Cutting Edge: Atypical PKCs Regulate T Lymphocyte Polarity and Scanning Behavior Eliana Real, Sophie Faure, Emmanuel Donnadieu and Jérôme Delon This information is current as of September 25, 2021. J Immunol 2007; 179:5649-5652; ; doi: 10.4049/jimmunol.179.9.5649 http://www.jimmunol.org/content/179/9/5649 Downloaded from References This article cites 18 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/179/9/5649.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY CUTTING EDGE Cutting Edge: Atypical PKCs Regulate T Lymphocyte Polarity and Scanning Behavior1 Eliana Real,*† Sophie Faure,*† Emmanuel Donnadieu,*† and Je´roˆme Delon2*† Leukocyte locomotion is a polarized process with diverse integrates adhesion to traction generation (5). The uropod, at regulatory assemblies segregating along an anterior-poste- the back of the cell, provides the contractile forces that allow the rior axis that defines two regions within the cell, the lead- cell body to translocate (6). ing edge and the uropod. However, the mechanisms that The Par6-PKC␨ complex (where PKC is protein kinase C) generate T cell asymmetry downstream of chemokine re- has been described in many cell types as a broad regulator of polarity (7). The function of this complex resides in the enzy- ceptors are ill defined. In this study we show that the atyp- ␨ ␫ ical protein kinases C (aPKCs), PKC␫ and PKC␨, are re- matic activity of the atypical PKCs (aPKCs), PKC or PKC / Downloaded from PKC␭, which are controlled by Par6. Interestingly, previous re- quired for an early symmetry breaking step. Once the sults have shown that a minute amount of PKC␨ is activated in polarity is established, aPKCs also drive uropod forma- leukocytes exposed to chemoattractants (8, 9), raising the pos- tion. These effects depend on the interaction between Par6 sibility that the Par6-PKC␨ complex might be an effector of and aPKCs. Finally, failure to transduce aPKC-depen- polarity in these cells. We have addressed this hypothesis and dent signals reduces T cell motility and their ability to scan provide evidence that aPKCs specify the topography of the two http://www.jimmunol.org/ dendritic cells. Altogether, our findings suggest that lym- poles of the cell. phocyte motor activity is regulated by a signaling cascade that relays chemokinetic input to aPKCs. The Journal of Materials and Methods Immunology, 2007, 179: 5649–5652. Plasmid constructs The FLAG-PKC␨ constructs were purchased from Addgene. The myc-Par6 and he adaptive immune system is composed of dispersed GST-PKC␫ constructs were described (10, 11). The pEGFPmax (Amaxa) or cellular elements that continuously survey peripheral pEGFP-C3 (Clontech Laboratories) vectors were used in cotransfection tissues for potential pathogenic agents and convey in- experiments. T by guest on September 25, 2021 formation to one another to effectively respond to infections. RT-PCR Naive T lymphocytes traffic through an extensive network of Total RNA from human T cells was extracted using TRIzol reagent (Invitrogen secondary lymphoid organs, dwelling for limited periods in spe- Life Technologies). cDNA was prepared using the Advantage RT-for-PCR kit cific compartments and then returning to the blood circulation (BD Biosciences). PCR amplification of cDNA samples was conducted using if no Ag is detected. specific primers and PCR products were resolved on a 1% agarose gel. The homeostatic chemokines CCL21 and CCL19 are the Stimulatory and staining reagents main high endothelial venule (HEV)3 signatures in peripheral CCL19 was provided by PeproTech and used for cell stimulation at 100 ng/ml lymph nodes (1). All naive T cells express the matching receptor in solution under stirring conditions at 37°C. F-actin was stained with phalloi- CCR7 and are able to arrest at HEVs. It has been recently din conjugated to TRITC or Texas Red (Molecular Probes). The anti-FLAG shown that dendritic cells (DCs) bear CCL21 at their surfaces Ab conjugated to FITC was purchased from Sigma-Aldrich. The anti-PKC␣ was from BD PharMingen. The anti-Myc and anti-PKC␨ (clone C-20) Abs (2) and can secrete CCL19 (3). Therefore, it is likely that DCs were from Santa Cruz Biotechnology. Anti-GST was from Amersham can also be a source of chemokines in vivo. Biosciences. Fluorescent anti-rabbit or anti-mouse IgG were all from Cell migration depends on the functional specialization of Jackson ImmunoResearch. discrete regions and is therefore intrinsically related to cell po- Cells larity (4). The protrusive forces for locomotion are generated at Human peripheral blood T lymphocytes (PBTs) were isolated by Ficoll density the lamellipodium, owing to an intense actin-polymerizing ac- gradient centrifugation from healthy blood donors. PBMCs were depleted of tivity concentrated at this site. The middle of the cell or lamella non-T cells with a human T lymphocyte enrichment set (BD Biosciences). DCs *Institut Cochin, Universite´Paris Descartes, Centre National de la Recherche Scientifique, 2 Address correspondence and reprint requests to Dr. Je´roˆme Delon, Institut Cochin, De´- Unite´Mixte de Recherche 8104 and †Institut National de la Sante´et de la Recherche partement de Biologie Cellulaire, Institut National de la Sante´et de la Recherche Me´dicale, Me´dicale, Unite´567, Paris, France Unite´567, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 8104, 22 Rue Me´chain, 75014 Paris, France. E-mail address: [email protected] Received for publication March 20, 2007. Accepted for publication September 4, 2007. 3 Abbreviations used in this paper: HEV, high endothelial venule; aPKC, atypical protein The costs of publication of this article were defrayed in part by the payment of page charges. kinase C; DC, dendritic cell; KD, kinase dead; NT, N terminal; PBT, peripheral blood T This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. lymphocyte; PKC, protein kinase C; WT, wild type. Section 1734 solely to indicate this fact. 1 This work was supported by Centre National de la Recherche Scientifique, Institut Na- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 tional de la Sante´et de la Recherche Me´dicale, and Ligue Nationale Contre le Cancer. E.R. is a recipient of a fellowship from the Portuguese Foundation for Science and Technology and a student from the Gulbenkian Ph.D. Program in Biomedicine. www.jimmunol.org 5650 CUTTING EDGE: CONTROL OF T CELL MIGRATION BY aPKCs GFPϩ cells cotransfected with the appropriate construct were compared. Cells were washed and added to the DC layer. Image acquisition was performed with the MetaFluor imaging software (Universal Imaging). Quantification of the tra- jectories of individual T cells was performed with the Metamorph (Universal Imaging) or Imaris (Bitplane) tracking software. A linear fit to the trace corre- sponding to the mean distance traveled vs the square root of time was overlaid. This slope represents the motility coefficient, M, given by M ϭ x2/4t where x ϭ mean distance from origin at time t. Results and Discussion FIGURE 1. T cells acquire a polarized morphology upon CCR7 signaling. Chemokine-driven T cell polarization is a multistep process PBTs were stimulated in suspension with CCL19, fixed, and stained with phal- We have analyzed cellular morphologies and F-actin distribu- loidin. The panel shows representative examples of T cell morphology and F- actin distribution in an inverted black and white scale (low fluorescence level in tion during T cell polarization induced by CCL19. Upon stim- white and high level in black) at successive time points. ulation, F-actin accumulated within seconds in multiple pseu- dopods around the periphery of cells (Fig. 1). One minute after the initial stimulus, F-actin had polarized to one side in most were provided by Immuno-Designed Molecules (IDM) (12). All cells were cul- cells and an axis of polarity was already clearly distinguishable. tured in complete RPMI 1640 medium containing 10% AB serum (Cambrex) During the minute that followed, an important fraction of the and were transfected as described (13). cells developed a uropod that was even reinforced at later times. Downloaded from Flow cytometry Thus, T cell polarization occurs through a series of sequential steps. T cells cotransfected with GFP together with indicated constructs were stimu- lated or not stimulated, fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and blocked. They were stained with relevant Abs and analyzed aPKCs drive T lymphocyte polarization on a FACScan (BD Biosciences). Studies in diverse model systems have converged into a model http://www.jimmunol.org/ Microscopy whereby Cdc42 and PI3K act in concert to control polarization For immunocytochemistry stainings, PBTs were processed as described in the with respect to external gradients (14). We initially hypothe- previous paragraph. Slides were then mounted on coverslips and samples were sized that PI3K could mediate these changes in T cells. How- visualized on an Eclipse TE2000-E inverted microscope (Nikon) equipped ever, an in-depth analysis performed in our laboratory has with a cooled charge-coupled device camera (CoolSNAPHQ; Photometrics).