Journal publications

2018
  • A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

    K. Ng, M. Yui, A. Mehta, S. Siu, B. Irwin, S. Pease, S. Hirose, M. Elowitz, E. Rothenberg,

    eLife, 2018 (7) e37851, doi: 10.7554/eLife.37851

    Keywords: Cell fate, time-lapse microscopy, single-cell imaging

    Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.
  • Drug-induced aneuploidy and polyploidy is a mechanism of disease relapse in MYC/BCL2-addicted diffuse large B-cell lymphoma

    S. Islam, A. Paek, M. Hammer, S. Rangarajan, R. Ruiitenbeek, L. Cooke, E. Weterings, D. Mahadevan,

    Oncotarget, 2018 (9) 35875, doi: 10.18632/oncotarget.26251

    Keywords: DLBCL, aurora kinase, aneuploidy-polyploidy, time-lapse microscopy, single-cell imaging

    Double-hit (DH) or double-expresser (DE) lymphomas are high-grade diffuse large B-cell lymphomas (DLBCL) that are mostly incurable with standard chemoimmunotherapy due to treatment resistance. The generation of drug-induced aneuploid/polyploid (DIAP) cells is a common effect of anti-DLBCL therapies (e.g. vincristine, doxorubicin). DIAP cells are thought to be responsible for treatment resistance, as they are capable of re-entering the cell cycle during off-therapy periods. Previously we have shown that combination of alisertib plus ibrutinib plus rituximab can partially abrogate DIAP cells and induce cell death. Here, we provide evidence that DIAP cells can reenter the cell cycle and escape cell death during anti-DLBCL treatment. We also discuss MYC/BCL2 mediated molecular mechanism that underlie treatment resistance. We isolated aneuploid/polyploid populations of DH/DE-DLBCL cells after treatment with the aurora kinase (AK) inhibitor alisertib. Time-lapse microscopy of single polyploid cells revealed that following drug removal, a subset of these DIAP cells divide and proliferate by reductive cell divisions, including multipolar mitosis, meiosis-like nuclear fission and budding. Genomic, proteomic, and kinomic profiling demonstrated that alisertib-induced aneuploid/polyploid cells up-regulate DNA damage, DNA replication and immune evasion pathways. In addition, we identified amplified receptor tyrosine kinase and T-cell receptor signaling, as well as MYC-mediated dysregulation of the spindle assembly checkpoints RanGAP1, TPX2 and KPNA2. We infer that these factors contribute to treatment resistance of DIAP cells. These findings provide opportunities to develop novel DH/DE-DLBCL therapies, specifically targeting DIAP cells.
  • Converse Smith-Martin cell cycle kinetics by transformed B Lymphocytes

    K. Pham, A. Kan, L. Whitehead, R. Hennessy, K. Rogers, P. Hodgkin,

    Cell Cycle, 2018 (17) 2041, doi: 10.1080/15384101.2018.1511511

    Keywords: Cell cycle, Smith-Martin model, FUCCI, time-lapse microscopy

    Recent studies using direct live cell imaging have reported that individual B lymphocytes have correlated transit times between their G1 and S/G2/M phases. This finding is in contradiction with the influential model of Smith and Martin that assumed the bulk of the total cell cycle time variation arises in the G1 phase of the cell cycle with little contributed by the S/G2/M phase. Here we extend these studies to examine the relation between cell cycle phase lengths in two B lymphoma cell lines. We report that transformed B lymphoma cells undergo a short G1 period that displays little correlation with the time taken for the subsequent S/G2/M phase. Consequently, the bulk of the variation noted for total division times within a population is found in the S/G2/M phases and not the G1 phase. Models that reverse the expected source of variation and assume a single deterministic time in G1 followed by a lag + exponential distribution for S/G2/M fit the data well. These models can be improved further by adopting two sequential distributions or by using the stretched lognormal model developed for primary lymphocytes. We propose that shortening of G1 transit times and uncoupling from other cell cycle phases may be a hallmark of lymphocyte transformation that could serve as an observable phenotypic marker of cancer evolution.
  • Nongenetic origins of cell-to-cell variability in B lymphocyte proliferation

    S. Mithcell, K. Roy, T. Zangle, A. Hoffman,

    PNAS, 2018 (115) E2888, doi: 10.1073/pnas.1715639115

    Keywords: Time-lapse microscopy, B cells, proliferation, cell lineage trees

    Rapid antibody production in response to invading pathogens requires the dramatic expansion of pathogen-derived antigen-specific B lymphocyte populations. Whether B cell population dynamics are based on stochastic competition between competing cell fates, as in the development of competence by the bacterium Bacillus subtilis, or on deterministic cell fate decisions that execute a predictable program, as during the development of the worm Caenorhabditis elegans, remains unclear. Here, we developed long-term live-cell microscopy of B cell population expansion and multiscale mechanistic computational modeling to characterize the role of molecular noise in determining phenotype heterogeneity. We show that the cell lineage trees underlying B cell population dynamics are mediated by a largely predictable decision-making process where the heterogeneity of cell proliferation and death decisions at any given timepoint largely derives from nongenetic heterogeneity in the founder cells. This means that contrary to previous models, only a minority of genetically identical founder cells contribute the majority to the population response. We computationally predict and experimentally confirm nongenetic molecular determinants that are predictive of founder cells’ proliferative capacity. While founder cell heterogeneity may arise from different exposure histories, we show that it may also be due to the gradual accumulation of small amounts of intrinsic noise during the lineage differentiation process of hematopoietic stem cells to mature B cells. Our finding of the largely deterministic nature of B lymphocyte responses may provide opportunities for diagnostic and therapeutic development.
  • 2017
    • Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

      A. Moussy, J. Cosette, R. Parmentier, C. da Silva, G. Corre, A. Richard, O. Gandrillon, D. Stockholm, A. Paldi,

      PLoS Biol, 2017 (15) e2001867, doi: 10.1371/journal.pbio.2001867

      Keywords: Time-lapse microscopy, single-cell imaging, cell fate

      Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34 + cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned).
    • Combination of imaging flow cytometry and time-lapse microscopy for the study of label-free morphology dynamics of hematopoietic cells

      J. Cosette, A. Moussy, A. Paldi, D. Stockholm,

      Cytometry Part A, 2017 (91) 254-260, doi: 10.1002/cyto.a.23064

      Keywords: Time-lapse microscopy

      Cell differentiation is a longitudinal and dynamic process. Studying and quantifying such a process require tools combining precise time resolution and statistical power. Imaging flow cytometry (IFC) provides statistically significant number of microscopy images of individual cells in a sample at a given time point. Time-lapse microscopy (TLM) is the method of choice for studying the dynamics of cell processes at a high temporal, but low statistical resolution. In this work, we show that the dynamic changes of cord-blood derived CD341 cells in response to cytokine stimulation can be successfully studied, in a label-free way, by the combination of the IFCs statistical power and the TLM’s high time resolution. Cell morphology phenotypes were quan- tified through roundness and surface area, measured both in IFC and with a home- made segmentation algorithm in TLM. Two distinct morphologies—polarized and round—were observed in cord-blood derived CD341. We show that some cells have the ability to fluctuate between these morphologies, suggesting that the apparent stable composition of round and polarized cells may actually represent a dynamic equi- librium. This example demonstrates that the different resolutions and modalities of IFC and TLM are complementary and allow the study of complex dynamic biological processes.
    • Imaging asymmetric T cell division

      M. Charnley, S. Russell,

      Methods and Protocols: The immune Synapse, Springer Protocols, New York, 2017 383-398, doi: 10.1007/978-1-4939-6881-7_23

      Keywords: Time-lapse imaging, fluorescence microscopy, asymmetric cell division, cell polarity

      Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving. Here we describe the methods currently applied to monitor and measure ACD of developing and activated T cells. We provide an overview of approaches for capturing cells in the process of cytokinesis in vivo, ex vivo, or during in vitro culture. We provide methods for in vitro fixed immunofluorescent staining and for time-lapse analysis. We provide an overview of the different approaches for quantification of ACD of lymphocytes, discuss the pitfalls and concerns in interpretation of these analyses, and provide detailed methods for the quantification of ACD in our group.
    • Comparative evaluation of performance measures for shading correction in time-lapse fluorescence microscopy

      L. Liu, A. Kan, P. Hodgkin,

      J. Microscopy, 2017 (266) 15-27, doi: 10.1111/jmi.12512

      Keywords: Time-lapse imaging, fluorescence microscopy

      Time-lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of in- tensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single-cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to com- pare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state-of-the-art shad- ing correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In or- der to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time-lapse fluorescence images. Based on this measure, we have pro- posed a novel shading correction method that performs better compared to well-established methods for a range of real data tested.
    2016
    • Single cell analysis of CD4+ T-cell differentiation reveals three major cell states and progressive acceleration of proliferation

      V. Prosperpio, A. Piccolo, L. Haim-Vilmovsky, G. Kar, T. Lonnberg, V. Svensson, J. Pramanik, K. Natarajan, W. Zhai, X. Zhang, G. Donati, M. Kayikci, J. Kotar, A. McKenzie, R. Montandon, O. Billker, S. Woodhouse, P. Cicuta, M. Nicodemi, S. Teichmann,

      Genome Biology, 2016 (17) 103, doi: 10.1186/s13059-016-0957-5

      Keywords: Live cell imaging, cell cycle differentiation, single cell RNA sequencing

      Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells.
    • Calcium signalling is required for erthroid enucliation

      C. Wolwer, L. Pase, S. Russell, P. Humbert,

      PLoS ONE, 2016 (11) e0146201, doi: 10.1371/journal.pone.0146201

      Keywords: Live cell imaging, immunofluorescence

      Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.
    • Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment

      H. Kueh, M. Yui, K. Ng, S. Pease, J. Zhang, S. Damle, G. Freedman, S. Siu, I. Bernstein, M. Elowitz, E. Rothenberg,

      Nature Immun., 2016 (16) 956-965, doi: 10.1038/ni.3514

      Keywords: Time-lapse imaging

      During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.
    • Elimination of HIV-1 infected cells by broadly neutralizing antibodies

      T. Bruel, F. Guivel-Benhassine, S. Amraoui, M. Malbec, L. Richard, K. Bourdic, D. A. Donahue, V. Lorin, N. Casartelli, N. Noel, O. Lambotte, H. Mouquet and O. Schwartz

      Nature Comm., 2016 (7) 10844, doi: 10.1038/ncomms10844

      Keywords:

      The Fc region of the HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for supressing viremia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through NK engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moierty. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most poetnt antibodies to clear the viral reservoir.
    2015
    • Asymmetric cell division during T cell development controls downstream fate

      K. Pham, R. Shimoni, M. Charnley, J. Ludford-Menting, E. Hawkins, K. Ramsbottom, J. Oliaro, D. Izon, S. Ting, J. Reynolds, G. Lythe, C. Molina-Paris, H. Melichar, E. Robey, P. Humbert, M. Gu, S. Russell

      JCB, 2015 (210) 933, doi: 10.1083/jcb.201502053

      Keywords: Live cell imaging, cell polarity, asymmetric cell division

      During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs speci cally during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to in uence the numbers of DN4 cells arising after the β-selection checkpoint. These ndings indicate that ACD enables the thymic microenvironment to orches- trate fate decisions related to differentiation and self-renewal.
    • Single cell dynamics causes Pareto-like effect in stimulated T cell populations

      J. Cosette, A. Moussy, F. Onodi, A. Auffret-Cariou, T. M. A. Neildez-Nguyen, A. Paldi and D. Stockholm

      Scientific Reports, 2015 (5) 17756, doi: 10.1038/srep17756

      Keywords: Time-lapse microscopy, single cell imaging, cell fate

      Cell fate choice during the process of di erentiation may obey to deterministic or stochastic rules. In order to discriminate between these two strategies we used time-lapse microscopy of individual murine CD4+T cells that allows investigating the dynamics of proliferation and fate commitment. We observed highly heterogeneous division and death rates between individual clones resulting in a Pareto-like dominance of a few clones at the end of the experiment. Commitment to the Treg fate was monitored using the expression of a GFP reporter gene under the control of the endogenous Foxp3 promoter. All possible combinations of proliferation and di erentiation were observed and resulted in exclusively GFP–, GFP+ or mixed phenotype clones of very di erent population sizes. We simulated the process of proliferation and di erentiation using a simple mathematical model of stochastic decision-making based on the experimentally observed parameters. The simulations show that a stochastic scenario is fully compatible with the observed Pareto-like imbalance in the nal population.
    • Individual human cytotoxic T Lymphocytes exhibit intraclonal heterogeneity during sustained killing

      Z. Vasconcelos, S. Muller, D. Guipouy, W. Yu, C. Christophe, S. Gadat, S. Valitutti and L. Dupre

      Cell Reports, 2015 (11) 1474-1485, doi: 10.1016/j.celrep.2015.05.002

      Keywords:

      The killing of antigen-bearing cells by clonal populations of cytotoxic T lymphocytes (CTLs) is thought to be a rapid phenomenon executed uniformly by individual CTLs. We combined bulk and single-CTL killing assays over a prolonged time period to provide the killing statistics of clonal human CTLs against an excess of target cells. Our data reveal efficiency in sustained killing at the population level, which relied on a highly heterogeneous multiple killing performance at the individual level. Although intraclonal functional heterogeneity was a stable trait in clonal populations, it was reset in the progeny of individual CTLs. In-depth mathematical analysis of individual CTL killing data revealed a substantial proportion of high-rate killer CTLs with burst killing activity. Importantly, such activity was delayed and required activation with strong antigenic stimulation. Our study implies that functional heterogeneity allows CTL populations to calibrate prolonged cytotoxic activity to the size of target cell populations.
    • Real-time tracking of cell cycle progression during CD+8 effector and memory T-cell differentiation

      I. Kinjyo, J. Qin, S. Tan, C. J. Wellard, P. Mrass, W. Ritchie, A. Doi, L. L. Cavanagh, M. Tomura, A. Sakaue-Sawano, O. Kanagawa, A. Miyawaki, P. Hodgkin and W. Weninger

      Nature Comm., 2015 (6) 7301, doi: 10.1038/ncomms7301

      Keywords:

      The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8+ T cells. During influezna virus infection in vivo, naive T cells enter CD62L intermediate state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L hi central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L hi memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.
    2014
    • Stretched cell cycle model for proliferating lymphocytes

      M. Dowling, A. Kan, S. Heinzel, J. H. S. Zhou, J. Marchingo, C. Wellard, J. Markham and P. Hodgkin

      PNAS, 2014 (111) 6377, doi: 10.1073/pnas.1322420111

      Keywords: time-lapse microscopy, FUCCI, lognormal distribution, Smith-Martin model

      Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G1 phase, and many mathematical models assume a constant time for traversing the S/G2/M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G2/M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.
    2013
    • Isolation of single mammalian cells from adherent cultures

      O. Guillaume-Gentil, T. Zambelli, J. Vorholt

      Lab Chip, 2013 (14) 402-414, doi: 10.1039/C3LC51174J

      Keywords: single-cell analysis

      The physical separation of individual cells from cell populations for single-cell analysis and proliferation is of wide interest in biology and medicine. Today, single-cell isolation is routinely applied to non-adherent cells, though its application to cells grown on a substrate remains challenging. In this report, a versatile approach for isolating single HeLa cells directly from their culture dish is presented. Fluidic force microscopy is first used to detach the targeted cell(s) via the tuneable delivery of trypsin, thereby achieving cellular detachment with single-cell resolution. The cell is then trapped by the microfluidic probe via gentle aspiration, displaced with micrometric precision and either transferred onto a new substrate or deposited into a microwell. An optimised non-fouling coating ensures fully reversible cell capture and the potential for serial isolation of multiple cells with 100% successful transfer rate (n=130) and a survival rate of greater than 95%. By providing an efficient means for isolating targeted adherent cells, the described approach offers exciting possibilities for biomedical research.
    • Divergent lymphocyte signalling revealed by a powerful new tool for analysis of time-lapse microscopy

      K. Pham, R. Shimoni, M. Ludford-Menting, C. Nowell, P. Lobachevsky, Z. Bomzon, M. Gu, T. Speed, C. McGlade, S. Russell

      Immunology and Cell Biology, 2013 (91) 70-81, doi: 10.1038/icb.2012.49

      Keywords: Time-lapse microscopy, image analysis, T-cell migration

      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.
    • Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity

      E. Hawkins, J. Oliaro, A. Kallies, G. Belz, A. Filby, T. Hogan, N. Haynes, K. Ramsbottom, V. van Ham, T. Kinwell, B. Seddon, D. Davies, D. Tarlinton, A. Lew, P. Humbert and S. Russell

      Nature Comm., 2013 (4) 1801, doi: 10.1038/ncomms2796

      Keywords:

      The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.
    • Divergent lymphocyte signalling revealed by a powerful new tool for analysis of time-lapse microscopy

      K. Pham, R. Shimoni, M. Ludford-Menting, C. Nowell, P. Lobachevsky, Z. Bomzon, M. Gu, T. Speed, C. McGlade and S. Russell

      Immunol. Cell Bio., 2013 (91) 70-81, doi: 10.1038/icb.2012.49

      Keywords: cell polarity, image analysis, T-cell migration, time-lapse microscopy, protein kinase

      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.
    2012
    • Activation-induced B cell fates are selected by intracellular stochastic competition

      K. Duffy, C. Wellard, J. Markham, J. Zhou, R. Holmberg, E. Hawkins, J. Hasbold, M. Dowling and P. Hodgkin

      Science, 2012 (335) 338-341, doi: 10.1126/science.1213230

      Keywords:

      In response to stimulation, B lymphocytes pursue a large number of distinct fates important for immune regulation. Whether each cell’s fate is determined by external direction, internal stochastic processes, or directed asymmetric division is unknown. Measurement of times to isotype switch, to develop into a plasmablast, and to divide or to die for thousands of cells indicated that each fate is pursued autonomously and stochastically. As a consequence of competition between these processes, censorship of alternative outcomes predicts intricate correlations that are observed in the data. Stochastic competition can explain how the allocation of a proportion of B cells to each cell fate is achieved. The B cell may exemplify how other complex cell differentiation systems are controlled.
    • Automated and semi-automated cell tracking: addressing portability challenges

      A. Kan, R. Chakravorty, J. Bailey, C. Leckie, J. Markham and M. Dowling

      J. Microscopy, 2011 (244) 194-213,

      Keywords:

      Cell tracking is a key task in the high-throughput quantitative study of important biological processes, such as immune system regulation and neurogenesis. Variability in cell density and dynamics in different videos, hampers portability of existing trackers across videos. We address these potability challenges in order to develop a portable cell tracking algorithm. Our algorithm can handle noise in cell segmentation as well as divisions and deaths of cells. We also propose a parameter-free variation of our tracker. In the tracker, we employ a novel method for recovering the distribution of cell displacements. Further, we present a mathematically justified procedure for determining the gating distance in relation to tracking performance. For the range of real videos tested, our tracker correctly recovers on average 96% of cell moves, and outperforms an advanced probabilistic tracker when the cell detection quality is high. The scalability of our tracker was tested on synthetic videos with up to 200 cells per frame. For more challenging tracking conditions, we propose a novel semi-automated framework that can increase the ratio of correctly recovered tracks by 12%, through selective manual inspection of only 10% of all frames in a video.
    • Femtosecond fabricated structures and devices for cell biology

      D. Day and M. Gu

      J. Opt. A, 2010 (12) 084005,

      Keywords:

      Microfabrication using femtosecond pulse lasers is enabling access to a range of structures, surfaces and materials that was not previously available for scientific and engineering applications. The ability to produce micrometre sized features directly in polymer and metal substrates is demonstrated with applications in cell biology. The size, shape and aspect ratio of the etched features can be precisely controlled through the manipulation of the fluence of the laser etching process with respect to the properties of the target material. Femtosecond laser etching of poly(methyl methacrylate) and aluminium substrates has enabled the production of micrometre resolution moulds that can be accurately replicated using soft lithography. The moulded surfaces are used in the imaging of T cells and demonstrate the improved ability to observe biological events over time periods greater than 10 h. These results indicate the great potential femtosecond pulse lasers may have in the future manufacturing of microstructured surfaces and devices.
    • Optical redistribution of microparticles and cells between microwells

      J. Baumgartl, G. M. Hannappel, D. Stevenson, D. Day, M. Gu and K. Dholakia

      Lab Chip, 2009 (9) 1334-1336,

      Keywords:

      The shaping of laser beams has developed into a powerful tool for optical micromanipulation. In this context, Airy and parabolic laser beams which follow curved trajectories have drawn considerable attention. These beams may allow clearing of microparticles through particle transport along curved paths, a concept termed optically mediated particle clearing (OMPC). In this communication we apply this concept to microparticles and cells within specially designed microwells. Our results open novel perspectives for the redistribution of cells between different media within a microfluidic environment.
    • A method for producing microgrid arrays for prolonged imaging of highly motile T cells

      D. Day, K. Pham, M. Ludford-Menting, J. Oliaro, D. Izon, S. M. Russell and M. Gu

      Immunol. Cell Bio., 2009 (87) 154-158,

      Keywords:antigen presentation, time-lapse microscopy, T-cells, lymphocytes, microfabrication

      With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non-adherent cells within the field of view. ‘Cell paddocks’ are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250x250 um2 with a height of 60 um. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte division through multiple generations of long-term interactions between T lymphocytes and dendritic cells, and of thymocyte–stromal cell interactions.