wild type ht1080  (ATCC)

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus

doi: 10.1098/rstb.2018.0225

Figure Lengend Snippet: Maintenance of migration through confining pores associates with shape change of the small and rigid G1 cell-cycle phase nucleus. ( a ) Sequence of a cell nucleus from a Fucci-positive HT1080 cell progressing through the cell cycle, as indicated by changing colour coding over 4 h. This sequence is part of electronic supplementary material, figure S2 and Movie S1. ( b ) Quantification of cell deformation (penetration) after 2 nN contact force by a bead-coupled cantilever connected to an atomic force microscope (quantified from the approach curve; electronic supplementary material, figure S1D). N = 3; 25–32 cells per indicated cell-cycle phase. ( c ) Top, example of collagen matrix (monitored by scanning electron microscopy as shown in Wolf et al. ) showing small pore areas (confining; marked in pink), adjacent to large pores (marked in yellow). Bottom, cartoon depicting cell and nucleus in collagen of heterogeneous, colour-coded pore areas, including open space for translocation of cell and nucleus (asterisk). ( d–f ) HT1080-Fucci cells migrated in collagen (1.7 mg ml −1 ) in the presence of matrix metalloproteinase (MMP) inhibitor GM6001 (except where the absence of GM6001 is indicated), as monitored by pathway microscopy. ( d ) Examples of migrating cells at different indicated cell-cycle phases. Image bar, 10 μM. ( e ) Averaged migration speed of single cells from Fucci-red or -green populations from movies of 5–24 h length at indicated conditions. Because the S-start phase covers a short, around 2 h, time period, from here on and in all further experiments G1-phase cells were compared with S/G2 cells only. N = 2–3; 40–65 cells per condition. ( f ) Nuclear segmentation of migrating HT1080-Fucci cells. Time in minutes as indicated. ( g–j ) Nuclear shape analysis. ( g ) Top, schematic illustration of the components used for the calculation of the nuclear irregularity index (NII), adapted from Filippi-Chiela . Bottom, calculation of all components that describe a circle, resulting in a NII of 2.2146. ( h ) Mean NII values per cell were computed and calculated from each nuclear shape sequence over time. ( i ) Left, scheme for calculation of the difference between subsequent NIIs as ΔNII, where rapid shape change of the nucleus results in a high ΔNII value. Right, mean values of ΔNII from each nuclear shape sequence over time. ( j ) Nuclear fluctuation analysis, sketching the analysis procedure (left), and mean values of the fluctuations from each nuclear sequence to the next (right). In ( h–j ), mean value per cell over 36–316 min; n = 3; 26–38 cells per condition. ( k ) Summary of speed as a function of nuclear shape change (ΔNII) in optimal and confining collagen conditions (data are from ( e,i ). Dots connected by red line represent G1 cycle cells; by green line, S/G2 cycle cells; by black line, G1 and S/G2 cycle cells together. ( b,e,h,i,j ) Black horizontal lines, boxes and whiskers show the medians, 25th/75th, and 5th/95th percentile ( b , 10th/90th percentile), and ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05; ns, non-significant (Mann–Whitney test).

Article Snippet: The following cells were used: human HT1080 wild-type fibrosarcoma cells (ACC315; DSMZ Braunschweig; [ ]); HT1080 dual-colour cells expressing cytoplasmic DsRed2 and nuclear histone-2B (H2B)–coupled EGFP [ ]; HT1080 cells stably transfected with NLS-GFP [ ] or H2B-mCherry; and HT1080 cells stably transfected with Fucci sensor.

Techniques: Migration, Sequencing, Microscopy, Electron Microscopy, Translocation Assay, MANN-WHITNEY

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus

doi: 10.1098/rstb.2018.0225

Figure Lengend Snippet: Speed oscillation and rapid nuclear rounding during cell migration in confining pores. HT1080 fibrosarcoma cells moved either in collagen ( a–d ) or in a synthetic microdevice ( e,f ). ( a ) Cells migrated in collagen of increasing density and the absence or presence of GM6001 as indicated, resulting in depicted decreasing effective pore areas (top; ). From each cell, speed of nucleus and cell body was quantified from the migration tracks from the centre of the nucleus as well as the cell body that often demonstrated synchronous speed peaks. Overall migration speed was measured as ‘beeline’ between the centre of the nucleus from the first and last image as depicted in electronic supplementary material, figure S4 and normalized over 55 min. ( b–d ) Oscillatory speed peaks of both cell and nucleus coincide with nuclear rounding. ( b ) Upper row, left, sequences of HT1080 dual-colour cell moving within high-density bovine collagen (3.3 mg ml −1 ) monitored by confocal microscopy at 37°C at 5 min intervals . White dots at first and last image and dotted white line indicate position and beeline of the migrated nucleus. Lower row, left, outlines of the nucleus generated from H2B-eGFP signal, with centroids (blue dots) and centroid-connecting movement trajectories in red. Long trajectories indicated in cyan, green and orange and by arrows represent peak movements, and respective nuclear roundings are marked by coloured outline and grey area. Right, overlay of the first and all rounded outlines, and trajectory of the nucleus over 5 h (corresponding to electronic supplementary material, Movie S4). ( c ) Upper graph, step-to-step and average speed quantification from the movement trajectories of moving cell body and nucleus from ( b ), as well as the beeline of the migrated nucleus. Repeated speed peaks (oscillations) of the nucleus are indicated by respective coloured dots. Lower graph, corresponding repeated phases of nuclear rounding measured as NII, indicated by respective colours and arrowheads. ( d ) Speed as a function of ΔNII per time step (=dot) of the moving nucleus shown in ( b ), and quantified in ( c ). All numbers and dotted lines in black and green indicate medians of speed and ΔNII, respectively, for either coloured oscillation peaks or all remaining dots. Dotted line and number in pink indicate speed median from all dots. ( e ) Migration in microdevice of 10 and 75 µm 2 pore areas (corresponding to electronic supplementary material, Movie S5, part 2). Left, upper row, sequence of migrating cell over indicated time. White arrowhead, deformed nucleus in pore. Arrowhead in cyan indicates rounding. Lower row, nuclear outlines with centroids as blue dots and trajectories in red. Arrow indicates long trajectory in cyan that corresponds to nucleus rounding. Right, kymogram visualizing rapid forward movement (arrow) after transmigration of narrow pore. ( f ) Speed and corresponding shape, with colour coding indicating respective pores in ( e ). Bottom, speed–nuclear shape change relationship; number in green, ΔNII value for peak oscillation. Reprinted modified images in ( b ) are with permission from Elsevier . All image bars, 10 µm.

Article Snippet: The following cells were used: human HT1080 wild-type fibrosarcoma cells (ACC315; DSMZ Braunschweig; [ ]); HT1080 dual-colour cells expressing cytoplasmic DsRed2 and nuclear histone-2B (H2B)–coupled EGFP [ ]; HT1080 cells stably transfected with NLS-GFP [ ] or H2B-mCherry; and HT1080 cells stably transfected with Fucci sensor.

Techniques: Migration, Confocal Microscopy, Generated, Sequencing, Transmigration Assay, Modification

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus

doi: 10.1098/rstb.2018.0225

Figure Lengend Snippet: Altered stiffness, migration and reshaping of HT1080 cells and nuclei after TSA treatment or lamin A/C downregulation. Cells were pre-treated with indicated TSA concentrations or DMSO alone (indicated as 0 ng ml −1 TSA), and were either measured for elastic modulus ( b ) or migrated in collagen (1.7 mg ml −1 ) in the presence of GM6001 ( a,c–g ). For all TSA experiments, except in ( b ) and ( c ), Fucci cells were used and G1-phase cells only were selected for analysis. ( a ) Nuclear areas after pre-treatment with TSA in collagen. Horizontal black lines show the medians. N = 1; 5–19 cells per TSA concentration. ( b ) Calculated stiffness at 1.5 nN contact force by bead-coupled cantilever probing using atomic force microscopy. N = 1–3; 14–37 cells per condition. ( c ) Mean cell migration efficacy per cell over 24 h with increasing concentrations of TSA. Cells that underwent mitosis during the recording were excluded from the analysis. N = 3; 66–90 cells per condition. ( b,c ) Horizontal black lines, boxes and whiskers show the medians, 25th/75th, and 10th and 90th ( b ) and 5th/95th ( c ) percentiles. ( d ) Correlation of stiffness with migration efficacy, using the medians and whiskers from ( a,b ). R 2 = 0.92. ( e ) Representative segmented nuclear sequences from migrating G1-phase (Fucci-red) cells at indicated time steps, in correspondence to Movie S6. Arrows indicate phase IV peak events based on speed increase and concomitant nucleus rounding. Bar, 10 µm. ( f ) Nuclear shape change during migration by TSA. Mean ΔNII per cell migrating over 0.3 to 10 h. Zero means no changes between subsequent nuclear shapes 5–19 cells per condition, shown as dots. Horizontal black lines show the medians. ( g ) Speed peaks, as in d , at indicated TSA concentrations. Graphs are superimposed from 22 to 38 respective events from each 7 to 19 cells per condition; n = 1. Mean (coloured solid lines) ± s.e.m. (shadowed coloured areas). Asterisk indicates decreased nuclear speed after TSA treatment before phase IV peak. ( h ) Left, lamin A/C expression intensity by western blot after transient downregulation by indicated siRNA (each 10 nM). Right, speed peaks in cells treated with non-targeting and lamin A/C siRNA after transmigration of 10 µm 2 pore in a microdevice. Asterisks indicate increased nuclear pore negotion speed after silamin A/C treatment before phase IV peak event. Each 21 cells per condition. ( g,h right) Dotted vertical lines, speed peak at nuclear rounding; grey-shadowed areas, phase IV events. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05; non-significant n .s. ( a,b,c,f , Mann–Whitney test; g,h Students t -test).

Article Snippet: The following cells were used: human HT1080 wild-type fibrosarcoma cells (ACC315; DSMZ Braunschweig; [ ]); HT1080 dual-colour cells expressing cytoplasmic DsRed2 and nuclear histone-2B (H2B)–coupled EGFP [ ]; HT1080 cells stably transfected with NLS-GFP [ ] or H2B-mCherry; and HT1080 cells stably transfected with Fucci sensor.

Techniques: Migration, Concentration Assay, Microscopy, Expressing, Western Blot, Transmigration Assay, MANN-WHITNEY