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Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein during late S/G2 phase of the cell cycle. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using either flow cytometry or live-cell imaging techniques. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.

The tethering together of sister chromatids by the cohesin complex ensures their accurate alignment and segregation during cell division. In vertebrates, sister chromatid cohesion requires the activity of the ESCO2 acetyltransferase, which modifies the Smc3 subunit of cohesin. It was shown recently that ESCO2 promotes cohesion through interaction with the MCM replicative helicase. However, ESCO2 does not significantly colocalize with the MCM complex, suggesting there are additional interactions important for ESCO2 function. Here we show that ESCO2 is recruited to replication factories, sites of DNA replication, through interaction with PCNA. We show that ESCO2 contains multiple PCNA-interaction motifs in its N terminus, each of which is essential to its ability to establish cohesion. We propose that multiple PCNA-interaction motifs embedded in a largely flexible and disordered region of the protein underlie the unique ability of ESCO2 to establish cohesion between sister chromatids precisely as they are born during DNA replication.

Sister chromatids are tethered together by the cohesin complex from the time they are made until their separation at anaphase. The ability of cohesin to tether sister chromatids together depends on acetylation of its Smc3 subunit by members of the Eco1 family of cohesin acetyltransferases. Vertebrates express two orthologs of Eco1, called Esco1 and Esco2, both of which are capable of modifying Smc3, but their relative contributions to sister chromatid cohesion are unknown. We therefore set out to determine the precise contributions of Esco1 and Esco2 to cohesion in vertebrate cells. Here we show that cohesion establishment is critically dependent upon Esco2. Although most Smc3 acetylation is Esco1 dependent, inactivation of the ESCO1 gene has little effect on mitotic cohesion. The unique ability of Esco2 to promote cohesion is mediated by sequences in the N terminus of the protein. We propose that Esco1-dependent modification of Smc3 regulates almost exclusively the noncohesive activities of cohesin, such as DNA repair, transcriptional control, chromosome loop formation, and/or stabilization. Collectively, our data indicate that Esco1 and Esco2 contribute to distinct and separable activities of cohesin in vertebrate cells.

During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.

Extracts prepared from the eggs of frogs, particularly Xenopus species, have provided critical material for seminal studies of nuclear and chromosome dynamics over several decades. Their usefulness for these types of analyses lies in several important characteristics: stockpiled nuclear components, absence of endogenous DNA, and intact and functioning signaling networks. These factors have allowed detailed molecular analyses of many aspects of chromosome biology, including DNA replication, checkpoint signaling, epigenetic control, and chromosome condensation, cohesion, and segregation. In this introduction, the preparation and application of Xenopus egg extracts for the study of chromosomes and chromatin are described in detail.

Chromosome structure in both interphase and M phase cells is strongly influenced by the action of the Cohesin and Condensin protein complexes. The Cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication, and promotes normal interphase chromosome structure and gene expression. In contrast, Condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system provides a uniquely suitable system with which to analyze the functions of both Cohesin and Condensin. Egg extracts, in which the cell cycle state can be manipulated, contain stockpiles of nuclear proteins, including Condensin and Cohesin, sufficient for the assembly of thousands of nuclei per microliter. Egg extract prepared from unfertilized eggs is arrested by the presence of cytostatic factor (CSF) in a state with high levels of M-phase kinase activity, but can be stimulated to enter interphase, in which DNA replication occurs spontaneously. For cohesion assays, demembranated sperm nuclei are incubated in interphase extract, where they undergo rapid and synchronous DNA replication and cohesion establishment through the recruitment of proteins and other factors (e.g. nucleotides) from the extract. Sister chromatid cohesion is assessed by then driving the extract into M phase by the addition of fresh CSF-arrested extract. Chromosome condensation occurs spontaneously in M phase extract extracts. Sperm nuclei are therefore added directly to CSF extracts to assay condensation. In the following protocols, we describe basic assays for Cohesin and Condensin function using Xenopus egg extracts (see Figure 1 for schematic overview).

Cornelia de Lange Syndrome is a pervasive developmental disorder characterized by limb truncations, craniofacial abnormalities, and cognitive delays. This syndrome is a member of a class of developmental disorders referred to ascohesinopathies, which result from mutations in the genes encoding subunits or regulators of the cohesin complex. The phenotypic consequences of these mutations may reflect the critical role that cohesin plays in chromosome structure, its ability to tether sister chromatids together during cell cycle progression, or some combination of both. Here we show that a sensitized assay for chromosome loss in budding yeast can be used to assess the impact of Cornelia de Lange syndrome (CdLS)-associated mutations in the core cohesin subunit Smc1 on cohesin function. We find that the CdLS-associated mutations can be grouped into two classes based on their impact on chromosome segregation. One class of mutations includes those that are defective in promoting accurate chromosome segregation, some no better than the null allele. Another class promotes both accurate chromosome cohesion and segregation. Strikingly, the mutations that have no impact chromosome dynamics in this assay are clustered near each other in the context of the folded SMC1 protein suggesting a previously uncharacterized region of functional importance in higher eukaryotes. This analysis illustrates how budding yeast can be used to elucidate mechanisms important in human health and development.

DNA replication origins in hyperacetylated euchromatin fire preferentially during early S phase. However, how acetylation controls DNA replication timing is unknown. TICRR/TRESLIN is an essential protein required for the initiation of DNA replication. Here, we report that TICRR physically interacts with the acetyl-histone binding bromodomain (BRD) and extraterminal (BET) proteins BRD2 and BRD4. Abrogation of this interaction impairs TICRR binding to acetylated chromatin and disrupts normal S-phase progression. Our data reveal a novel function for BET proteins and establish the TICRR–BET interaction as a potential mechanism for epigenetic control of DNA replication.

Cells depleted of Ska arrest at metaphase. Chromosome-associated APC/C shows high ubiquitylation activity. Ska complex helps to localize APC/C on chromosomes. Forced localization of Ska complex to kinetochores enhances APC/C localization to chromosomes and promotes APC/C activity., The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

Timely dissolution of sister-chromatid cohesion in mitosis ensures accurate chromosome segregation to guard against aneuploidy and tumorigenesis. The complex of shugoshin and protein phosphatase 2A (Sgo1–PP2A) protects cohesin at centromeres from premature removal by mitotic kinases and Wapl in prophase. Here we address the regulation and mechanism of human Sgo1 in centromeric cohesion protection, and show that cyclin-dependent kinase (Cdk)-mediated, mitosis-specific phosphorylation of Sgo1 activates its cohesion-protection function and enables its direct binding to cohesin. The phospho-Sgo1-bound cohesin complex contains PP2A, Pds5, and hypophosphorylated sororin, but lacks Wapl. Expression of non-phosphorylatable sororin bypasses the requirement for Sgo1–PP2A in centromeric cohesion. Thus, mitotic phosphorylation of Sgo1 targets Sgo1–PP2A to cohesin, promotes dephosphorylation of Pds5-bound sororin, and protects centromeric cohesin from Wapl. PP2A-orchestrated, selective removal of a specific subset of phosphorylation from cohesin and its regulators underlies centromeric cohesion protection.

Sister chromatids are tethered together from the time they are formed in S-phase until they separate at anaphase. A protein complex called cohesin is responsible for holding the sister chromatids together and serves important roles in chromosome condensation, gene regulation, and the repair of DNA damage. Cohesin contains an open central pore and becomes topologically engaged with its DNA substrates. Entrapped DNA can be released either by the opening of a gate in the cohesin ring or by proteolytic cleavage of a component of the ring. This review summarizes recent research that provides important new insights into how DNA enters and exits the cohesin ring and how the rings behave on entrapped DNA molecules to provide functional cohesion.

Summary Background Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit. Results Here we show that during experimentally induced metaphase delay, spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term "cohesion fatigue." Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the "prophase pathway" of cohesin release from chromosomes, or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids. Conclusion In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.

Anogenital cancers and head and neck cancers are causally associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live cell imaging data indicate that these changes do not arise from cell–cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude that HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation.

In mitotically dividing cells, the cohesin complex tethers sister chromatids, the products of DNA replication, together from the time they are generated during S phase until anaphase. Cohesion between sister chromatids ensures accurate chromosome segregation, and promotes normal gene regulation and certain kinds of DNA repair. In somatic cells, the core cohesin complex is composed of four subunits: Smc1, Smc3, Rad21 and an SA subunit. During meiotic cell divisions meiosis-specific isoforms of several of the cohesin subunits are also expressed and incorporated into distinct meiotic cohesin complexes. The relative contributions of these meiosis-specific forms of cohesin to chromosome dynamics during meiotic progression have not been fully worked out. However, the localization of these proteins during chromosome pairing and synapsis, and their unique loss-of-function phenotypes, suggest non-overlapping roles in controlling meiotic chromosome behavior. Many of the proteins that regulate cohesin function during mitosis also appear to regulate cohesin during meiosis. Here we review how cohesin contributes to meiotic chromosome dynamics, and explore similarities and differences between cohesin regulation during the mitotic cell cycle and meiotic progression. A deeper understanding of the regulation and function of cohesin in meiosis will provide important new insights into how the cohesin complex is able to promote distinct kinds of chromosome interactions under diverse conditions.

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.

A promoter-probe strategy was devised to identify genes specifically expressed byLegionella pneumophiladuring growth within the macrophage. Random fragments from theL. pneumophilachromosome were inserted upstream of a promoterless phage T4tdgene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes theL. pneumophilaortholog ofahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis ofahpCgene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate ofL. pneumophilain cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.

Activated Xenopus laevis eggs undergo a series of surface contractions in response to cell-cycle progression but fall to cleave unless the sperm centrosome is present. These surface contraction waves (SCWs) begin at the animal pole and progress around the egg, occur every cell cycle and precede cleavage [1–3]. The SCWs are biphasic, comprising a relaxation phase (SCWa) and a contraction phase (SCWb). To investigate how these events are linked to the underlying cell cycle, we studied the temporal and spatial relationship between the SCWs and previously characterized biochemical markers of cell-cycle progression. We found that the relaxation phase was a response to activated maturation-promoting factor (MPF). In contrast, the contraction phase required inactivation of MPF and was blocked when MPF activity was maintained at elevated levels. We also found that a wave of MPF activity traveled within the cell from the animal to the vegetal hemisphere. Taken together, these experiments suggest that the SCWs are a local response to a wave of MPF activation and inactivation. The egg cytoplasm, therefore, is metachronous in terms of cell-cycle progression; multiple cell-cycle states are present and spatially distinct within the egg at the same time.

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl–Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1–SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.

In most eukaryotic organisms, chiasmata, the connections formed between homologous chromosomes as a consequence of crossing over, are important for ensuring that the homologues move away from each other at meiosis I. Some organisms have the capacity to partition the rare homologues that have failed to experience reciprocal recombination. The yeast Saccharomyces cerevisiae is able to correctly partition achiasmate homologues with low fidelity by a mechanism that is largely unknown. It is possible to test which parameters affect the ability of achiasmate chromosomes to segregate by constructing strains that will have three achiasmate chromosomes at the time of meiosis. The meiotic partitioning of these chromosomes can be monitored to determine which ones segregate away from each other at meiosis I. This approach was used to test the influence of homologous yeast DNA sequences, recombination intiation sites, chromosome size and crossing over on the meiotic segregation of the model chromosomes. Chrome some size had no effect on achiasmate segregation. The influence of homologous yeast sequences on the segregation of noncrossover model chromosomes was negligible. In meioses in which two of the three model chromosomes experienced a crossover, they nearly always disjoined at meiosis I.

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.

The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, whereas resorption of the cilium precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein Nde1 (nuclear distribution gene E homologue 1) as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 have longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer's vesicle and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications for understanding the transition from a quiescent to a proliferative state.

The number of identified E3 ubiquitin ligases has dramatically increased in recent years. However, the substrates targeted for degradation by these particular ligases have not been easily identified. One reason for the inability of matching substrates and ligases is the finding that E3 recognition elements in substrates are often poorly defined. This minimizes the likelihood that bioinformatic approaches will lead to the identification of E3 substrates. For example, the multi-subunit complex the anaphase promoting complex (APC) is an E3 that recognizes destruction boxes (RXXLXXXXD/N/E) or KEN motifs within substrates (Glotzer et al., 1991; Pfleger and Kirschner, 2000). However, many proteins that contain either a potential destruction or a KEN motif are not recognized by the APC in vitro or in vivo, suggesting that there are other, less well-defined characteristics of substrates that contribute to their ability to serve as APC substrates (Ayad, Rankin, and Kirschner, unpublished observations). Aside from bioinformatic approaches of identifying APC substrates, several groups have also attempted to use affinity techniques to discover novel APC substrates. This has not been widely successful, because many APC substrates are not abundant. Also, as is the case with many ligase-substrate interactions, the affinity of substrates for the APC is likely to be very low. All these considerations have motivated a search for other techniques to assist in identifying substrates of this particular E3 ligase. Here, we describe the use of in vitro expression cloning to identify novel APC substrates.

Sister chromatid cohesion is essential for the maintenance of genome integrity. Errors in regulation of cohesion result in increased sensitivity to DNA damage, mis-segregation of chromosomes, and loss of genetic information. We recently showed that sororin is an essential regulator of sister chromatid cohesion in vertebrates. Interestingly, we identified sororin in a screen for proteins whose levels are controlled by the Anaphase Promoting Complex (APC), a cell cycle-regulated ubiquitin ligase. Ubiquitination by the APC and the resulting degradation ensure that sororin levels are low throughout G1 and only rise during S phase. We speculate that this regulation is an essential part of the mechanism that ensures that cohesion is established only after there are in fact two sister chromatids to tie together. Cohesion thus established can then be used both to mediate recombinational DNA repair, as well as to ensure accurate sister chromatid segregation in anaphase. Both of these roles are essential to genome stability.

The number of identified E3 ubiquitin ligases has dramatically increased in recent years. However, the substrates targeted for degradation by these particular ligases have not been easily identified. One reason for the inability of matching substrates and ligases is the finding that E3 recognition elements in substrates are often poorly defined. This minimizes the likelihood that bioinformatic approaches will lead to the identification of E3 substrates. For example, the multi-subunit complex the anaphase promoting complex (APC) is an E3 that recognizes destruction boxes (RXXLXXXXD/N/E) or KEN motifs within substrates (Glotzer et al., 1991; Pfleger and Kirschner, 2000). However, many proteins that contain either a potential destruction or a KEN motif are not recognized by the APC in vitro or in vivo, suggesting that there are other, less well-defined characteristics of substrates that contribute to their ability to serve as APC substrates (Ayad, Rankin, and Kirschner, unpublished observations). Aside from bioinformatic approaches of identifying APC substrates, several groups have also attempted to use affinity techniques to discover novel APC substrates. This has not been widely successful, because many APC substrates are not abundant. Also, as is the case with many ligase-substrate interactions, the affinity of substrates for the APC is likely to be very low. All these considerations have motivated a search for other techniques to assist in identifying substrates of this particular E3 ligase. Here, we describe the use of in vitro expression cloning to identify novel APC substrates.

Publisher Summary This chapter reveals that the genetic analysis of outer membrane protein function in gram-negative bacteria may be hampered by the inability to achieve proper localization of potentially informative mutant proteins. The technique described allows functional analysis of an outer membrane protein with mutant derivatives, which cannot be properly expressed on the cell surface. The proteins are purified and subsequently attached to the bacterial cell surface, which allows easy manipulation of the final protein coating concentration. The technique is therefore particularly useful in studying the relative ability of a surface-exposed protein that mediates binding to and uptake by eukaryotic cells. The apparent obstacles to this genetic analysis, and the availability of the maltose-binding protein (MBP)-invasin hybrid proteins, led to the design of a new protocol for the analysis of invasin function. By attaching MBP-Inv hybrid proteins to the surface of Staphylococcus aureus , an organism that is not ordinarily able to enter mammalian cells, it is possible to test various C-terminal fragments of invasin for their ability to mediate bacterial uptake using the previously standardized gentamicin protection assay. This approach could be broadly applicable in the analysis of potential adhesins or invasins from a wide range of unrelated organisms.

Yersinia pseudotuberculosis is able to enter normally nonphagocytic host cells by multiple pathways, the most efficient of which is mediated by invasin, a 986-amino-acid bacterial outer membrane protein. It has previously been shown that the C-terminal 192 amino acids of invasin are sufficient to bind mammalian cells. To determine if additional regions of the invasin protein are necessary to promote entry, we developed a novel assay that tests the ability of various invasin derivatives to confer on Staphylococcus aureus the ability to enter animal cells. We determined that the 192-amino-acid cell-binding region of invasin, when used to coat the bacterial cell surface, was also sufficient to promote cellular penetration. These results suggest that the simple binding of invasin to its receptors is sufficient to mediate entry and that the bacterium plays a largely passive role in the entry process.

We have suggested that in the nonhuman primate endometrium, stromal cells might play a role in mediating the effects of estrogen on the epithelium, especially during the luteal—follicular transition (LFT) when target cells normally escape from the inhibitory influence of progesterone (P). We now report that like estrogen receptors (ER), endometrial progestin receptors (PR) are detectable only in stromal cells until the fifth day of the LFT. With a technique that combined immunocytochemistry and autoradiography on the same sections, we characterized the cellular distribution of ER or PR coincidentally with the localization of [ 3 H]thymidine taken up in vitro by endometria from monkeys undergoing an LFT. DNA synthesis in the glands of the upper endometrium was E 2 -dependent, but the distribution of [ 3 H]thymidine was not positively correlated with the presence of ER or PR. Readministration of P to animals on days 3 or 4 of the LFT significantly reduced the [ 3 H]thymidine labeling index of the glandular epithelium and caused stromal ER to decline, but P did not block the eventual appearance of ER in epithelial cells on day 5 of the LFT. Thus, E 2 stimulated DNA synthesis in epithelial cells that lacked ER, and P suppressed DNA synthesis in these cells even though PR was only detected in the stroma when P treatment began. These data are consistent with a role for endometrial stromal cells in mediating the effects of E 2 and P on the epithelium during the LFT.

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.

Sister chromatids are held together by the cohesin complex from the time they are made until cell division. In recent articles published in Cell and Current Biology , the characterization of Wapl, a newly identified cohesin-interacting protein, suggests that a dynamic interaction between the cohesin complex and chromatin is important for normal regulation of sister chromatid cohesion.