Technical Reference #1623

1623.

The Distribution of Polar Ejection Forces Determines the Amplitude of Chromosome Directional Instability Kevin Ke; Jun Cheng; Alan Hunt, University of Michigan, Current Biology, 19(1623), (2009)
Link To Paper

Abstract:
Background: Polar ejection forces have often been hypothesized to guide directional instability of mitotic chromosomesbut a direct link has never been established. This has ledin part to the resurgence of alternative theories. By takingadvantage

Materials & Methods:
Tissue Culture Newt lung cultures are prepared as previously described [42]. In brief redspotted newts (Notophthalmus viridescens) obtained from Connecticut Valley Biological Supply Co. (Southampton MA) are euthanized by immersion for 30 min in 1 mg/ml tricaine (Sigma #A5040 St. Louis MO). The lungs are sterilely dissected cut into 1 mm2 pieces and soaked in L-15 medium supplemented with FBS and antibiotics for 24 hr. Explants are then lightly squashed onto the Petri dish glass covered with a piece of dialysis filter held down by a Teflon ring and incubated at room temperature (22C– 25C). After about 7 days when a few rows of epithelial cells surround the lung fragment the dialysis filter is removed. Experiments are performed about 2 weeks after dissection. Cells are selected based on flatness during mitosis the visibility of centrosomes and the ability to identify individual chromosomes. Femtosecond Laser Microsurgery For all experiments we use a Zeiss 1003/1.30 NA Neofluar Phase 3 objective and matching condenser. High-precision ultrafast laser surgery is performed with a diode-pumped Nd:glass chirped pulse amplification (CPA) laser system (Intralase Corp. Irvine CA) that generates 600 to 800 fs pulses at a repetition rate of up to 3 kHz. Dichroic and polarized mirrors couple the laser pulses into the illumination light path of an inverted microscope (Figure 2; Zeiss Axiovert 200 Thornwood NY). To achieve high precision the laser is focused to a diffraction-limited spot by completely filling the back focal aperture of the objective. A tri-axial computer-controlled piezoelectric stage (Mad City Labs Inc. Madison WI) is mounted on the manual stage to achieve nanometer precision while allowing scanning across a 35 mm Petri dish with 14 mm microwell (MatTek Corp. Ashland MA). Custom software and design electronics allows control of laser pulse delivery specimen targeting and image capture. Chromosome movements before and after ablation are tracked by phasecontrast video microscopy time-lapse recorded at 2 to 3 s intervals. Phase contrast is used instead of DIC because the greater depth of focus simplifies tracking and assures that chromosomes are severed completely as both sister chromatids are visible if either is in focus. This was verified by scanning the focus in the z-axis. With a pulse energy of 2 to 3 nJ the laser is scanned in a raster pattern to slice across a chromosomearm. Scans are repeated once from top to bottom (estimated by the diameter of the chromosome) then again from bottom to top at 10 nm steps to ensure complete ablation. The total volume of ablated material is approximately 3 mm3 less than 0.1% of the cell volume. Chromosome Tracking Wedeveloped semiautomatic tracking software with an algorithmand operating procedure similar to that described in Skibbens et al. [1] implementing National Instruments pattern-matching routines. Additional processing to enhance tracking accuracy includes contrast equalization image thresholding and calculating the centroid of the cross-correlations. Reproducibility and accuracy were demonstrated by tracking stationary objects and by verifying that consistent tracking data was obtained by independent operators. The accuracy depends on the variability of the chromosome image during the course of an experiment. Rolls yaws and pitches decrease the accuracy especially if a chromosome moves out of the imaging plane. If significant changes in the region of interest occurred tracking was terminated or if the chromosome was still clearly identifiable a new region of interest was selected.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HeLa