presentation_1.pdf

<p>Time-lapse imaging of cell colonies in microfluidic chambers provides time series of bioimages, i.e., biomovies. They show the behavior of cells over time under controlled conditions. One of the main remaining bottlenecks in this area of research is the analysis of experimental data and the extraction of cell growth characteristics, such as lineage information. The extraction of the cell line by human observers is time-consuming and error-prone. Previously proposed methods often fail because of their reliance on the accurate detection of a single cell, which is not possible for high density, high diversity of cell shapes and numbers, and high-resolution images with high noise. Our task is to characterize subpopulations in biomovies. In order to shift the analysis of the data from individual cell level to cellular groups with similar fluorescence or even subpopulations, we propose to represent the cells by two new abstractions: the particle and the patch. We use a three-step framework: preprocessing, particle tracking, and construction of the patch lineage. First, preprocessing improves the signal-to-noise ratio and spatially aligns the biomovie frames. Second, cell sampling is performed by assuming particles, which represent a part of a cell, cell or group of contiguous cells in space. Particle analysis includes the following: particle tracking, trajectory linking, filtering, and color information, respectively. Particle tracking consists of following the spatiotemporal position of a particle and gives rise to coherent particle trajectories over time. Typical tracking problems may occur (e.g., appearance or disappearance of cells, spurious artifacts). They are effectively processed using trajectory linking and filtering. Third, the construction of the patch lineage consists in joining particle trajectories that share common attributes (i.e., proximity and fluorescence intensity) and feature common ancestry. This step is based on patch finding, patching trajectory propagation, patch splitting, and patch merging. The main idea is to group together the trajectories of particles in order to gain spatial coherence. The final result of CYCASP is the complete graph of the patch lineage. Finally, the graph encodes the temporal and spatial coherence of the development of cellular colonies. We present results showing a computation time of less than 5 min for biomovies and simulated films. The method, presented here, allowed for the separation of colonies into subpopulations and allowed us to interpret the growth of colonies in a timely manner.</p

Inhibition of actin-bundling using Fascin-specific nanobodies displays a significant reduction of virus transmission.

(A-C) 293T cells were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmid pCMVHT1M-ΔEnv encoding all HTLV-1 proteins except env (Δenv) pseudotyped with VSV-G. Cells were co-transfected with V5-tagged expression plasmids encoding a mitochondrial outer membrane (MOM) sequence and nanobodies (Nb) targeting Fascin (MOMFASNb2 (FASNb2), 0.5μg; or MOMFASNb5 (FASNb5), 0.25; 0.5; 1μg), or a control nanobody (MOMGFPNb (GFPNb), 0.5μg). At 48h post transfection, luciferase assays, ELISA and western blot were performed as described in Fig 1A. The means of four independent experiments ± standard error (SE) are shown and were compared to the control (GFPNb) using Student’s t-test (**: p(A) Luciferase activity (cell-to-cell transmission). (B) Detection of gag p19 release by ELISA. (C) Detection of Fascin, Tax-1, V5 (MOM-Nanobodies) and gag by western blot. β-actin (ACTB) served as control. (D-F) Confocal laser scanning microscopy of 293T cells co-transfected with pCMVHT1M-ΔEnv and with V5-tagged expression plasmids encoding a MOM-sequence and nanobodies FASNb2, FASNb5 or GFPNb (control). Stains of nanobodies (V5; green) and (D) mitochondria (red), (E) Fascin (turquoise), and (F) gag p19 (turquoise) and the merges of the respective stains are shown. DAPI (blue) served as control.</p

Knockdown of Fascin does not influence cell aggregation between Jurkat T-cells and Raji/CD4⁺ B-cells.

(A-C) Conjugate formation between transfected Jurkat T-cells and co-cultured Raji/CD4+ B-cells was quantitated by flow cytometry. Jurkat T-cells were transfected with one of two different Tax-expression constructs (GFP-Tax; pEFTax) and one of two different shRNAs encoding IRES-EGFP and targeting Fascin (shFascin5, shFascin4) or a control (shNonsense). After 24h, Jurkat T-cells were co-cultured with Raji/CD4+ B-cells (ratio 1:1) for 1h at 37°C. Co-cultures were fixed and stained with anti-CD3-AlexaFluor700 (for Jurkat T-cells) and anti-HLA-DR-PacificBlue antibodies (for Raji/CD4+ B-cells) to differentiate between the two cell types. (A) Detection of GFP encoded by GFP-Tax and/or the shRNA construct (shNonsense) was used to differ between transfected (GFP+) and untransfected (GFP-) cells. Cell-cell conjugates were identified in GFP+ (Tax-positive) and GFP- (Tax-negative) gates as double-positive signals (HLA-DR+CD3+) and normalized on the total number of Jurkat T-cells (CD3+; see S6 Fig). The means of four independent experiments ± standard error (SE) are shown. Student’s t-tests were performed (*: p(B) Control cells (shNonsense) compared to Fascin-repressed cells (shFascin5, shFascin4) within Tax-positive cells of A). (C) Detection of Fascin, GFP-Tax and Tax-1 in transfected Jurkat T-cells by western blot. β-actin (ACTB) served as control.</p

Repression of endogenous Fascin impairs virus release and HTLV-1 reporter activity independent of the envelope type used.

<p><b>(A)</b> Scheme of experimental setup using single-cycle replication-dependent reporter vectors with 293T cells. <b>(A-D)</b> Stable 293T cells (sh293T) that carry one of two different shRNAs targeting Fascin (shFascin5, shFascin4) or the control (shNonsense) were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmids pCMVHT1M containing either HTLV-1 wildtype env (wt) or lacking env (Δenv). The latter were pseudotyped with VSV-G or supplemented with pcDNA3 (control). Cells were co-transfected with pEFTax or pEF (mock). Luciferase assays, ELISA and western blot were performed as shown in <b>A)</b>. Values were normalized on those obtained from shNonsense 293T cells transfected with inluc+wt, and the mean of four independent experiments ± standard error (SE) is shown. Values were compared to the respective mock (shNonsense+pEF or shNonsense+pEFTax) using Student’s t-test (*: p<0.05, **: p<0.01). <b>(B)</b> Luciferase activity (cell-to-cell transmission). Right panel: enlargement of dotted box. <b>(C)</b> Detection of gag p19 release by ELISA. <b>(D)</b> Detection of Fascin, Tax-1 and gag by western blot. Hsp90 α/β and β-actin (ACTB) served as control. Numbers indicate densitometric analysis of Fascin detection normalized on Hsp90 α/β.</p

Fascin knockdown in chronically HTLV-1-infected T-cells impairs virus release and infection of co-cultured T-cells.

<p><b>(A)</b> Scheme of co-culture experiments using HuT-102 cells and reporter Jurkat T-cells. <b>(A-C)</b> Chronically HTLV-1-infected HuT-102 cells with stable repression of Fascin (shFascin5) or control cells (untreated, shNonsense) were co-cultured with Jurkat T-cells that had been transfected 24h earlier with luciferase reporter vectors carrying the core promoter U3R of HTLV-1 (pGL3-U3R) or a control (pGL3-Basic). After 48h, relative light units (RLU) normalized on protein content and on background activity (pGL3-Basic) were determined. <b>(B)</b> Luciferase activity of co-cultures. The means of four independent experiments ± standard error (SE) are shown and compared to shNonsense using Student’s t-test (*: p<0.05). <b>(C)</b> Detection of Fascin and Tax-1 by western blot. β-actin (ACTB) served as control. <b>(D)</b> Scheme of infection experiments using MT-2 cells and Jurkat T-cells. <b>(D-F)</b> Chronically HTLV-1-infected MT-2 cells with stable repression of Fascin (shFascin5) or control cells (untreated, shNonsense) were co-cultured with Jurkat T-cells for 1h at 37°C. Thereafter, cells were stained for CD25 and gag p19 and analyzed by flow cytometry to detect the number of newly-infected Jurkat T-cells (CD25^- gagp19⁺). <b>(E)</b> Gag-positive Jurkat T-cells (%) co-cultured with the respective MT-2 cells. The means of four independent experiments ± standard error (SE) are shown and compared to shNonsense using Student’s t-test (*: p<0.05). <b>(F)</b> Detection of Fascin and Tax-Env by western blot. β-actin (ACTB) served as control. <b>(G-H)</b> Gag p19 ELISA using supernatants of <b>(G)</b> stable MT-2 cells (shNonsense, shFascin5) and <b>(H)</b> differently treated MT-2 cells. Equal numbers of cells (10⁵ cells/ml) were seeded and treated with cytochalasin D, nocodazole (each 5μM), or DMSO (control) for 48h. The means of at least four independent experiments ± standard error (SE) are shown and were compared to control cells (shNonsense or untreated) using Student’s t-test (*: p<0.05, **: p<0.01). <b>(I)</b> Detection of Fascin, Tax-Env and gag by western blot. Hsp90 α/β served as control.</p

Repression of Tax-induced Fascin results in reduction of both virus release and cell-to-cell transmission.

<p><b>(A)</b> Scheme of experimental setup using single-cycle replication-dependent reporter vectors in Jurkat T-cells and Raji/CD4⁺ B-cells. <b>(A-D)</b> Jurkat T-cells were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmid pCMVHT1M encoding HTLV-1 with wildtype env (wt). Cells were co-transfected with pEFTax or pEF (mock) and one of two different shRNAs targeting Fascin (shFascin5, shFascin4) or the control (shNonsense). Luciferase assays, ELISA and western blot were performed as depicted in <b>A)</b>. <b>(B)</b> Luciferase activity (cell-to-cell transmission). The means of four independent experiments ± standard error (SE) are shown and were compared to the respective mock (shNonsense+pEF or shNonsense+pEFTax) using Student’s t-tests (*: p<0.05). <b>(C)</b> Detection of gag p19 release by ELISA. A representative experiment is shown. <b>(D)</b> Detection of Fascin, Tax-1 and gag by western blot. Hsp90 α/β served as control. Numbers indicate densitometric analysis of Fascin detection normalized on Hsp90 α/β. <b>(E-F)</b> Jurkat T-cells were co-transfected with the reporter vector pCRU5HT1M-inluc (inluc), the packaging plasmid pCMVHT1M (wt), pEFTax, and one of three different V5-tagged expression plasmids encoding a MOM-sequence and nanobodies FASNb2, FASNb5 or GFPNb (control). Luciferase assays and western blot were performed as depicted in <b>A)</b>. <b>(E)</b> Luciferase activity (cell-to-cell transmission). The means of six independent experiments ± standard error (SE) were normalized and compared to control samples (GFPNb) using Student’s t-tests (**: p<0.01). <b>(F)</b> Detection of Fascin, Tax-1, V5-tagged nanobodies and gag by western blot. β-actin (ACTB) served as control.</p

Model of Fascin’s role during HTLV-1 transmission.

<p>HTLV-1-infected T-cells express the transactivator Tax that upregulates Fascin expression via the NF-κB signaling pathway. Not only Tax-induced Fascin, but also endogenous (endog.) Fascin is required for virus release and cell-to-cell transmission. Beyond, adhesion of infected cells occurs Fascin-dependently, which may favor dissemination of infected cells <i>in vivo</i>. Functionally, Fascin makes room for gag clusters reminiscent of viral biofilms at the virological synapse (VS) and Fascin-containing short-distance membrane extensions clutch uninfected T-cells. Additionally, Fascin localizes with gag in long-distance connections between chronically infected and newly infected T-cells. A “mini VS” may be shaped at the tip of the long-distance connection towards the target cell. Overall, Fascin seems to be important for the transport of viral proteins to foster polarized budding, virus release and cell-to-cell transmission of HTLV-1.</p

Knockdown of Fascin does not affect cell aggregation but decreases cell adhesion of MT-2 cells in co-cultures with Jurkat T-cells.

<p><b>(A)</b> Scheme of immunofluorescence stains with MT-2 cells and Jurkat T-cells. <b>(A-D)</b> MT-2 cells with stable repression of Fascin (shFascin5) or control cells (shNonsense) were co-cultured with Calcein-stained Jurkat T-cells for 1h at 37°C either on poly-L-lysine- (a-e, k-o) or on fibronectin-coated (f-j, p-t) coverslips. <b>(B)</b> Immunofluorescence stainings of co-cultures. Jurkat cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Stainings of gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments are shown. <b>(C-D)</b> Results of automatic image analysis (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005916#ppat.1005916.s001" target="_blank">S1 Fig</a>). The means of three independent experiments ± standard error (SE) are shown and were compared to shNonsense using Student’s t-tests (*: p<0.05, **: p<0.01). <b>(C)</b> Percentage of MT-2 cells (shNonsense, shFascin5) with 0 to ≥5 cell-to-cell contacts to co-cultured Jurkat T-cells on the different coatings. <b>(D)</b> Percentage of adherent MT-2 cells (shNonsense, shFascin5) on the different coatings normalized on MT-2 shNonsense.</p

Fascin and gag localize at cell-cell contacts and in long-distance connections between infected and uninfected T-cells.

<p><b>(A)</b> Confocal laser scanning microscopy of HTLV-1-infected MS-9 cells co-cultured with Jurkat T-cells. Jurkat T-cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Cells were co-cultured for 0 (k-p), 30 (a-j) or 60min (q-w) on poly-L-lysine-coated coverslips prior to drying (20min), fixation, and staining. <b>(A)</b> Stainings of Calcein (green), gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments showing clusters of Fascin (a-e) and gag (a-j), Fascin clutches (f-j) or long-distance connections (k-w) are depicted. Thin white arrows indicate gag of an infected cell clustering at the cell-cell contact towards an uninfected cell; framed white arrows indicate short-distance Fascin-containing membrane extensions; and thick white arrows indicate long-distance protrusions between uninfected and infected cells. Protrusions (k-o; q-u) were examined in more detail, and (p) the stains of gag and Fascin within the protrusion shown in (n) were enlarged; further, a region of interest (v-w) was analyzed showing the intensities of gag- (blue) and Fascin- specific (red) fluorescences shown in (t). <b>(B)</b> Detection of Fascin, Tax-1 and gag in HTLV-1-infected MS-9 cells and uninfected Jurkat T-cells by western blot. Hsp90 α/β served as control.</p