Assembly of kinetochores in vertebrate cells

The centromere is a specialized region of each chromosome that is essential for faithful chromosome segregation during mitosis and meiosis in eukaryotic cells. Centromeres are the site at which kinetochores are formed. The kinetochore is responsible for microtubule binding and chromosome movement. In this review, I will focus on recent advances in our understanding of centromere DNAs as sites for kinetochore assembly and the mechanism underlying kinetochore assembly in vertebrate cells.     

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.     

Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.     

Grp1 plays a key role in linking insulin signaling to glut4 recycling

The glucose transporter type 4 (glut4) is critical for metabolic homeostasis. Insulin regulates glut4 by modulating its expression on the cell surface. This regulation is mainly achieved by targeting the endocytic recycling of glut4. We identify general receptor for 3-phosphoinositides 1 (Grp1) as a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6) that promotes glut4 vesicle formation. Grp1 also promotes the later steps of glut4 recycling through ARF6. Insulin signaling regulates Grp1 through phosphorylation by Akt. We also find that mutations that mimic constitutive phosphorylation of Grp1 can bypass upstream insulin signaling to induce glut4 recycling. Thus, we have uncovered a major mechanism by which insulin regulates glut4 recycling. Our findings also reveal the complexity by which a single small GTPase in vesicular transport can coordinate its multiple steps to accomplish a round of transport.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Depletion of Drad21/Scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression

BACKGROUND: The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells.RESULTS: We have shown that a bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 non-SMC subunits associate more intimately with one another than they do with the SMCs. We have observed defects in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. We have also made the surprising observation that SA is unstable in the absence of Drad21; however, we have shown that the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression. CONCLUSIONS: We conclude that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex will be tightly regulated.     

Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.     

The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

Parental provisioning behaviour plays a key role in linking personality with reproductive success

Repeatable behavioural traits (‘personality’) have been shown to covary with fitness, but it remains poorly understood how such behaviour–fitness relationships come about. We applied a multivariate approach to reveal the mechanistic pathways by which variation in exploratory and aggressive behaviour is translated into variation in reproductive success in a natural population of blue tits, Cyanistes caeruleus. Using path analysis, we demonstrate a key role for provisioning behaviour in mediating the link between personality and reproductive success (number of fledged offspring). Aggressive males fed their nestlings at lower rates than less aggressive individuals. At the same time, their low parental investment was associated with increased female effort, thereby positively affecting fledgling production. Whereas male exploratory behaviour was unrelated to provisioning behaviour and reproductive success, fast-exploring females fed their offspring at higher rates and initiated breeding earlier, thus increasing reproductive success. Our findings provide strong support for specific mechanistic pathways linking components of behavioural syndromes to reproductive success. Importantly, relationships between behavioural phenotypes and reproductive success were obscured when considering simple bivariate relationships, underlining the importance of adopting multivariate views and statistical tools as path analysis to the study of behavioural evolution.     

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase.     

Substitution F659G in the Irr1p/Scc3p cohesin influences the cell wall of Saccharomyces cerevisiae

The sister chromatid cohesion complex of Saccharomyces cerevisiae is composed of proteins termed cohesins. The complex forms a ring structure that entraps sister DNAs, probably following replication. The mechanism of cohesion is universal and the proteins participating in this process are evolutionarily highly conserved. We investigated the Irr1p/Scc3p cohesin subunit, an under-studied protein. We show that the presence of a mutated copy of IRR1 gene, encoding the F658G substitution in Irr1p, changes the sensitivity of the heterozygous irr1-1/IRR1 diploid to cell wall-affecting compounds. Microscopic images indicate that chitin distribution in the mutant cell wall is affected, although the biochemical composition of the cell wall is not drastically changed. This observation suggests that irr1-1 mutation in heterozygous state may influence the cell wall integrity and indicates a possible link between mechanisms regulating the cell wall biosynthesis, nuclear migration and chromosome segregation.     

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.     

The role of hydrogen peroxide produced by polychlorinated biphenyls in PMR1-deficient yeast cells

Polychlorinated biphenyls (PCBs) are well-known recalcitrant environmental pollutants. Although the metabolism of the PCBs has been intensively studied, very little is known about their mechanism of toxicity in living organisms or how they are degraded. We have examined the effects of PCBs on two different yeast strains to determine their mechanism of action. One yeast strain (K601, wild type) is resistant to the growth-inhibitory effect of PCBs, whereas the other strain (AA542, PMR1 mutant) is susceptible. PCBs increased the level of intracellular hydrogen peroxide in AA542 cells but not in K601 cells. In the presence of alpha-tocopherol or ursolic acid the growth of AA542 cells was not inhibited by treatment with PCBs. These results suggest that PCBs block cell growth through production of hydrogen peroxide in the PMR1 mutant strain, AA542. We compared superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase activities in both strains. The catalase activity in K601 cells was 10 times higher than that in AA542 cells. In contrast, there was no difference in activities of SOD and GPx between the two strains. Collectively, these observations indicate that oxidative stress causes the inhibition of cell growth observed in catalase-deficient yeast cells exposed to PCBs.     

Additional copies of the NOG2 and IST2 genes suppress the deficiency of cohesin Irr1p/Scc3p in Saccharomyces cerevisiae

The protein encoded by the IRR1/SCC3 gene is an element of the cohesin complex of Saccharomyces cerevisiae, responsible for establishing and maintaining sister chromatid cohesion during mitotic cell division. We noticed previously that lowering the level of expression of IRR1/SCC3 affects colony formation on solid support. Here we describe two dosage suppressors (IST2, NOG2) overcoming the inability to form colonies of an Irr1p-deficient strain. Ist2 is probably involved in osmotolerance, Nog2p is a putative GTPase required for 60S ribosomal subunit maturation, but may also participate in mRNA splicing.     

Crucial role of vHNF1 in vertebrate hepatic specification

Mouse liver induction occurs via the acquisition of ventral endoderm competence to respond to inductive signals from adjacent mesoderm, followed by hepatic specification. Little is known about the regulatory circuit involved in these processes. Through the analysis of vHnf1 (Hnf1b)-deficient embryos, generated by tetraploid embryo complementation, we demonstrate that lack of vHNF1 leads to defective hepatic bud formation and abnormal gut regionalization. Thickening of the ventral hepatic endoderm and expression of known hepatic genes do not occur. At earlier stages, hepatic specification of vHnf1(-/-) ventral endoderm is disrupted. More importantly, mutant ventral endoderm cultured in vitro loses its responsiveness to inductive FGF signals and fails to induce the hepatic-specification genes albumin and transthyretin. Analysis of liver induction in zebrafish indicates a conserved role of vHNF1 in vertebrates. Our results reveal the crucial role of vHNF1 at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification.     

Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion

The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.     

The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells

When late prophase PtK₁ cells are chilled to 6 ° C the nuclear envelope (NE) breaks down as in normal cells but the spindle is inhibited from forming. When these cells are subsequently warmed to 18 ° C the spindle slowly forms and pro-metaphase congression ensues. Using this approach we have been able to experimentally eliminate the influence of asynchronous NE breakdown on the formation and development of the spindle, and also to slow down (and thus increase the temporal separation of) the subsequent events which occur during the initial stages of spindle formation. Correlative light and high voltage electron microscopic studies on these cells, fixed after various times of recovery, reveal the following results: 1) the centrosomes generate microtubules (MTs) well before MTs are seen to be associated with the kinetochores; 2) as in untreated PtK₁ cells (Roos, 1973a, 1976) the order in which chromosomes attach to the forming spindle is influenced by their proximity to a centrosome-kinetochores closest to a centrosome appear stretched towards the centrosome at a time during recovery when other kinetochores, more distal to the centrosome appear unstretched and unoriented; 3) as in untreated cells (Heneen, 1970; Roos, 1976) the predominant behavior during recovery is for a chromosome to initially mono-orient and associate with the near centrosome and only later to develop a bipolar association; and 4) MTs associated with early pro-metaphase kinetochores are almost always oriented towards a centrosome. — From our results we conclude that the proximity effect and the tendency of pro-metaphase chromosomes in PtK₁ to initially mono-orient and associate with the near centrosome cannot be ascribed, as suggested by Roos (1976), to influences arising during the asynchronous breakdown of the NE. Rather, our data clearly demonstrate that a kinetochore-centrosome interaction occurs during spindle formation which cannot be attributed to transient influences. The proximity effect and the predominant tendency of PtK₁ pro-metaphase chromosomes to mono-orient to the near pole are taken to signify the existance of a centrosomal influence on the attachment and orientation of chromosomes. Two possible mechanisms for this influence, both involving a structural interaction between the centrosome and the kinetochore, are outlined.     

Stearoyl-CoA Desaturase 1 (SCD1) is a key factor mediating diabetes in MyD88-deficient mice

Saturated fatty acids, acting as ligands for toll-like receptor 4 (TLR4), induce inflammation and mediate the development of insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adaptor protein for TLR4. Previously, we found MyD88-deficient mice fed a high-fat diet (HFD) exhibited a severe diabetic phenotype. Stearoyl-CoA Desaturase 1 (SCD1) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and known as a risk factor of diabetes. In the present study, we found SCD1 was dramatically increased in HFD-fed MyD88-deficient mice liver. This finding showed the novel linkage between MyD88 and SCD1 in the development of diabetes mellitus.