According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilageto-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.osteoblast ontogeny | chondrocyte lineage | bone repair I n vertebrates, the endochondral bones of the axial and appendicular skeleton (1) develop from mesenchymal progenitors that form condensations in the approximate shape of the future skeletal elements. These progenitors differentiate into chondrocytes, which proliferate, mature, and undergo hypertrophy, forming an avascular cartilaginous template surrounded by a perichondrium. The first osteoblasts differentiate from mesenchymal precursors in the perichondrium and produce a bone collar, which will become the future cortical bone (1). Blood vessels then invade through the bone collar into the hypertrophic cartilage, bringing in osteoblast progenitors from the perichondrium (2), which lay down bone matrix to form the primary ossification center (POC); the cartilage matrix is degraded; and the proximal and distal growth plates, comprising layers of differentiating chondrocytes and spongy/trabecular bone (the primary spongiosa), form (2). Thereafter, linear bone growth continues by endochondral ossification mediated by the growth plate, whereas osteoblasts in the perichondrium form cortical bone on the outer circumference.Chondrocytes and osteoblasts are regarded as separate lineages in development, being derived from common mesenchymal progenitors that express the transcription factors sex determining region Y (SRY)-box 9 (Sox9) and runt related transcription factor 2 (Runx2) (1). Lineage determination toward the chondrocyte or osteoblast fate is controlled by the relative expression of Sox9 and Runx2 (3-5) (Fig. 1A). Sox9 controls chondrocyte proliferation and their progression into hypertrophy (6). Collagen X is the most specific marker of hypertrophic chondrocytes (HCs), the Col10a1 gene being expressed only in prehypertrophic and hypertrophic chondrocytes in the growth plate (7). By contrast, Runx2 is essential for specifying the osteoblast lineage an...

The role of cyclin B-CDC2 as M phase-promoting factor (MPF) is well established, but the precise functions of cyclin A remain a crucial outstanding issue. Here we show that down-regulation of cyclin A induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation. The phenotype is rescued by expressing cyclin A resistant to the RNA interference. In contrast, down-regulation of cyclin B disrupts mitosis without inactivating cyclin A-CDK, indicating that cyclin A-CDK acts upstream of cyclin B-CDC2. Even when ectopically expressed, cyclin A cannot replace cyclin B in driving mitosis, indicating the specific role of cyclin B as a component of MPF. Deregulation of WEE1, but not the PLK1-CDC25 axis, can override the arrest caused by cyclin A knockdown, suggesting that cyclin A-CDK may tip the balance of the cyclin B-CDC2 bistable system by initiating the inactivation of WEE1. These observations show that cyclin A cannot form MPF independent of cyclin B and underscore a critical role of cyclin A as a trigger for MPF activation. INTRODUCTIONClassic cell cycle studies in the 1980s culminated in the purification of M phase-promoting factor (MPF) from Xenopus eggs and starfish oocytes and the identification of its components as cyclin B and CDC2 (also called cyclin-dependent kinase [CDK]-1; reviewed in Doree and Hunt, 2002). Cyclin B accumulates from S phase and forms a complex with CDC2. The complex is kept inactive by phosphorylation of CDC2 Thr14/Tyr15 by MYT1 and WEE1 until it is abruptly activated by CDC25 during mitosis. As the cell exits mitosis cyclin B is destroyed via an ubiquitin-mediated mechanism that is catalyzed by the APC/C (reviewed in .Cyclin B-CDC2 catalyzes its own activation by an intricate network of feedback loops. WEE1 is phosphorylated by CDKs, facilitating its degradation by the ubiquitin ligases SCF ␤-TrCP and SCF Tome-1 (Ayad et al., 2003;Watanabe et al., 2004Watanabe et al., , 2005Harvey et al., 2005). The CDC25C phosphatase is activated by multiple CDK phosphorylation (reviewed in Hutchins and Clarke, 2004) and inactivated by phosphorylation on Ser216 (enforced by several kinases, including C-TAK1, CHK1, and CHK2). This phosphorylation creates a 14-3-3 binding site, which masks a proximal nuclear localization sequence and excludes CDC25C from the nucleus. Furthermore, phosphorylation of CDC25C Ser214 inhibits the phosphorylation of CDC25C Ser216 (Bulavin et al., 2003). Thus MPF is essentially a bistable system (Ferrell, 2002) that becomes autocatalytic once a critical portion is activated.What triggers the initial activation of MPF has been the subject of immense speculation. PLK1 is a candidate as it can phosphorylate CDC25C at the nuclear-exporting sequence (NES) and promote nuclear accumulation of CDC25C (Toyoshima-Morimoto et al., 2002). PLK1 likewise phosphorylates the NES of cyclin B and increases its nuclear accumulation (Toyoshima-Morimoto et al., 2001;Yuan et al., 2002), coordinating the localization of both cyclin B and CDC25C t...

Biochemical studies have indicated that p31(comet) and TRIP13 are critical for inactivating MAD2. To address unequivocally whether p31(comet) and TRIP13 are required for mitotic exit at the cellular level, their genes were ablated either individually or together in human cells. Neither p31(comet) nor TRIP13 were absolutely required for unperturbed mitosis. MAD2 inactivation was only partially impaired in p31(comet)-deficient cells. In contrast, TRIP13-deficient cells contained MAD2 exclusively in the C-MAD2 conformation. Our results indicate that although p31(comet) enhanced TRIP13-mediated MAD2 conversion, it was not absolutely necessary for the process. Paradoxically, TRIP13-deficient cells were unable to activate the spindle-assembly checkpoint, revealing that cells lacking the ability to inactivate MAD2 were incapable in mounting a checkpoint response. These results establish a paradigm of the roles of p31(comet) and TRIP13 in both checkpoint activation and inactivation.

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

Inhibition of cyclin-dependent kinases (CDKs) by Thr14 /Tyr 15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G 2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr 14 /Tyr 15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle. In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions: cyclin D-CDK4

A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.

HeLa is one of the oldest and most commonly used cell lines in biomedical research. Owing to the ease of which they can be effectively synchronized by various methods, HeLa cells have been used extensively for studying the cell cycle. Here, we describe several protocols for synchronizing HeLa cells from different phases of the cell cycle, including G phase using the HMG-CoA reductase inhibitor lovastatin, S phase with a double thymidine block procedure, and G phase with the CDK1 inhibitor RO-3306. Cells can also be enriched in mitosis using nocodazole and mechanical shake-off. Releasing the cells from these blocks enables researchers to follow gene expression and other events through the cell cycle. We also describe several protocols, including flow cytometry, BrdU labeling, immunoblotting, and time-lapse microscopy, for validating the synchrony of the cells and monitoring the progression of the cell cycle.

Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G 2 arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and ␤-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCF ␤-TrCP1 -EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.