Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase.

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T 1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green¯uorescent proteintagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase,~30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin speci®cation in geminin-supplemented extracts; sitespeci®c initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modi®cations of pre-existing proteins, and is not suf®-cient to select speci®c origin sites. A subsequent, as yet unde®ned, step selects which pre-RCs will function as replication origins.

Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif. INTRODUCTIONSuccessive waves of cyclin-dependent kinase (Cdk) activity control progress through the eukaryotic cell cycle. Cdks are activated by binding a member of the cyclin family and phosphorylation by Cdk-activating kinase. The cyclin-Cdks that have been most strongly implicated in controlling entry into, and progress through, DNA replication are cyclins A and E. Both cyclins bind to Cdk2 (Elledge and Spottswood, 1991;Tsai et al., 1991;Koff et al., 1992), and the levels of both cyclins are strictly regulated throughout the cell cycle, by transcriptional and proteolytic mechanisms. Cyclin E levels are primarily dictated by the rate of its transcription because it is an unstable protein that is rapidly degraded by two different pathways of ubiquitin-dependent proteolysis (Clurman et al., 1996;Won and Reed, 1996;Singer et al., 1999;Wang et al., 1999;Winston et al., 1999;Nakayama et al., 2000). In contrast, cyclin A is stable until cells enter mitosis. Cyclin A levels first start to increase at the beginning of S phase and continue to rise throughout S and G2 phases until prometaphase, when they are rapidly degraded by ubiquitin-dependent proteolysis (Pines and Hunter, 1990;Hunt et al., 1992).There is extensive evidence to indicate that the activation of cyclin E/Cdk2 leads to the initiation of DNA replication. Cyclin E/Cdk2 activity is maximal at G1/S (Koff et al., 1992), replication of DNA in vitro is dependent on cyclin E/Cdk2 activity (Jackson et al., 1995;Strausfeld et al., 1996;Krude et al., 1997) and, in vivo, cyclin E is essential for DNA replication in Drosophila (Sauer et al., 1995). Cyclin A/Cdk2 has also been demonstrated to promote DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Strausfeld et al., 1996). However, cyclin A may be more significant in regulating progression through S phase (Connell-Crowley et al., 1998), perhap...

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherinbased bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts. INTRODUCTIONCadherins are a family of calcium-dependent cell-cell adhesion molecules that regulate the actin cytoskeleton. On productive ligation of their adhesive ectodomains, classical cadherins activate cellular responses necessary for a range of morphogenetic processes, including cell sorting and tissue cohesion (Tepass et al., 2000), cell-upon-cell locomotion (Brieher and Gumbiner, 1994), and the coordination of cell migration in dorsal closure and wound repair (Danjo and Gipson, 1998). These morphogenetic responses likely reflect complex functional and biochemical interactions between the cadherin, its associated catenins, cell signaling pathways, and the cytoskeleton. Dynamic activity of the actin cytoskeleton, in particular, must be coordinated with surface adhesion to provide local protrusive force and mechanical stability at cell-cell contacts (Takeichi, 1991;Gumbiner, 1996;Yap et al., 1997). The challenge, then, is to define key molecular regulators responsible for these functional interrelationships.The capacity for cadherins to regulate actin was first suggested by the extensive alterations in actin filament distribution that occur when epithelial cells adhere to one another (Yonemura et al., 1995;Adams et al., 1996). It subsequently became evident that multiple forms of actin activity occur at cadherin contacts, including de novo nucleation and crosslinking, that must presumably be stringently coordinated with the dynamic state of adhesive contacts (Yonemura et al., 1995;Adams et al., 1996;Vasioukhin et al., 2000; WatermanStorer et al., 2000;Kobielak et al., 2004;Verma et al., 2004). Indeed, a growing corpus of actin-regulatory proteins have been identified at cadherin-b...

Oriented cell division is a fundamental determinant of tissue organization. Simple epithelia divide symmetrically in the plane of the monolayer to preserve organ structure during epithelial morphogenesis and tissue turnover. For this to occur, mitotic spindles must be stringently oriented in the Z-axis, thereby establishing the perpendicular division plane between daughter cells. Spatial cues are thought to play important roles in spindle orientation, notably during asymmetric cell division. The molecular nature of the cortical cues that guide the spindle during symmetric cell division, however, is poorly understood. Here we show directly for the first time that cadherin adhesion receptors are required for planar spindle orientation in mammalian epithelia. Importantly, spindle orientation was disrupted without affecting tissue cohesion or epithelial polarity. This suggests that cadherin receptors can serve as cues for spindle orientation during symmetric cell division. We further show that disrupting cadherin function perturbed the cortical localization of APC, a microtubule-interacting protein that was required for planar spindle orientation. Together, these findings establish a novel morphogenetic function for cadherin adhesion receptors to guide spindle orientation during symmetric cell division.