independent experiments.
FOXP3+ regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function1; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites2, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage3. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients4, 5, 6, 7, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis8, 9, 10, 11, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.
Figure 1:ST2-expressing Treg cells are enriched in the colon.
a, Change in gene expression in colonic (c)Treg cells versus mesenteric lymph node (MLN) Treg cells (n = 3 per group) presented as volcano plot. b, Top differentially upregulated transcripts in colonic Treg versus MLN Treg cells. c, ST2 protein expression on Treg cells from indicated organs. d, Phenotypic analysis of ST2 or ST2+ colonic Treg cells. e, Expression of transcription factors in colonic Treg cells. f, Representative histograms gated on colonic Treg cells from control or Gata3fl/fl-Foxp3-cre mice.
Figure 2:Effects of IL-33 on iTreg and thymus-derived Treg cells.
a, Naive CD4+ T cells were cultured with anti-CD3/CD28 plus the indicated cytokines and the frequencies and absolute numbers of Foxp3+ T cells were determined 3 days later (mean ± standard error of the mean (s.e.m.) of three independent experiments). b, Naive CD4+ T cells were cultured for 48 h with anti-CD3/CD28 plus TGF-1, followed by stimulation with IL-33 for 45 min. Blots are representative of two independent experiments. p, phosphorylated. c, d, Cells were cultured and stimulated as in b and recruitment of GATA3 or RNA Pol II to the indicated regions was assessed by ChIP-qPCR. Data are from one experiment representative of two (mean ± standard deviation (s.d.)). Pro., promoter. , no IL-33 added. e, Representative plots of Treg cells cultured with anti-CD3/CD28 plus indicated cytokines and analysed after 3 days. Data are representative of three independent experiments. f, Treg cells were cultured with anti-CD3/CD28 for 24 h followed by stimulation with IL-33. Blots are representative of three independent experiments. g, Mixed chimaeras were generated containing wild-type (WT) and St2/ bone marrow cells. Reconstituted mice were analysed at steady state or 2 weeks after infection with H. hepaticus and anti-IL-10R treatment (inflamed). Absolute numbers of wild-type or St2/ Treg cells in steady state (n = 3) and inflamed (n = 6) hosts (mean ± s.e.m.). h, Analysis of Foxp3 expression in Treg cells in spleen (SPN) and colon from inflamed chimaeric hosts presented as geometric mean fluorescence intensity (gMFI). *P < 0.05, **P < 0.01, ***P < 0.001 as calculated by one-way analysis of variance (ANOVA) with Bonferroni post-test or paired Student’s t-test.
Figure 3:IL-33 promotes Treg-cell stability and function in vivo.
a, C57BL/6 Rag1/ mice were injected with CD45.1+ naive T cells alone (RBhi; n = 4) or in combination with wild-type (WT; n = 4) or St2/ (n = 6) CD45.1 Treg cells. Mice were killed 6–8 weeks after transfer and colitis scores are shown (mean ± s.e.m.). b, Absolute numbers of colon lamina propria (LP) cells from mice in a (mean ± s.e.m.). c, C57BL/6 Rag1/ mice were injected as in a and killed at 2 weeks post-injection. Representative plots are gated on colonic Treg-cell progeny (CD45.1). d, Ratio of RBhi T-cell progeny (CD45.1+) to wild-type or St2/ Foxp3+ Treg-cell progeny (CD45.1) in the colon (n = 5 per group) from mice in c (mean ± s.e.m.). e, C57BL/6 Rag1/ mice were injected as in a and killed at 8 weeks post-injection. Representative plots are gated on colonic Treg-cell progeny (CD45.1). f, Ratio of RBhi T-cell progeny (CD45.1+) to wild-type or St2/ Treg-cell progeny (CD45.1) in the colon from mice in e (mean ± s.e.m.). g, Absolute numbers of
Two studies provide evidence that the protein complex PTEX is needed for export of malaria-parasite proteins into the cytoplasm of infected cells, and that such export is essential for parasite survival.
Mutants in the described crossover interference pathway all confer coordinate changes in crossover interference, which is reduced, and the total number of crossovers, which is increased, by about 20% on chromosome XV. There are the expected consequences of a single defect in crossover interference, as illustrated by corresponding beam-film simulations, which quantitatively explain these results by a change in a single parameter, the interference length (LBF) (Figs 2 and 3). This interference defect could comprise a defect in generation and spreading of the inhibitory signal and/or of the ability of unreacted precursors to respond to that signal (see text and Methods (section ‘Beam-film simulations’)). An increase in the number of crossovers can also occur as the result of either (1) prolongation of the crossover-designation period or (2) an increase in the number of DSBs8. Neither of these effects can explain the mutant phenotypes described in the text. (1) Crossover designation precedes synaptonemal complex formation and thus the pachytene stage14. Time-course analysis of representative mutant strains reveals that, in sir2 mutants and in top2SNM, meiosis proceeds through pachytene and the two meiotic divisions normally (Extended Data Fig. 8a; ref. 14; data not shown). slx5/8 mutants and PCLB2-TOP2 mutants show no delay in progressing through prophase to pachytene (data not shown) but show a delay in meiosis I (slx5) or pachytene arrest (PCLB2-TOP2) (Extended Data Fig. 8a; data not shown). The pCLB2-TOP2 top2YF mutant does show a delay in achieving pachytene, as well as pachytene arrest, but exhibits the same crossover patterning phenotype as all other mutants, which show no pre-pachytene delay. Thus, prolonged crossover designation is not the basis for these phenotypes. (2) An increase in DSBs, without any change in crossover interference, does increase the number of crossovers; however, it has very little effect on crossover interference relationships (coefficient of coincidence curves) in budding yeast8. Correspondingly, two lines of evidence show that the mutant defects described here cannot be attributed to an increase in DSBs. a, A tel1 mutant exhibits increased DSBs but no change in coefficient of coincidence relationships. TEL1 encodes the yeast homologue of ATM. Absence of Tel1 confers a 50% increase in DSBs62 and a 10% increase in number of Zip3 foci (Supplementary Fig. 7 in ref. 8; reproduced in Extended Data Fig. 7a left, red colour). However, (1) there is no change in coefficient of coincidence relationships relative to WT (Extended Data Fig. 7a left), (2) the increase in crossovers is precisely that predicted on the basis of crossover homeostasis (ref. 8; text Fig. 2d, filled black circle at 19 DSBs/precursors per chromosome XV) and (3) beam-film simulation accurately describes the tel1 phenotype, relative to WT, by a change in a single parameter: the level of DSBs (n = 19, grey, versus 13, gold, in WT). The last point is documented in Extended Data Fig. 7a middle and right. The middle panel in Extended Data Fig. 7a shows the beam-film best-fit simulation for WT chromosome XV, where n = 13 (gold), compared with the experimental coefficient of coincidence curve (black; from Fig. 1); the right panel shows the beam-film best-fit simulation for tel1 chromosome XV, where n = 19 (grey) and all other parameters are the same as for WT, compared with the experimental coefficient of coincidence curve (red) from the left panel. b, Beam-film simulations predict no/little change in coefficient of coincidence with increasing DSBs for yeast chromosome XV (data not shown). More specifically, to explain the increased number of crossovers observed in the analysed mutants, for example pCLB2-TOP2, the value of N required for beam-film simulations of chromosome XV would be 26 (double the WT value of N = 13). If beam-film simulations are performed under the same parameter values used for WT except that N = 26 instead of N = 13, the predicted coefficient of coincidence curve is unchanged compared with that predicted for WT (left panel, compare gold for N = 13 with green for N = 26). Correspondingly, the coefficient of coincidence curve predicted for N = 26 (green) matches the WT coefficient of coincidence curve (black) and is unlike the coefficient of coincidence curve for the mutant (pink) (right panel). Additional evidence that DSB number is not altered in pCLB2-TOP2 versus TOP2 is presented in Extended Data Figs 4 and 8.