Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B.
BACKGROUND
During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe.


RESULTS
We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles.


CONCLUSIONS
These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.
New York State Department of Health, Wadsworth Center, Albany 12201, USA. khodj@wadsworth.org
Alexey
A
Khodjakov
Sabrina
S
La Terra
Fred
F
Chang
D000705:Anaphase / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins; D007734:Kymography; D053685:Laser Therapy / Q000379:methods; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008866:Microsurgery / Q000379:methods; D008870:Microtubules / Q000502:physiology*; D012568:Schizosaccharomyces / Q000166:cytology* / Q000235:genetics; D008941:Spindle Apparatus / Q000502:physiology* / Q000528:radiation effects; D014404:Tubulin / Q000378:metabolism
D008164:Luminescent Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins