Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit

CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis

Macrophages abundantly found in the tumor microenvironment enhance malignancy(1). At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promote tumor cell extravasation, seeding and persistent growth(2). Our study has defined the origin of these macrophages by showing Gr1+ inflammatory monocytes (IMs) are preferentially recruited to pulmonary metastases but not primary mammary tumors, a process also found for human IMs in pulmonary metastases of human breast cancer cells. The recruitment of these CCR2 (receptor for chemokine CCL2) expressing IMs and subsequently MAMs and their interaction with metastasizing tumor cells is dependent on tumor and stromal synthesized CCL2 (FigS1). Inhibition of CCL2/CCR2 signaling using anti-CCL2 antibodies blocks IM recruitment and inhibits metastasis in vivo and prolongs the survival of tumor-bearing mice. Depletion of tumor cell-derived CCL2 also inhibits metastatic seeding. IMs promote tumor cell extravasation in a process that requires monocyte-derived VEGF. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer (Fig S2)(3-6). Our data provides the mechanistic link between these two clinical associations and indicates new therapeutic targets for treating metastatic breast disease

A Novel Endothelial L-Selectin Ligand Activity in Lymph Node Medulla That Is Regulated by α(1,3)-Fucosyltransferase-IV

Lymphocytes home to peripheral lymph nodes (PLNs) via high endothelial venules (HEVs) in the subcortex and incrementally larger collecting venules in the medulla. HEVs express ligands for L-selectin, which mediates lymphocyte rolling. L-selectin counterreceptors in HEVs are recognized by mAb MECA-79, a surrogate marker for molecularly heterogeneous glycans termed peripheral node addressin. By contrast, we find that medullary venules express L-selectin ligands not recognized by MECA-79. Both L-selectin ligands must be fucosylated by α(1,3)-fucosyltransferase (FucT)-IV or FucT-VII as rolling is absent in FucT-IV+VII−/− mice. Intravital microscopy experiments revealed that MECA-79–reactive ligands depend primarily on FucT-VII, whereas MECA-79–independent medullary L-selectin ligands are regulated by FucT-IV. Expression levels of both enzymes paralleled these anatomical distinctions. The relative mRNA level of FucT-IV was higher in medullary venules than in HEVs, whereas FucT-VII was most prominent in HEVs and weak in medullary venules. Thus, two distinct L-selectin ligands are segmentally confined to contiguous microvascular domains in PLNs. Although MECA-79–reactive species predominate in HEVs, medullary venules express another ligand that is spatially, antigenically, and biosynthetically unique. Physiologic relevance for this novel activity in medullary microvessels is suggested by the finding that L-selectin–dependent T cell homing to PLNs was partly insensitive to MECA-79 inhibition

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality

Targeting the Neurokinin Receptor 1 with Aprepitant: A Novel Antipruritic Strategy

Chronic pruritus is a global clinical problem with a high impact on the quality of life and lack of specific therapies. It is an excruciating and frequent symptom of e.g. uncurable renal, liver and skin diseases which often does not respond to conventional treatment with e.g. antihistamines. Therefore antipruritic therapies which target physiological mechanisms of pruritus need to be developed. Substance P (SP) is a major mediator of pruritus. As it binds to the neurokinin receptor 1 (NKR1), we evaluated if the application of a NKR1 antagonist would significantly decrease chronic pruritus.Twenty hitherto untreatable patients with chronic pruritus (12 female, 8 male; mean age, 66.7 years) were treated with the NKR1 antagonist aprepitant 80 mg for one week. 16 of 20 patients (80%) experienced a considerable reduction of itch intensity, as assessed by the visual analog scale (VAS, range 0 to 10). Considering all patients, the mean value of pruritus intensity was significantly reduced from 8.4 VAS points (SD +/-1.7) before treatment to 4.9 VAS points (SD +/-3.2) (p<0.001, CI 1.913-5.187). Patients with dermatological diseases (e.g. atopic diathesis, prurigo nodularis) had the best profit from the treatment. Side-effects were mild (nausea, vertigo, and drowsiness) and only occurred in three patients.The high response rate in patients with therapy refractory pruritus suggests that the NKR1 antagonist aprepitant may indeed exhibit antipruritic effects and may present a novel, effective treatment strategy based on pathophysiology of chronic pruritus. The results are promising enough to warrant confirming the efficacy of NKR1 antagonists in a randomized, controlled clinical trial

Kinetics of Eotaxin Generation and Its Relationship to Eosinophil Accumulation in Allergic Airways Disease: Analysis in a Guinea Pig Model In Vivo

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12–24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge

Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation

Fission Yeast Cells Undergo Nuclear Division in the Absence of Spindle Microtubules

Through a previously undescribed mechanism, fission yeast cells can undergo nuclear division and enter the next cell cycle, even in the absence of spindle microtubules

A quantitative systems view of the spindle assembly checkpoint

The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway

Bub1-Mediated Adaptation of the Spindle Checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore–microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest