Aim： In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm＇s plasma membrane. We have addressed questions suc...Aim： In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm＇s plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Methods： Atomic force microscopy identified a novel region （EqSS） within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Results： Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. Conclusion： A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg. （Asian JAndrol 2007 July; 9： 438-444）展开更多
Germinal centers(GCs)are essential for the establishment of long-lasting antibody responses.GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into funct...Germinal centers(GCs)are essential for the establishment of long-lasting antibody responses.GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome.However,the critical proteins driving these key mechanisms are still unknown.Here,we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses.TIA1-and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection,expansion and differentiation into B-cell clones producing high-affinity antibodies.Mechanistically,TIA1 and TIAL1 control the transcriptional identity of dark-and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1.Thus,we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.展开更多
Mammalian cloning has been one of the most active research topics in the world. Cioning with in vitro culured foetal fibroblast cells, in comparison with embryonic cells, can be used not only to theoretically study th...Mammalian cloning has been one of the most active research topics in the world. Cioning with in vitro culured foetal fibroblast cells, in comparison with embryonic cells, can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals, but also to utilize the unlimited fibroblast cells to produce large numbers of clonings. The preliminary results are as follows: (i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%, 77% and 35%, 31%, respectively. There is no significant statistical difference between them. (? These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast celi lines, which are the first cloned mammals from somatic cells in China. This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development, and a novel technique for the展开更多
Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca^(2+)) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP_(3)R), a...Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca^(2+)) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP_(3)R), a homo- or heterotetramer of the IP_(3)R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca^(2+) from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP_(3)R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP_(3)R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca^(2+) signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca^(2+) signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP_(3)R3 in IP_(3)R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca^(2+) channels and immunodeficiency and identify IP_(3)Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca^(2+)-associated immunodeficiency from store-operated entry to impaired Ca^(2+) mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca^(2+) signaling.展开更多
MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to ME...MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to MEK1/2 inhibitors,including selumetinib(AZD6244/ARRY-142866),can arise through amplification of BRAF^(V600E) or KRAS^(G13D) to reinstate ERK1/2 signalling.We have found that BRAF^(V600E) amplification and selumetinib resistance are fully reversible following drug withdrawal.This is because resistant cells with BRAF^(V600E) amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling(2%-3%of total ERK1/2 active),that is optimal for cell proliferation and survival.Selumetinib withdrawal drives ERK1/2 activation outside of this critical“sweet spot”(~20%-30%of ERK1/2 active)resulting in a p57^(KIP2)-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy;these terminal responses select against cells with amplified BRAF^(V600E).ERK1/2-dependent p57^(KIP2) expression is required for loss of BRAF^(V600E) amplification and determines the rate of reversal of selumetinib resistance.Growth of selumetinib-resistant cells with BRAF^(V600E) amplification as tumour xenografts also requires the presence of selumetinib to“clamp”ERK1/2 activity within the sweet spot.Thus,BRAF^(V600E) amplification confers a selective disadvantage or“fitness deficit”during drug withdrawal,providing a rationale for intermittent dosing to forestall resistance.Remarkably,selumetinib resistance driven by KRAS^(G13D) amplification/upregulation is not reversible.In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents.Our results reveal that the emergence of drug-addicted,MEKi-resistant cells,and the opportunity this may afford for intermittent dosing schedules(“drug holidays”),may be determined by the nature of the amplified driving oncogene(BRAF^(V600E) vs.KRAS^(G13D)),further exemplifying the difficulties of targeting KRAS mutant tumour cells.展开更多
文摘Aim： In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm＇s plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Methods： Atomic force microscopy identified a novel region （EqSS） within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Results： Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. Conclusion： A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg. （Asian JAndrol 2007 July; 9： 438-444）
基金We thank all personnel from the Toulouse animal facility CREFRE and from the flow cytometry,imaging,transcriptomics and bioinformatics technical platforms of INFINITy.M.D.D-M.is supported by ATIP-Avenir-Plan Cancer(C18003BS),ANR(ANR-20-CE15-0007)foundation ARSEP R19201BB,foundation ARC,La Ligue Contre Le Cancer and INSPIRE(Region Occitanie,Inserm and CHU Toulouse)M.T.is supported with a BBSRC core funding grant and a Wellcome Investigator award(200823/Z/16/Z).D.C.-S.is supported by Boehringer Ingelheim Fonds.
文摘Germinal centers(GCs)are essential for the establishment of long-lasting antibody responses.GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome.However,the critical proteins driving these key mechanisms are still unknown.Here,we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses.TIA1-and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection,expansion and differentiation into B-cell clones producing high-affinity antibodies.Mechanistically,TIA1 and TIAL1 control the transcriptional identity of dark-and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1.Thus,we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.
文摘Mammalian cloning has been one of the most active research topics in the world. Cioning with in vitro culured foetal fibroblast cells, in comparison with embryonic cells, can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals, but also to utilize the unlimited fibroblast cells to produce large numbers of clonings. The preliminary results are as follows: (i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%, 77% and 35%, 31%, respectively. There is no significant statistical difference between them. (? These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast celi lines, which are the first cloned mammals from somatic cells in China. This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development, and a novel technique for the
基金supported by the VIB Grand Challenges Program,the KU Leuven C1 program,the European Union’s Horizon 2020 research and innovation program under grant agreement No 779295(to AL)the Biotechnology and Biological Sciences Research Council(BBSRC)through Institute Strategic Program Grant funding BBS/E/B/000C0427 and BBS/E/B/000C0428 and the KU Leuven BOFZAP start-up grant(to SH-B)+7 种基金Work in the Bultynck team was supported by grants from the Research Council of the KU Leuven(C14/19/99 and AKUL/19/34)Research Foundation-Flanders(G.0818.21NG.0945.22N)DIY is supported by the National Institutes of Health(NIH)R01-DE0014756 grant.MRB and IIS are supported by the NIH R01GM072804 grant(to IIS)the Welch Foundation Research Grant AU-2014-20190331(to IIS)the American Heart Association grant 18CDA34110086(to MRB)IIS,DIY,and GB are in the FWO Scientific Research Network CaSign(W0.019.17N)IM and RS are FWO senior clinical investigator fellows.IM and RS are members of the European Reference Network for Rare Immunodeficiency,Autoinflammatory and Autoimmune Diseases(project ID No.739543).
文摘Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca^(2+)) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP_(3)R), a homo- or heterotetramer of the IP_(3)R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca^(2+) from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP_(3)R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP_(3)R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca^(2+) signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca^(2+) signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP_(3)R3 in IP_(3)R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca^(2+) channels and immunodeficiency and identify IP_(3)Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca^(2+)-associated immunodeficiency from store-operated entry to impaired Ca^(2+) mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca^(2+) signaling.
基金Work in the Cook laboratory relevant to this article was supported by Cancer Research UK A14867,a Cambridge Cancer Centre PhD Studentship,a BBSRC PhD studentship,to Sale MJ and Cook SJan AstraZeneca-Cambridge Cancer Centre Collaborative Award,to Sale MJ and Cook SJInstitute Strategic Programme(BB/J004456/1,BB/P013384/1)from BBSRC to Balmanno K and Cook SJ,and AstraZeneca.
文摘MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma,where they are used in combination with BRAF inhibitors,and are undergoing evaluation in other malignancies.Acquired resistance to MEK1/2 inhibitors,including selumetinib(AZD6244/ARRY-142866),can arise through amplification of BRAF^(V600E) or KRAS^(G13D) to reinstate ERK1/2 signalling.We have found that BRAF^(V600E) amplification and selumetinib resistance are fully reversible following drug withdrawal.This is because resistant cells with BRAF^(V600E) amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling(2%-3%of total ERK1/2 active),that is optimal for cell proliferation and survival.Selumetinib withdrawal drives ERK1/2 activation outside of this critical“sweet spot”(~20%-30%of ERK1/2 active)resulting in a p57^(KIP2)-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy;these terminal responses select against cells with amplified BRAF^(V600E).ERK1/2-dependent p57^(KIP2) expression is required for loss of BRAF^(V600E) amplification and determines the rate of reversal of selumetinib resistance.Growth of selumetinib-resistant cells with BRAF^(V600E) amplification as tumour xenografts also requires the presence of selumetinib to“clamp”ERK1/2 activity within the sweet spot.Thus,BRAF^(V600E) amplification confers a selective disadvantage or“fitness deficit”during drug withdrawal,providing a rationale for intermittent dosing to forestall resistance.Remarkably,selumetinib resistance driven by KRAS^(G13D) amplification/upregulation is not reversible.In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents.Our results reveal that the emergence of drug-addicted,MEKi-resistant cells,and the opportunity this may afford for intermittent dosing schedules(“drug holidays”),may be determined by the nature of the amplified driving oncogene(BRAF^(V600E) vs.KRAS^(G13D)),further exemplifying the difficulties of targeting KRAS mutant tumour cells.
