Divergent lymphocyte signalling revealed by a powerful new tool for analysis of time‐lapse microscopy

We describe a new approach for interactive analysis of time‐lapse microscopy, and apply this approach to elucidating whether polarity regulation is conserved between epithelial cells and lymphocytes. A key advantage of our analysis platform, ‘TACTICS’, is the capacity to visualize individual data points in the context of large data sets, similar to standard approaches in flow cytometry. Scatter plots representing microscopic parameters or their derivations such as polarity ratios are linked to the original data such that clicking on each dot enables a link to images and movies of the corresponding cell. Similar to flow cytometric analysis, subsets of the data can be gated and reanalyzed to explore the relationships between different parameters. TACTICS was used to dissect the regulation of polarization of the cell fate determinant, Numb, in migrating lymphocytes. We show here that residues of Numb that are phosphorylated by atypical protein kinase C (aPKC) to mediate apicobasal polarity in epithelial cells are not required for polarization of Numb in T cells, indicating that the role of aPKC is not conserved between lymphocytes and epithelia.

[1]  M. Ciofani,et al.  Notch promotes survival of pre–T cells at the β-selection checkpoint by regulating cellular metabolism , 2005, Nature Immunology.

[2]  Thomas M. Schmitt,et al.  Obligatory Role for Cooperative Signaling by Pre-TCR and Notch during Thymocyte Differentiation1 , 2004, The Journal of Immunology.

[3]  Min Zhang,et al.  Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells , 2005, The Journal of experimental medicine.

[4]  Sudha Kumari,et al.  Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function , 2012, Proceedings of the National Academy of Sciences.

[5]  K. Kaibuchi,et al.  Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. , 2007, Developmental cell.

[6]  A. Zlotnik,et al.  A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. , 1993, Journal of immunology.

[7]  Y. Jan,et al.  The Notch Regulator Numb Links the Notch and TCR Signaling Pathways1 , 2005, The Journal of Immunology.

[8]  Michael D. Cahalan,et al.  The Immunological Synapse: a Dynamic Platform for Local Signaling , 2010, Journal of Clinical Immunology.

[9]  H. Piwnica-Worms,et al.  The Polarity Protein Par1b/EMK/MARK2 Regulates T Cell Receptor-Induced Microtubule-Organizing Center Polarization1 , 2009, The Journal of Immunology.

[10]  M. Takeichi,et al.  Willin and Par3 cooperatively regulate epithelial apical constriction through aPKC-mediated ROCK phosphorylation , 2011, Nature Cell Biology.

[11]  L. Couturier,et al.  Endocytosis by Numb breaks Notch symmetry at cytokinesis , 2012, Nature Cell Biology.

[12]  S. Russell,et al.  The Scribble and Par complexes in polarity and migration: friends or foes? , 2006, Trends in cell biology.

[13]  A. Bhandoola,et al.  Eliciting the T cell fate with Notch. , 2010, Seminars in immunology.

[14]  J. Shabanowitz,et al.  Par3 Controls Epithelial Spindle Orientation by aPKC-Mediated Phosphorylation of Apical Pins , 2010, Current Biology.

[15]  Daniel Day,et al.  A method for prolonged imaging of motile lymphocytes , 2009, Immunology and cell biology.

[16]  M. Baron Endocytic routes to Notch activation. , 2012, Seminars in cell & developmental biology.

[17]  G. Sauvageau,et al.  An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity. , 2010, Cell stem cell.

[18]  Philippe Bousso,et al.  Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo. , 2012, Immunity.

[19]  Peter Kovesi,et al.  Phase Preserving Denoising of Images , 1999 .

[20]  Min Zhang,et al.  Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NF-κB transcription factors , 2007, Nature Immunology.

[21]  L. Guilgur,et al.  Drosophila aPKC is required for mitotic spindle orientation during symmetric division of epithelial cells , 2012, Development.

[22]  D. Rodeberg,et al.  Antiparallel Segregation of Notch Components in the Immunological Synapse Directs Reciprocal Signaling in Allogeneic Th:DC Conjugates1 , 2007, The Journal of Immunology.

[23]  Mario Niepel,et al.  Adaptive informatics for multi-factorial and high content biological data , 2011, Nature Methods.

[24]  S. Valitutti,et al.  Activation of the Ancestral Polarity Regulator Protein Kinase Cζ at the Immunological Synapse Drives Polarization of Th Cell Secretory Machinery toward APCs , 2010, The Journal of Immunology.

[25]  S. Russell How polarity shapes the destiny of T cells , 2008, Journal of Cell Science.

[26]  L. G. Morelli,et al.  Computational Approaches to Developmental Patterning , 2012, Science.

[27]  D. Grier,et al.  Methods of Digital Video Microscopy for Colloidal Studies , 1996 .

[28]  Matthew F Krummel,et al.  Maintenance and modulation of T cell polarity , 2006, Nature Immunology.

[29]  Thomas M. Schmitt,et al.  Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. , 2002, Immunity.

[30]  N. Martín-Blanco,et al.  The endocytic adaptor Numb regulates thymus size by modulating pre-TCR signaling during asymmetric division. , 2010, Blood.

[31]  N. Otsu A threshold selection method from gray level histograms , 1979 .

[32]  J. Ahringer,et al.  Cell Polarity in Eggs and Epithelia: Parallels and Diversity , 2010, Cell.

[33]  B. Roysam,et al.  Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues , 2011, Immunology and cell biology.

[34]  A. Durán,et al.  Atypical protein kinase C (aPKCζ and aPKCλ) is dispensable for mammalian hematopoietic stem cell activity and blood formation , 2011, Proceedings of the National Academy of Sciences.

[35]  John T. Chang,et al.  Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division. , 2011, Immunity.

[36]  Michael D. Cahalan,et al.  A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System , 2012, Science.

[37]  John T. Chang,et al.  Asymmetric T Lymphocyte Division in the Initiation of Adaptive Immune Responses , 2007, Science.

[38]  M. Smyth,et al.  A network of PDZ-containing proteins regulates T cell polarity and morphology during migration and immunological synapse formation. , 2005, Immunity.

[39]  Pilar Martín,et al.  Control of T helper 2 cell function and allergic airway inflammation by PKCzeta. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Alex Mogilner,et al.  Cell Polarity: Tension Quenches the Rear , 2012, Current Biology.

[41]  J. Feramisco,et al.  Targeted Deletion of Protein Kinase C λ Reveals a Distribution of Functions between the Two Atypical Protein Kinase C Isoforms1 , 2004, The Journal of Immunology.

[42]  John T. Chang,et al.  Asymmetric Cell Division of T Cells upon Antigen Presentation Uses Multiple Conserved Mechanisms , 2010, The Journal of Immunology.

[43]  P. Thibault,et al.  aPKC‐mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb , 2007, The EMBO journal.

[44]  Robert Spence,et al.  The Attribute Explorer: information synthesis via exploration , 1998, Interact. Comput..

[45]  H. Petrie,et al.  Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4, and CD8 acquisition in vivo and in vitro , 1990, The Journal of experimental medicine.

[46]  Pilar Martín,et al.  Crosstalk between PKCζ and the IL4/Stat6 pathway during T‐cell‐mediated hepatitis , 2004, The EMBO journal.

[47]  M. Perez-Moreno,et al.  Epithelial cell polarity, stem cells and cancer , 2011, Nature Reviews Cancer.

[48]  T. Pawson,et al.  Immature T-cell clustering and efficient differentiation require the polarity protein Scribble , 2010, Proceedings of the National Academy of Sciences.

[49]  Roy A. Ruddle,et al.  Visualization of Parameter Space for Image Analysis , 2011, IEEE Transactions on Visualization and Computer Graphics.

[50]  J. Oliaro,et al.  Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function , 2012, Clinical & developmental immunology.

[51]  T Aach,et al.  Automated segmentation and tracking for large‐scale analysis of focal adhesion dynamics , 2011, Journal of microscopy.

[52]  R. Hopkins,et al.  Spontaneous release of a factor with properties of T cell growth factor from a continuous line of primate tumor T cells. , 1981, Journal of immunology.

[53]  C. Redon,et al.  γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis. , 2011, Mutation research.

[54]  J. Delon,et al.  Cutting Edge: Atypical PKCs Regulate T Lymphocyte Polarity and Scanning Behavior1 , 2007, The Journal of Immunology.

[55]  Arup K Chakraborty,et al.  Understanding the Structure and Function of the Immunological Synapse , 2010 .

[56]  K. Mechtler,et al.  The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl , 2003, Nature.

[57]  B. Fowlkes,et al.  Early T lymphocytes. Differentiation in vivo of adult intrathymic precursor cells , 1985, The Journal of experimental medicine.

[58]  K. Harvey,et al.  Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms , 2010, Current Biology.