The nature of cell-cycle checkpoints: facts and fallacies

The concept of checkpoint controls revolutionized our understanding of the cell cycle. Here we revisit the defining features of checkpoints and argue that failure to properly appreciate the concept is leading to misinterpretation of experimental results. We illustrate, using the mitotic checkpoint, problems that can arise from a failure to respect strict definitions and precise terminology

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle

Regulation of cytokinesis by spindle-pole bodies

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 8 (2006): 891-893, doi:10.1038/ncb1449.In the fission yeast Schizosaccharomyces pombe, cytokinesis is thought to be controlled by the daughter spindle pole body (SPB) through a regulatory pathway, the Septation Initiation Network (SIN). Here we demonstrate that laser ablation of both but not a single SPB results in cytokinesis failure. Ablation of just the daughter SPB often leads to activation of the SIN on the mother and successful cytokinesis. Thus, either SPB can drive cytokinesis.This work was supported by National Institutes of Health grants GMS 59363 (to A.K.), GMS 69670 (to F.C), and by the Human Frontiers Science Program grant RGP0064 (to AK)

Laser microsurgery in the GFP era : a cell biologist's perspective

Author Posting. © The Author(s), 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Methods in Cell Biology 82 (2007): 237, 239-266, doi:10.1016/S0091-679X(06)82007-8.Modern biology is based largely on a reductionistic ‘dissection’ approach – most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter we review some of the applications for laser microsurgery at the subcellular level, and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our lab. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope or software, but instead only in defining creative questions and in visualizing the target to be destroyed.Our work is sponsored by grants from the NIH (GM59363 to AK and GM40198 to CLR) and HFSP (RGP0064 to AK). Construction of the laser microsurgery workstation was supported in par by Summer Research Fellowship from Nikon/Marine Biological Laboratory (2003 to AK)

Laser Microsurgery in Fission Yeast Role of the Mitotic Spindle Midzone in Anaphase B

AbstractIntroduction: 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

Chromosome Segregation Is Biased by Kinetochore Size

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore—the critical chromosomal interface with spindle microtubules—impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity

Comment on "A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint"

Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature