Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany
Isabel Raabe, Sven K. Vogel, Jan Peychl, Iva Tolic-Nørrelykke
Schizosaccharomyces pombe strain SP837 (h90 leu-32 ura4-D18 ade6- 216) was used that contains the GFP-α2-tubulin fusion construct pDQ105 (Ding et al., 1998). Cells were grown on Edinburgh Minimal Medium (EMM) agar plates with appropriate supplements and 200 µM thiamine at 30°C. In order to induce expression of GFP, the cells were grown at 2 µM thiamine using the media as described above. Before microscopy, the cells were resuspended in liquid EMM + supplements + 2 µM thiamine and transferred to a glass-bottom microwell dish (MatTek, Ashland) coated with 4 µl of 2 mg/ml lectin BS-1 (Sigma) in PBS.
Laser Scanning Confocal Microscopy
Imaging of the cells was performed on the OLYMPUS FluoView 1000 laser scanning confocal system using a Plan Apo 60x/1.10 oil objective. Emission of GFP was collected using a spectral detector of the FluoView 1000 (500 nm -600 nm). All time-lapse fluorescence images (512x512 pixels) were acquired with unidirectional scanning mode at a 2 µs/pixel scanning speed. For laser ablation of the mitotic spindle the tornado mode of the SIM scanner of the FluoView 1000 and a PDL 800-B picosecond pulsed diode laser with a LDH-P-C-405B were used. The 405 nm picosecond laser for cutting was triggered manually, hence the given exposure times are an approximation. The scan speed of the SIM scanner was 10 µs/pixel.
Movie 1: Spindle cutting followed by unilateral elongation
Movie of a dividing fission yeast cell expressing GFP-labeled tubulin. The mitotic spindle in anaphase B was laser-irradiated in the middle for 2 seconds ("tornado"). The spindle breaks into two segments that move towards each other while the astral microtubules gradually disassemble. The spindle fragments are positioned parallel to each other. New tubulin molecules are polymerized only at the tip of the right fragment, which therefore elongates. This is called unilateral elongation since only one fragment elongates whereas the other one gradually disassembles. The genetic material does not get separated towards the cell tips, but remains close to the cell center instead. The equatorial microtubule ring is visible at the cell center.
Movie 2: Spindle cutting followed by crossing of fragments and subsequent recovery
Movie of a dividing fission yeast cell with GFP-labeled tubulin. The medium-length mitotic spindle in the cell at left was laser-irradiated in the middle for 2 seconds. The
spindle breaks into two segments that move towards each other and ultimately cross each other. The astral microtubules gradually disassemble. In this cell the spindle fragments
later fuse spontaneously and form a functional spindle that elongates and segregates the chromosomes towards the cell tips. The equatorial microtubule ring is visible at the cell
Movie 3: Bleaching of the spindle mid-zone
Movie of a dividing fission yeast cell with GFP-labeled tubulin. The mitotic spindle in anaphase B was laser-irradiated in the middle for 2 seconds. In this case the spindle was
not cut but only photo-bleached. The GFP-tubulin fluorescence recovers in the spindle mid-zone and two bleach marks reappear on the spindle later during spindle elongation.
The bleach marks remain at a constant distance from the spindle poles, while the central fluorescent part of the spindle grows. This experiment shows that the spindle elongates
by addition of new tubulin molecules in the spindle mid-zone.