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1.
Science ; 380(6647): 818-823, 2023 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-37228189

RESUMO

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.


Assuntos
Diglicerídeos , Exocitose , Sinapses Imunológicas , Receptores de Antígenos de Linfócitos T , Linfócitos T Citotóxicos , Divisão Celular , Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Exocitose/imunologia , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/ultraestrutura , Micropartículas Derivadas de Células/imunologia , Diglicerídeos/metabolismo
2.
J Cell Biol ; 220(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34292303

RESUMO

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca2+ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.


Assuntos
Linfócitos T Citotóxicos/imunologia , Animais , Cálcio/imunologia , Células Cultivadas , Centrossomo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia , Sinapses/imunologia
3.
J Clin Invest ; 129(12): 5600-5614, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31710310

RESUMO

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Citotoxicidade Imunológica , Linfócitos T Citotóxicos/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/análise , Actinas/análise , Antígenos CD8/análise , Polaridade Celular , Transportador de Glucose Tipo 1/análise , Células HEK293 , Humanos , Sinapses Imunológicas/fisiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T alfa-beta/análise
4.
Methods Mol Biol ; 1584: 473-486, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255720

RESUMO

Here, we describe 4D imaging of effector CD8+ T cells as they conjugate and kill live targets in vitro and analyze the polarization dynamics of intracellular compartments to this cell-cell interface.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imageamento Tridimensional , Sinapses Imunológicas/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Humanos , Microscopia Confocal/métodos
5.
J Cell Sci ; 129(15): 2881-6, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27505426

RESUMO

The immune synapse provides an important structure for communication with immune cells. Studies on immune synapses formed by cytotoxic T lymphocytes (CTLs) highlight the dynamic changes and specialised mechanisms required to facilitate focal signalling and polarised secretion in immune cells. In this Cell Science at a Glance article and the accompanying poster, we illustrate the different steps that reveal the specialised mechanisms used to focus secretion at the CTL immune synapse and allow CTLs to be such efficient and precise serial killers.


Assuntos
Sinapses Imunológicas/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Membrana Celular/metabolismo , Cílios/metabolismo , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
6.
Nat Rev Immunol ; 16(7): 421-32, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27265595

RESUMO

Cytotoxic T lymphocytes (CTLs) kill virus-infected and tumour cells with remarkable specificity. Upon recognition, CTLs form a cytolytic immune synapse with their target cell, and marked reorganization of both the actin and the microtubule cytoskeletons brings the centrosome up to the plasma membrane to the point of T cell receptor signalling. Secretory granules move towards the centrosome and are delivered to this focal point of secretion. Such centrosomal docking at the plasma membrane also occurs during ciliogenesis; indeed, striking similarities exist between the cytolytic synapse and the primary cilium that throw light on the possible origins of immune synapses.


Assuntos
Citotoxicidade Imunológica/imunologia , Sinapses Imunológicas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Humanos , Sinapses Imunológicas/ultraestrutura , Linfócitos T Citotóxicos/ultraestrutura
7.
Immunity ; 42(5): 864-76, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25992860

RESUMO

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.


Assuntos
Actinas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Sinapses Imunológicas/metabolismo , Linfócitos T Citotóxicos/citologia , Membrana Celular/química , Células Cultivadas , Grânulos Citoplasmáticos/química , Imunofluorescência , Humanos , Modelos Imunológicos , Fosfolipídeos/metabolismo , Linfócitos T Citotóxicos/metabolismo
8.
Elife ; 3: e01310, 2014 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-24596147

RESUMO

T cell receptor (TCR) activation leads to a dramatic reorganisation of both membranes and receptors as the immunological synapse forms. Using a genetic model to rapidly inhibit Zap70 catalytic activity we examined synapse formation between cytotoxic T lymphocytes and their targets. In the absence of Zap70 catalytic activity Vav-1 activation occurs and synapse formation is arrested at a stage with actin and integrin rich interdigitations forming the interface between the two cells. The membranes at the synapse are unable to flatten to provide extended contact, and Lck does not cluster to form the central supramolecular activation cluster (cSMAC). Centrosome polarisation is initiated but aborts before reaching the synapse and the granules do not polarise. Our findings reveal distinct roles for Zap70 as a structural protein regulating integrin-mediated control of actin vs its catalytic activity that regulates TCR-mediated control of actin and membrane remodelling during formation of the immunological synapse. DOI: http://dx.doi.org/10.7554/eLife.01310.001.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Sinapses Imunológicas/metabolismo , Linfócitos T Citotóxicos/fisiologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/enzimologia , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores
9.
EMBO J ; 30(7): 1311-23, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21336257

RESUMO

Tob is a member of the anti-proliferative protein family, which functions in transcription and mRNA decay. We have previously demonstrated that Tob is involved in the general mechanism of mRNA decay by mediating mRNA deadenylation through interaction with Caf1 and a general RNA-binding protein, PABPC1. Here, we focus on the role of Tob in the regulation of specific mRNA. We show that Tob binds directly to a sequence-specific RNA-binding protein, cytoplasmic polyadenylation element-binding protein 3 (CPEB3). CPEB3 negatively regulates the expression of a target by accelerating deadenylation and decay of its mRNA, which it achieves by tethering to the mRNA. The carboxyl-terminal RNA-binding domain of CPEB3 binds to the carboxyl-terminal unstructured region of Tob. Tob then binds Caf1 deadenylase and recruits it to CPEB3 to form a ternary complex. The CPEB3-accelerated deadenylation was abrogated by a dominant-negative mutant of either Caf1 or Tob. Together, these results indicate that Tob mediates the recruitment of Caf1 to the target of CPEB3 and elicits deadenylation and decay of the mRNA. Our results provide an explanation of how Tob regulates specific biological processes.


Assuntos
Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo
10.
Cell Motil Cytoskeleton ; 65(12): 923-34, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18814278

RESUMO

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity.


Assuntos
Citoesqueleto de Actina/metabolismo , Quimiotaxia , Dictyostelium/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Citoesqueleto de Actina/genética , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/genética , Cromonas/farmacologia , Citoesqueleto/metabolismo , Dictyostelium/genética , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Morfolinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tiazolidinas/farmacologia
11.
J Am Chem Soc ; 126(38): 12112-20, 2004 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-15382947

RESUMO

The mechanism of the photochromic cycloreversion reactions is theoretically examined in a model system of dithienylethenes by means of the CASSCF and CASPT2 methods. The structures of its conical intersections (CIs), which are the branching points of the internal conversions, were obtained. The analyses of the minimum energy paths from the Franck-Condon states and the CI points suggest that the cycloreversion reaction occurs during the intramolecular vibrational energy redistribution (IVR) toward the quasi-equilibrium on the 2A state. The current study of the model system will provide a basic insight for the photochromic molecular design.

12.
Cell Motil Cytoskeleton ; 59(1): 17-27, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15259052

RESUMO

Starved Dictyostelium amoebae continuously change their shape and they are elongated along the front-rear axis during locomotion. In contrast, we found that disruption of the amiB gene, which had been identified as a gene required for the aggregation process during development, caused these cells to move in a manner similar to fish keratocytes. Starved amiB- cells were elongated laterally and had one large lamellipodium along the front side arc of the cell. These cells moved unidirectionally for long distances maintaining the half-moon shape, and this movement followed the predictions of the graded radial extension model, which was originally developed to describe the keratocyte movements. Furthermore, the distributions of actin, Arp2, and myosin II in amiB- cells were similar to those in keratocytes. Therefore, locomotion by keratocytes and amiB- cells appears to be driven by similar mechanisms of cytoskeletal regulation. Double knockout cells lacking both AmiB and myosin II were still able to move unidirectionally in a keratocyte-like manner, although the frequency of those movements was lower. Thus, myosin II is dispensable for the unidirectional movement, though it likely functions in the maintenance of the characteristic half-moon shape. This mutant cell can be a useful tool for further molecular genetic analysis of the mechanism of cell locomotion.


Assuntos
Dictyostelium/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Proteínas do Citoesqueleto/genética , Dictyostelium/genética , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Protozoários/genética
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