View metadata, citation and similar papers at core.ac.uk brought to you by CORE providedDispatch by ElsevierR613 - Publisher Connector Cell cycle: Checkpoint proteins and kinetochores Aaron F. Straight Vertebrate homologs of yeast spindle assembly spindle and the alignment of the chromosomes. Cells with checkpoint proteins are localized to kinetochores and an intact mitotic spindle will still arrest in mitosis if a may act as a sensor for proper chromosome attachment single chromosome is not properly attached to the spindle to the mitotic spindle. (Figure 1b) [11]. This arrest is caused by an inhibitory signal emanating from kinetochores that are not attached Address: Department of Physiology, Box 0444, School of Medicine, University of California, San Francisco, California 94143-0444, USA. to the spindle apparatus [12]. E-mail: [email protected] Genetic studies in yeast have identified some of the Current Biology 1997, 7:R613–R616 molecules involved in the spindle assembly checkpoint. http://biomednet.com/elecref/09609822007R0613 Like vertebrate cells, budding yeast cells arrest in mitosis © Current Biology Ltd ISSN 0960-9822 in response to spindle depolymerization [13]. Yeast mutants were isolated that died rapidly when treated with Cells carefully monitor the alignment of chromosomes on the spindle-depolymerizing drug benomyl. These the mitotic spindle to ensure that their chromosomes are mutants — mad1, mad2 and mad3, and bub1, bub2 and bub3 properly segregated at anaphase. Detachment of kineto- — all fail to activate the spindle assembly checkpoint and chores from microtubules or depolymerization of the continue through mitosis without a spindle. Although the mitotic spindle activates a checkpoint mechanism that mutants die rapidly in the presence of benomyl, the mad arrests the cell cycle prior to the initiation of anaphase. This and bub gene products do not appear to be directly ‘spindle assembly checkpoint’ allows the cell time to involved in spindle integrity, as the mutant strains have correct the offending lesion so that it can proceed through normal spindles [3,5]. Instead, mad and bub mutants are mitosis without chromosome missegregation [1,2]. Proteins thought to be defective in the signalling mechanism that involved in the spindle assembly checkpoint have been iso- arrests the cell cycle in response to spindle depolymeriza- lated in budding yeast and in vertebrates [3–7]. Cells tion. Wells and Murray [8] showed that excess kineto- lacking components of the spindle assembly checkpoint can chores in budding yeast cells cause pronounced cell cycle proceed through mitosis in the absence of a spindle or in delays in mitosis. These cell cycle delays are absent in the the presence of unattached chromosomes [3,5,8]. mad and bub mutants, suggesting that the Mad and Bub proteins are involved in monitoring chromosome attach- Recent work has shown that two of the spindle assembly ment to kinetochores as well as spindle integrity. checkpoint proteins, Mad2 and Bub1, localize to the kine- tochores of vertebrate cells for part of mitosis. Prophase The kinetochore as the mechanism and the sensor for and prometaphase chromosomes show kinetochore local- mitotic spindle attachment ization of Mad2 and Bub1, but as chromosomes align on Kinetochores mediate attachment of the chromosomes to the metaphase plate the kinetochore staining disappears the mitotic spindle. When a microtubule contacts a kinet- and does not reappear until the next cell cycle (Figure 1a). ochore, the kinetochore captures the microtubule [14,15]. Misaligned chromosomes show Mad2 and Bub1 localiza- These kinetochore–microtubule attachments rearrange tion at the kinetochore even when all other chromosomes until sister kinetochores are attached to opposite poles of reside on the metaphase plate (Figure 1b,c) [4,6,7]. These the spindle. In insect cells, tension on the kinetochores results suggest that the kinetochore both monitors the further stabilizes kinetochore–microtubule interactions, attachment of the chromosome to the spindle and signals thus promoting bipolar attachment of the chromosomes to the cell to arrest until chromosomes are properly aligned. the spindle [16,17]. Once kinetochores are attached to the spindle microtubules, the kinetochores mediate congres- The spindle assembly checkpoint sion of the chromosomes to the metaphase plate [18] and Early experiments in the 1930s showed that, when verte- cells do not initiate anaphase until all chromosomes are brate cells are treated with drugs that depolymerize the properly aligned. mitotic spindle, they arrest in mitosis [9,10]. The mitotic arrest in these cells was attributed to the removal of the Kinetochores can sense proper attachment to the mitotic mechanical apparatus that separates the chromosomes — spindle. Proteins at the kinetochore are specifically phos- the mitotic spindle. Recent work has shown that the phorylated dependent upon their proper attachment to absence of the spindle itself is not the cause of the mitotic the mitotic spindle. In insect cells, kinetochores that are arrest, but that the arrest is mediated through the activa- not attached to the mitotic spindle, and kinetochores tion of a checkpoint that monitors the integrity of the that have only attached to one pole of the spindle, stain R614 Current Biology, Vol 7 No 10 Figure 1 (a)Interphase Prophase Metaphase Anaphase Mad Mad Bub Bub Mad Bub Mad Bub (b) Misaligned chromosome Error correction Mad Mad Bub Bub Mad Bub Mad Bub Mad Bub Mitotic arrest (c) Spindle depolymerization Adaptation Mad Mad Bub Bub Mad Mad Mad Bub Bub Bub Mad Mad Bub Bub Mad Bub Mitotic arrest © 1997 Current Biology Mad2 and Bub1 proteins reside at kinetochores and mediate checkpoint (c) Spindle depolymerization causes mitotic arrest. In the absence of a arrest of the cell cycle. (a) During a normal mitosis, Mad2 and Bub1 spindle, Mad2 and Bub1 remain at the kinetochores of chromosomes localize to kinetochores in prophase and prometaphase. Once all and the cell arrests in mitosis. The checkpoint arrest can be overcome by chromosomes are attached to the spindle, cells proceed through two mechanisms: by error correction, the cell can correct the offending anaphase. (b) Misaligned chromosomes signal spindle assembly lesion and process through a normal mitosis; by adaptation, the cell can checkpoint arrest. Chromosomes that are not properly attached to the override the checkpoint arrest, in the presence of the defect, resulting in spindle maintain Mad2 at the kinetochore and the cell arrests in mitosis. an abnormal mitosis with the potential for chromosome loss. brightly with the 3F3/2 antibody, which recognizes a spindle by modifying the resident kinetochore proteins phospho-epitope at the kinetochores of mammalian and (reviewed in [19]). insect cells. When chromosomes achieve a bipolar attach- ment to the spindle and the sister kinetochores come The spindle assembly checkpoint meets the kinetochore under tension, the staining of the 3F3/2 epitope is dimin- Three recent papers report that spindle assembly check- ished. This loss of 3F3/2 staining can be mimicked by point proteins reside at the kinetochore and regulate pulling on a mono-attached chromosome so as artificially passage through mitosis. Chen et al. [6] and Li and to apply tension to the kinetochore. Cells that are Benezra [4] isolated, respectively, the Xenopus (XMAD2) microinjected with the 3F3 antibody delay dephosphory- and human (hsMAD2) homologs of the yeast Mad2, and lation of the kinetochore and pause in mitosis. Thus, the Taylor and McKeon [7] isolated the mouse homolog kinetochore responds to proper attachment to the (mBub1) of yeast Bub1. All of these proteins localize to Dispatch R615 the kinetochores of chromosomes in cultured cells of the Does the lack of a spindle assembly checkpoint affect the organisms from which they were isolated. Interestingly, in timing of a normal cell cycle? Taylor and McKeon [7] all three organisms, the checkpoint proteins only localize investigated this question by examining the effect of to the kinetochores of cells in prophase and prometaphase, overexpressing the mBub1 amino-terminal domain on the prior to proper chromosome alignment on the metaphase timing of the cell cycle. They found that the overexpres- plate, or of cells that have been treated with nocodazole to sion had no effect on the timing of G1, S or G2 phases of depolymerize the mitotic spindle (Figure 1a,c). the cell cycle, but delayed the exit from mitosis by approximately 25 minutes. Yeast cells that lack the spindle To investigate further the relationship between chromo- assembly checkpoint protein Mad1 are also accelerated in some attachment to the spindle and XMAD2 staining, their exit from mitosis (P. Dann and A. Rudner, personal Chen et al. [6] took advantage of cells that had attached all communication). Thus, the spindle checkpoint may have but one or two chromosomes to both spindle poles. Chro- a role during the normal cell cycle in restraining the mosomes that had only attached a single kinetochore to passage through mitosis. the spindle showed no XMAD2 staining on the attached kinetochore, but bright XMAD2 staining on the unat- Other proteins involved in the spindle assembly check- tached sister kinetochore (Figure 1b). Thus, the check- point have been found to be resident components of the point proteins at the kinetochore respond to microtubule kinetochore. Studies with budding yeast have shown that attachment to the kinetochore. the Mad2 protein interacts tightly with another spindle assembly checkpoint protein, Mad1, and studies with Mad2 and Bub1 are required for the spindle assembly Xenopus extracts have shown that XMAD2 interacts with a checkpoint in vertebrate cells. Chen et al. [6] and Li and Mad1-like protein (R. Chen and K. Hardwick, personal Benezra [4] showed that antibodies against Mad2 blocked communication). The Mad1 protein has also been shown the spindle assembly checkpoint. The spindle assembly to be phosphorylated by the protein kinase Mps1, and checkpoint can be reconstituted in vitro using extracts of overexpression of Mps1 causes constitutive activation of Xenopus laevis eggs [20]. Using X.