ex-vivo-imaging-t-cells-murine-lymph-node-slices-with-widefield

<em>Ex vivo</em> Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

phagosome-closure-assay-to-visualize-phagosome-formation-three

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

intravital-longitudinal-imaging-vascular-dynamics-calvarial-bone

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the ...

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by alphaMbeta2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis.

Microtubule Dynamik werden durch regulatorische Proteine moduliert, die ihre plus enden (+ Tipps [plus Ende verfolgen Proteine]), z. B. zytoplasmatischen Linker Protein 170 (CLIP-170) oder Ende-verbindliches Protein 1 (ES1) zu binden. Wir untersuchten die Rolle von + Tipps w&auml;hrend der Phagozytose in Makrophagen. RNA-Interferenz und Dominant-negativen Ans&auml;tze verwenden, zeigen wir, dass die CLIP-170 speziell f&uuml;r effiziente Phagozytose, ausgel&ouml;st durch alphaMbeta2 Integrin/Komplement-Rezeptor-Aktivierung erforderlich ist. Diese Eigenschaft wird nicht f&uuml;r ES1 und EB3 beobachtet. Folglich w&auml;hrend CLIP-170 dynamisch auf dem Gel&auml;nde der Phagozytose bereichert wird, ist ES1 keine. Dar&uuml;ber hinaus beobachten wir, dass die CLIP-170 steuert die Rekrutierung von formin mDia1, eines Proteins Aktin-Nukleierungsmittel zu Beginn der Phagozytose und damit steuert Aktin-Polymerisation-Ereignisse, die f&uuml;r die Phagozytose unverzichtbar sind. CLIP-170 interagiert direkt mit der formin Homologie 2-Dom&auml;ne des mDia1. Die Interaktion zwischen CLIP-170 und mDia1 wird w&auml;hrend der alphaMbeta2-vermittelte Phagozytose negativ reguliert. Unsere Ergebnisse zu entwirren eine neue Microtubule/Aktin-Kooperation, bei der CLIP-170 und mDia1 und die Funktionen stromabw&auml;rts des alphaMbeta2 Integrine.

Die Microtubule-verbindliches Protein CLIP-170 Koordinaten mDia1 und Aktin Reorganisation w&auml;hrend CR3-vermittelte Phagozytose.

Cross-presentation is an essential mechanism that allows dendritic cells (DCs) to efficiently present exogenous antigens to CD8(+) T cells. Among cellular antigen sources, apoptotic cells are commonly considered as the best for cross-presentation by DCs. However, the potential of live cells as a source of antigen has been overlooked. Here we explored whether DCs were able to capture and cross-present antigens from live cells. DCs internalized cytosolic and membrane material into vesicles from metabolically labeled live cells. Using time-lapse confocal microscopy in whole spleens, we showed that DCs internalized material from live cells in vivo. After ovalbumin uptake from live cells, DCs cross-primed ovalbumin-specific naive OT-I CD8(+) T cells in vitro. Injected into mice previously transferred with naive OT-I T cells, they also cross-primed in vivo, even in the absence of endogenous DCs able to present the epitope in the recipient mice. Interestingly, DCs induced stronger natural CD8(+) T-cell responses and protection against a lethal tumor challenge after capture of antigens from live melanoma cells than from apoptotic melanoma cells. The potential for cross-presentation from live cells uncovers a new type of cellular intercommunication and must be taken into account for induction of tolerance or immunity against self, tumors, grafts, or pathogens.

Cross-presentation by dendritic cells from live cells induces protective immune responses in vivo.

Cross-Pr&auml;sentation ist ein wesentlicher Mechanismus, der dendritische Zellen (DCs) effizient exogene Antigene CD8(+) T-Zellen pr&auml;sentieren zu k&ouml;nnen. Unter zellul&auml;re Antigen Quellen gelten Apoptotic Zellen h&auml;ufig als das beste f&uuml;r Kreuz-Pr&auml;sentation von DCs. Das Potenzial der lebenden Zellen als eine Quelle von Antigen wurde jedoch &uuml;bersehen. Hier wir gepr&uuml;ft, ob DCs Capture und Kreuz-heute Antigene aus lebenden Zellen konnten. DCs verinnerlicht Zytosolische und Membranmaterial in Vesikeln von metabolisch beschriftet lebenden Zellen. Mit Time-Lapse confocal Mikroskopie in ganze Milzen, zeigten wir dass DCs verinnerlichte Material von lebenden Zellen in-vivo. Nach Eieralbumin Aufnahme aus lebenden Zellen, DCs Kreuz-grundiert Eieralbumin-spezifische naiv OT-ich CD8(+) T-Zellen in vitro. In M&auml;usen, die zuvor mit naiven OT &uuml;bertragen injiziert-ich T Zellen, sie auch Kreuz-grundiert in Vivo, sogar in Ermangelung einer endogenen DCs die Epitop in die Empf&auml;nger-M&auml;use zu pr&auml;sentieren. Interessanterweise induzierte DCs st&auml;rker nat&uuml;rliche CD8(+) T-Zell-Antworten und Schutz vor einem t&ouml;dlichen Tumor-Herausforderung nach Einnahme von Antigenen aus live Melanomzellen als von Apoptotic Melanomzellen. Das Potenzial f&uuml;r Kreuz-Pr&auml;sentation von lebenden Zellen entdeckt eine neue Art der zellul&auml;ren Kommunikation untereinander und muss f&uuml;r die Induktion von Toleranz oder Immunit&auml;t gegen selbst, Tumoren, Transplantate oder Krankheitserreger ber&uuml;cksichtigt werden.

Cross-Pr&auml;sentation von dendritischen Zellen aus lebenden Zellen induziert sch&uuml;tzende Immunantworten in-vivo.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilien und Flagellen sind eukaryotische Organellen mehrere Zellfunktionen beteiligt. Die prim&auml;re Zilie ist prinzipiell nicht beweglich und in zahlreichen vertebrate Zelltypen gefunden wo es wichtige Signalwege steuert. Trotz einer gemeinsamen Architektur deuten ultrastrukturelle Daten einige Unterschiede in ihrer Organisation. Wir berichten hier, die erste detaillierte Charakterisierung der Zilien Tasche, eine Depression der Plasmamembran in die prim&auml;re Zilie verwurzelt ist. Diese Struktur findet sich in geringer Anzahl in Epithelzellen der Niere (IMCD3), aber praktisch alle prim&auml;ren Zilien Retinales Epithelzellen (RPE1) zugeordnet ist. &Uuml;bertragung und Rasterelektronenmikroskopie, Immunofluoreszenz-Analyse und Videomicroscopy ergab, dass die Zilien Tasche geschlossene Beziehungen auf der Grundlage von Aktin-Zytoskeletts richtet und dass es in aktiven und dynamischen Clathrin-coated-Pits angereichert ist. Die Existenz der Zilien Tasche wurde in Maus-Geweben, wobei prim&auml;re Zilien (Cumulus), sowie bewegliche Cilien und Flagellen (ependymalen Zellen und Spermatids) best&auml;tigt. Die Zilien Tasche teilt auffallende morphologische und funktionelle &Auml;hnlichkeiten mit der Flimmerband Tasche von Trypanosomatids, einer Dom&auml;ne der Drogenhandel spezialisierten Membran an der Basis der Gei&szlig;el. Unsere Daten markieren daher die konservierte Rolle der Membran des Menschenhandels in der N&auml;he von Zilien.

Die Zilien Tasche: einer endocytic Membran-Dom&auml;ne an der Basis der Prim&auml;r- und bewegliche Zilien.

The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.

Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites.

Die Pathologie verursacht Stufen von der menschlichen Malaria Parasit Plasmodium Falciparum befinden sich innerhalb der roten Blutk&ouml;rperchen, die frei von jedem geregelten Transportsystem sind. Der Parasit, daher ist vollst&auml;ndig verantwortlich f&uuml;r die Vermittlung der vesikul&auml;ren Transport in sich und im Zytoplasma infizierten Erythrozyten, und es tut sich also teilweise &uuml;ber seine Familie von 11 Rab-GTPasen. Vermeintliche Funktionen haben wurden zugeschrieben Plasmodium Rabs aufgrund ihrer Homologie mit Rabs Hefe, besonders mit Saccharomyces, die eine entsprechende Anzahl von Rab/Ypt Gene hat und wo Analysen der Ypt-Funktion ist gut gekennzeichnet.

Bau einer Plasmodium Falciparum Rab-Interaktom identifiziert CK1 und PKA als Rab-Effektor-Kinasen in Malaria-Parasiten.

Studying the immunological processes taking place during the initial steps of acute hepatitis C virus (HCV) infection has been a challenge in patients. Shin et al. have recently reported that delayed induction, not impaired recruitment of specific CD8(+) T cells, causes the late onset of acute hepatitis C in chimpanzees (Gastroenterology, 2011). However, further elucidation of the underlying mechanisms is difficult in vivo. We made observations consistent with their conclusions in human liver slices inoculated ex vivo with HCV produced in cell culture (HCVcc). Autologous immune cells were purified from blood and differentially stained prior to their incubation with the slices for 2 hours. A two-photon confocal microscopic analysis revealed that many more stained dendritic and T cells contracted interactions within two-day infected slices than non-inoculated ones (p<0.001). While in the first instance some dendritic and T cells entered into closer interactions, they never did in the latter case. These results suggest that ex vivo infection of human liver slices with HCVcc may be useful for gaining experimental insight regarding the immunological processes taking place at early steps of HCV infections.

Recruitment and interaction of human dendritic and T cells in autologous liver slices experimentally infected with HCV produced in cell culture.

The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2-specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs.

Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection.

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles.

Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene.

3D-FISH analysis reveals chromatid cohesion defect during interphase in Roberts syndrome.

Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1-infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1-infected macrophages, leading to strong alterations in phagolysosome biogenesis.

ArticleTotal : 3	Year
Publication title Cited by 21	2011
Publication title Cited by 8	2016
Publication title Cited by 1	2025

Article	Year
The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis. The Journal of cell biology\| PubMed ID: 19114595	2008
Cross-presentation by dendritic cells from live cells induces protective immune responses in vivo. Blood\| PubMed ID: 20308597	2010
The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia. Journal of cell science\| PubMed ID: 20427320	2010
Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites. Biology of the cell / under the auspices of the European Cell Biology Organization\| PubMed ID: 22188458	2012
Recruitment and interaction of human dendritic and T cells in autologous liver slices experimentally infected with HCV produced in cell culture. Journal of immunological methods\| PubMed ID: 22349126	2012
Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection. Hepatology (Baltimore, Md.)\| PubMed ID: 22454196	2012
The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles. Developmental cell\| PubMed ID: 23153494	2012
3D-FISH analysis reveals chromatid cohesion defect during interphase in Roberts syndrome. Molecular cytogenetics\| PubMed ID: 25320640	2014
The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking. The Journal of cell biology\| PubMed ID: 26504171	2015

Pierre Bourdoncle

Biography

JoVE Journal Publications

Other Publications