Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

doi: 10.1073/pnas.1402087111

Figure Lengend Snippet: Microscopic analysis of HL-60 neutrophil polarization and cell migration in response to CNO. (A) HL-60 neutrophils coelectroporated with Di and GFP were plated on a fibronectin-coated glass surface and observed by time-lapse microscopy in the presence of a steep, micropipette-generated gradient of CNO. Di- and GFP-expressing cells migrated directionally toward the micropipet. Fluorescent dye Alexa 594 tracer is mixed with CNO solution in micropipet to visualize the diffusive gradient. The micropipet gradient source is marked by a magenta asterisk. Track start locations are marked by black squares, and red triangles mark cell location and direction in each frame. Traces (black and gray) connect track start locations (black squares) and cell location (red triangles). Drug concentration used (at source): 1μM CNO. See Movies S2–S4 for full movies. (B) HL-60 neutrophils stably expressing Di were placed in the fibronectin-coated viewing area of a microfluidic chemotaxis assay device capable of generating a smooth, stable gradient of CNO. Time-lapse microscopy was used to track cell migration, and cell-tracking software was used to quantitate various migration metrics. Cells migrated toward the CNO gradient (trajectories plotted with cell start locations at origin) and show increased track velocity, displacement rate, and directionality compared with basal motility in the presence of vehicle control. Drug concentration used (at source): 200 nM CNO. Mean ± SEM is shown for n = 61 cells tracked (**P < 0.0001 by Student t test). See Movie S5 for full movie.

Article Snippet: The ONIX microfluidic platform with M04G gradient generator plate (CellASIC/EMD Biosciences) was used to study HL-60 neutrophil migration.

Techniques: Migration, Time-lapse Microscopy, Generated, Expressing, Concentration Assay, Stable Transfection, Chemotaxis Assay, Cell Tracking Assay, Software, Control

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: Wild type (AM16065), cdc14-1 (AM16066), slk19Δ (AM16110) and spo12Δ (AM15985) cells carrying TUB1-GFP and PDS1-tdTomato were induced to sporulate, released from prophase I arrest and imaged at 10 min intervals for a total of 12 h in a microfluidics device. ( A ) Representative images are shown. The black arrow denotes Pds1 degradation, marking entry of cells into anaphase I. The white asterisk marks the time of spindle breakdown. ( B ) The time taken for complete spindle disassembly after Pds1 degradation was recorded for individual cells and plotted for a total of 67 wild type cells and 100 of each of cdc14-1 , slk19Δ and spo12Δ cells. Mean rates of spindle breakdown are shown (red line), with error bars representing standard deviation. The two-tailed Student’s t -test was used to calculate p values. ( C ) Inactivation of Cdc14 results in abnormal spindle behaviour. Cells were categorised based on spindle morphology as indicated in the legend.

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Standard Deviation, Two Tailed Test

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: ( A ) Sporulating CDC14-GFP SPC42-tdTomato cells (AM11517) were imaged in a microfluidics chamber at 15 min intervals for a total of 12 h. Example of a cell in which Cdc14-GFP localizes asymmetrically to one SPB during anaphase I (arrows). ( B and C ) Wild type cells as in ( A ) were released from a prophase I arrest and imaged at 30 min intervals on agarose pads. ( B ) The ratio of Cdc14-GFP/Spc42-tdTomato signal per SPB is shown with error bars representing standard error. Cells were classified into different cell cycle stages (metaphase I, anaphase I, metaphase II, anaphase II) by scoring the number of SPBs, the distance between them and Cdc14 nucleolar sequestration. The two-tailed Student’s t- test was used to calculate p values (* p<0.001) n= 50 cells. ( C ) Co-localization of Cdc14-GFP with SPBs was scored in the indicated cell cycle stages. ( D and E ) Cdc14 localises prematurely to SPBs in the absence of Cdc55. pCLB2-3HA-CDC55 cells carrying CDC14-GFP and SPC42-tdTomato (AM13123) were induced to sporulate, released from pGAL-NDT80 block and imaged 3h later on agarose pads. Cdc14 localisation was classified into three categories: sequestered in nucleolus (1), partially released from nucleolus (2) and completely released from nucleolus (3). ( D ) Example images of with numbered arrows showing examples of each category. ( E ) Co-localisation of Cdc14 with SPBs was scored in 100 cells of each category.

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Two Tailed Test, Blocking Assay

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: ( A ) Wild type (AM13989) cells containing SPC42-tdTomato were induced to sporulate, released from pGAL-NDT80 block and imaged at 15 minute intervals for a total of 12 hours in a microfluidics chamber. Representative images of wild type cells are shown. ( B ) SPBs are duplicated at meiotic prophase. Wild type cdc14-1 (AM16163), and pCLB2-3HA-CDC55 (AM15984) cells containing SPC42-tdTomato and pGAL-NDT80 and were resuspended in sporulation medium in the absence of β-oestradiol and imaged immediately (t=0) or after 3 and 6h as they progress into the prophase I arrest due to the absence of Ndt80. Individual SPB foci were quantified and mean total SPB fluorescence intensity (F i ) per cell was plotted, with error bars (obscured by the markers) representing standard error, n=100 per timepoint. ( C – E ) SPB fluorescence was quantified as in B from movies of live cells after release from the prophase I arrest by addition of β-oestradiol as in ( A ). The first time point at which a cell contained 2 Spc42-tdTomato foci was defined as 1h and SPB fluorescence in the preceding 4 (1 focus) and following 4 time points was quantified (n=10 cells). Note that at the 1.5h time point 7 wild type cells carried 2 Spc42-tdTomato foci, while 3 wild type cells carried 4 Spc42-tdTomato foci. ( F and G ) LFQ Proteomic analysis of SPB composition and environment in wild type and cdc14-1 cells. Strains used were AM1835 (no tag), AM11444 ( SPC42-3FLAG ), AM9459 ( cdc14-1 ) and AM11443 ( cdc14-1 SPC42-3FLAG ) After 4 h, cells were harvested and SPBs purified by anti-FLAG immunoprecipitation. Peptides were generated by in-gel trypsin digestion and LC-MS data sets for 3 biological replicas was analysed using MaxQuant software. ( F ) Example silver stained SDS-PAGE showing Spc42-3FLAG immunoprecipitates of one of three biological replicas used in LFQ proteomic analysis. BSA standards were used to estimate protein concentration. ( G ) Statistical analysis of relative LFQ intensity output was carried out using Perseus. Volcano plot shows –log of P values versus ratio of wild type/c dc14-1 for all 254 proteins in >5 columns. No significant change in composition and environment was observed between wild type and cdc14-1 SPBs. (FDR = 0.05, s0 = 1). Proteins of interest are highlighted as follows: red = SPB components; blue = Cdc14; green = Bfa1/Bub2. See also https://osf.io/g5cmh/ .

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Blocking Assay, Fluorescence, Purification, Immunoprecipitation, Generated, Liquid Chromatography with Mass Spectroscopy, Software, Staining, SDS Page, Protein Concentration