E-cadherin variants associated with oral facial clefts trigger aberrant cell motility in a REG1A-dependent manner.

BACKGROUND: Germline mutations of E-cadherin contribute to hereditary diffuse gastric cancer (HDGC) and congenital malformations, such as oral facial clefts (OFC). However, the molecular mechanisms through which E-cadherin loss-of-function triggers distinct clinical outcomes remain unknown. We postulate that E-cadherin-mediated disorders result from abnormal interactions with the extracellular matrix and consequent aberrant intracellular signalling, affecting the coordination of cell migration. METHODS: Herein, we developed in vivo and in vitro models of E-cadherin mutants associated with either OFC or HDGC. Using a Drosophila approach, we addressed the impact of the different variants in cell morphology and migration ability. By combining gap closure migration assays and time-lapse microscopy, we further investigated the migration pattern of cells expressing OFC or HDGC variants. The adhesion profile of the variants was evaluated using high-throughput ECM arrays, whereas RNA sequencing technology was explored for identification of genes involved in aberrant cell motility. RESULTS: We have demonstrated that cells expressing OFC variants exhibit an excessive motility performance and irregular leading edges, which prevent the coordinated movement of the epithelial monolayer. Importantly, we found that OFC variants promote cell adhesion to a wider variety of extracellular matrices than HDGC variants, suggesting higher plasticity in response to different microenvironments. We unveiled a distinct transcriptomic profile in the OFC setting and pinpointed REG1A as a putative regulator of this outcome. Consistent with this, specific RNAi-mediated inhibition of REG1A shifted the migration pattern of OFC expressing cells, leading to slower wound closure with coordinated leading edges. CONCLUSIONS: We provide evidence that E-cadherin variants associated with OFC activate aberrant signalling pathways that support dynamic rearrangements of cells towards improved adaptability to the microenvironment. This proficiency results in abnormal tissue shaping and movement, possibly underlying the development of orofacial malformations.

Mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

DNA damage response (DDR) during interphase involves active signaling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy, it was found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but ataxia telangiectasia mutated-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, it is shown that mitotic DNA damage causes missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Importantly, with the exception of DNA damage induced by etoposide-a topoisomerase II inhibitor-this outcome was independent of a general effect on kinetochore microtubule stability. Colony formation assays in pan-cancer cell line models revealed that mitotic DNA damage causes distinct cytotoxic effects, depending on the nature and extent of the damage. Overall, these findings unveil and raise awareness that therapeutic DNA damage regimens may contribute to genomic instability through a surprising link with chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

Mechanical Properties of Colorectal Cancer Cells Determined by Dynamic Atomic Force Microscopy: A Novel Biomarker.

Colorectal cancer (CRC) has been addressed in the framework of molecular, cellular biology, and biochemical traits. A new approach to studying CRC is focused on the relationship between biochemical pathways and biophysical cues, which may contribute to disease understanding and therapy development. Herein, we investigated the mechanical properties of CRC cells, namely, HCT116, HCT15, and SW620, using static and dynamic methodologies by atomic force microscopy (AFM). The static method quantifies Young's modulus; the dynamic method allows the determination of elasticity, viscosity, and fluidity. AFM results were correlated with confocal laser scanning microscopy and cell migration assay data. The SW620 metastatic cells presented the highest Young's and storage moduli, with a defined cortical actin ring with distributed F-actin filaments, scarce vinculin expression, abundant total focal adhesions (FAK), and no filopodia formation, which could explain the lessened migratory behavior. In contrast, HCT15 cells presented lower Young's and storage moduli, high cortical tubulin, less cortical F-actin and less FAK, and more filopodia formation, probably explaining the higher migratory behavior. HCT116 cells presented Young's and storage moduli values in between the other cell lines, high cortical F-actin expression, intermediate levels of total FAK, and abundant filopodia formation, possibly explaining the highest migratory behavior.

Integrin ß1 orchestrates the abnormal cell-matrix attachment and invasive behaviour of E-cadherin dysfunctional cells.

BACKGROUND: Tumour progression relies on the ability of cancer cells to penetrate and invade neighbouring tissues. E-cadherin loss is associated with increased cell invasion in gastric carcinoma, and germline mutations of the E-cadherin gene are causative of hereditary diffuse gastric cancer. Although E-cadherin dysfunction impacts cell-cell adhesion, cell dissemination also requires an imbalance of adhesion to the extracellular matrix (ECM). METHODS: To identify ECM components and receptors relevant for adhesion of E-cadherin dysfunctional cells, we implemented a novel ECM microarray platform coupled with molecular interaction networks. The functional role of putative candidates was determined by combining micropattern traction microscopy, protein modulation and in vivo approaches, as well as transcriptomic data of 262 gastric carcinoma samples, retrieved from the cancer genome atlas (TCGA). RESULTS: Here, we show that E-cadherin mutations induce an abnormal interplay of cells with specific components of the ECM, which encompasses increased traction forces and Integrin ß1 activation. Integrin ß1 synergizes with E-cadherin dysfunction, promoting cell scattering and invasion. The significance of the E-cadherin-Integrin ß1 crosstalk was validated in Drosophila models and found to be consistent with evidence from human gastric carcinomas, where increased tumour grade and poor survival are associated with low E-cadherin and high Integrin ß1 levels. CONCLUSIONS: Integrin ß1 is a key mediator of invasion in carcinomas with E-cadherin impairment and should be regarded as a biomarker of poor prognosis in gastric cancer.

A guide to adhesion GPCR research.

Adhesion G protein-coupled receptors (aGPCRs) are a class of structurally and functionally highly intriguing cell surface receptors with essential functions in health and disease. Thus, they display a vastly unexploited pharmacological potential. Our current understanding of the physiological functions and signaling mechanisms of aGPCRs form the basis for elucidating further molecular aspects. Combining these with novel tools and methodologies from different fields tailored for studying these unusual receptors yields a powerful potential for pushing aGPCR research from singular approaches toward building up an in-depth knowledge that will facilitate its translation to applied science. In this review, we summarize the state-of-the-art knowledge on aGPCRs in respect to structure-function relations, physiology, and clinical aspects, as well as the latest advances in the field. We highlight the upcoming most pressing topics in aGPCR research and identify strategies to tackle them. Furthermore, we discuss approaches how to promote, stimulate, and translate research on aGPCRs 'from bench to bedside' in the future.

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK i: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK i: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK d values comparable to the pK i values. The fluorescent probes 21, 23, and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Orally administrated chitosan microspheres bind Helicobacter pylori and decrease gastric infection in mice.

Persistent Helicobacter pylori (H. pylori) infection is related to 90% of gastric cancers. With bacterial resistance rising and treatment inefficiency affecting 15% of the patients, alternative treatments urge. Chitosan microspheres (ChMics) have been proposed as an H. pylori-binding system. This work evaluates ChMics biocompatibility, mucopenetration and capacity to treat H. pylori infection in mice after oral administration. ChMics of different size (XL, ∼120 µm and XS, ∼40 µm) and degree of acetylation (6% and 16%) were developed and revealed to be able to adhere both human and mouse-adapted H. pylori strains without cytotoxicity towards human gastric cells. Ex vivo studies showed that smaller (XS) microspheres penetrate further within the gastric foveolae, suggesting their ability to reach deeply adherent bacteria. In vivo assays showed 88% reduction of infection when H. pylori-infected mice (C57BL/6) were treated with more mucoadhesive XL6 and XS6 ChMics. Overall, ChMics clearly demonstrate ability to reduce H. pylori gastric infection in mice, with chitosan degree of acetylation being a dominant factor over microspheres' size on H. pylori removal efficiency. These results evidence the strong potential of this strategy as an antibiotic-free approach to fight H. pylori infection, where microspheres are orally administered, bind H. pylori in the stomach, and remove them through the gastrointestinal tract. STATEMENT OF SIGNIFICANCE: Approximately 90% of gastric cancers are caused by the carcinogenic agent Helicobacter pylori, which infects >50% of the world population. Bacterial resistance, reduced antibiotic bioavailability, and the intricate distribution of bacteria in mucus and within gastric foveolae hamper the success of most strategies to fight H. pylori. We demonstrate that an antibiotic-free therapy based on bare chitosan microspheres that bind and remove H. pylori from stomach can achieve 88% reduction of infection from H. pylori-infected mice. Changing size and mucoadhesive properties, microspheres can reach different areas of gastric mucosa: smaller and less mucoadhesive can penetrate deeper into the foveolae. This promising, simple and inexpensive strategy paves the way for a faster bench-to-bedside transition, therefore holding great potential for clinical application.

Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp.

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages.

Differential GIP/GLP-1 intestinal cell distribution in diabetics' yields distinctive rearrangements depending on Roux-en-Y biliopancreatic limb length.

As incretins are known to play an important role in type 2 diabetics (T2D) improvement observed after Roux-en-Y gastric bypass (RYGB), our aim was to assess whether increasing the length of RYGB biliopancreatic limb in T2D would modify the incretin staining cell density found after the gastric outlet. Small intestine biopsies (n = 38) were harvested during RYGB at two different distances from the duodenal angle; either 60-90 cm (n = 28), from non-diabetic (n = 18) patients, and T2D (n = 10), or 200 cm (n = 10) from T2D. GIP and GLP-1 staining cells were identified by immunohistochemistry and GLP-1/GIP co-staining cells by immunofluorescence. Incretin staining cell density at the proximal small intestine of T2D and non-diabetic individuals was similar. At 200 cm, T2D patients depicted a significantly lower GIP staining cell density (0.181 ± 0.016 vs 0.266 ± 0.033, P = 0.038) with a similar GLP-1 staining cell density when compared to the proximal gut. GIP/GLP-1 co-staining cells was similar in all studied groups. In T2D patients, the incretin staining cells density in the distal intestine is significantly different from the proximal gut. Thus, a longer RYGB biliopancreatic limb produces a distinctive incretin cell pattern at the gastro-enteric anastomosis that can result in different endocrine profiles.

A genome-scale RNAi screen for genetic interactors of the dynein co-factor nud-2 in Caenorhabditis elegans.

Cytoplasmic dynein 1 (dynein) is the predominant microtubule minus end-directed motor in animals and participates in a wide range of cellular processes, including membrane trafficking, nuclear migration, and cell division. Dynein's functional diversity depends on co-factors that regulate its subcellular localization, interaction with cargo, and motor activity. The ubiquitous co-factor nuclear distribution gene E (NudE) is implicated in many of dynein's functions, and mutations in NudE cause the brain developmental disease microcephaly. To identify genetic interactors of the Caenorhabditis elegans NudE homolog nud-2, we performed a genome-wide RNAi screen with the null allele nud-2(ok949), which compromises dynein function but leaves animals viable and fertile. Using bacterial feeding to deliver dsRNAs in a 96-well liquid format and a semi-automated fluorescence microscopy approach for counting parents and progeny, we screened 19762 bacterial clones and identified 38 genes whose inhibition caused enhanced lethality in nud-2(ok949) relative to the nud-2(+) control. Further study of these genes, many of which participate in cell division, promises to provide insight into the function and regulation of dynein.

Immunomodulation of Human Mesenchymal Stem/Stromal Cells in Intervertebral Disc Degeneration: Insights From a Proinflammatory/Degenerative Ex Vivo Model.

STUDY DESIGN: Ex vivo experimental study. OBJECTIVE: To investigate the effect of proinflammatory/degenerative intervertebral disc (IVD) microenvironment on the regenerative and immunomodulatory behavior of mesenchymal stem/stromal cells (MSCs), using an ex vivo model from bovine origin. SUMMARY OF BACKGROUND DATA: Low back pain is a cause of disability worldwide, most frequently associated with IVD degeneration and inflammation, and characterized by increased levels of inflammatory mediators, often disregarded. MSC-based therapies to low back pain have been advocated, but the involvement of inflammation in IVD remodeling mechanism, promoted by MSCs has not yet been explored. METHODS: Bovine IVD organ cultures of nucleus pulposus punches were stimulated with needle puncture and culture medium supplementation with 10 ng/mL of interleukin (IL)-1ß, to induce a proinflammatory/degenerative environment, as previously established. Human bone marrow-derived MSCs were cultured on top of transwells, placed above nucleus pulposus punches, for up to 16 days. MSCs were analyzed by screening cell viability/apoptosis, metabolic activity, migration, and inflammatory cytokines production in response to the proinflammatory environment. IVD extracellular matrix (ECM) remodeling, gene expression profile of IVD cells, and inflammatory cytokine profile in the presence of MSCs in basal versus proinflammatory conditions were also evaluated. RESULTS: Proinflammatory/degenerative IVD conditions did not affect MSCs viability, but promoted cell migration, while increasing IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 and reducing transforming growth factor-ß1 production by MSCs. MSCs did not stimulate ECM production (namely type II collagen or aggrecan) in neither basal nor inflammatory conditions, instead MSCs downregulated bovine proinflammatory IL-6, IL-8, and TNF-α gene expression levels in IL-1ß-stimulated IVDs. CONCLUSION: The present study provides evidence for an immunomodulatory paracrine effect of MSCs in degenerated IVD without an apparent effect in ECM remodeling, and suggest an MSCs mechanism-of-action dependent on a cytokine feedback loop. LEVEL OF EVIDENCE: 5.

Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment.

Orchestration of bone repair processes requires crosstalk between different cell populations, including immune cells and mesenchymal stem/stromal cells (MSC). Extracellular vesicles (EV) as mediators of these interactions remain vastly unexplored. Here, we aimed to determine the mechanism of MSC recruitment by Dendritic Cells (DC), hypothesising that it would be mediated by EV. Primary human DC-secreted EV (DC-EV), isolated by ultracentrifugation, were characterized for their size, morphology and protein markers, indicating an enrichment in exosomes. DC-EV were readily internalized by human bone marrow-derived MSC, without impacting significantly their proliferation or influencing their osteogenic/chondrogenic differentiation. Importantly, DC-EV significantly and dose-dependently promoted MSC recruitment across a transwell system and enhanced MSC migration in a microfluidic chemotaxis assay. DC-EV content was analysed by chemokine array, indicating the presence of chemotactic mediators. Osteopontin and matrix metalloproteinase-9 were confirmed inside EV. In summary, DC-EV are naturally loaded with chemoattractants and can contribute to cell recruitment, thus inspiring the development of new tissue regeneration strategies.

Reed-Sternberg cells form by abscission failure in the presence of functional Aurora B kinase.

Large multinucleated Reed-Sternberg cells (RS) and large mononucleated Hodgkin cells (H) are traditionally considered to be the neoplastic population in classical Hodgkin lymphoma, (cHL) and postulated to promote the disease. However, the contribution of these larger cells to the progression of cHL remains debatable. We used established cHL cell lines and cHL cellular fractions composed of small mononucleated cells only or enriched in large RS/H cells to investigate RS/H cell origin and to characterize the cells which they derive from. We confirm that the small mononucleated cells give rise to RS/H cells, and we show that the latter proliferate significantly more slowly than the small cells. By using live-cell imaging, we demonstrate that binucleated RS cells are generated by failure of abscission when a few small cells attempt to divide. Finally, our results reveal that the small mononucleated cells are chromosomally unstable, but this is unlikely to be related to a malfunctioning chromosomal passenger protein complex. We propose that the small mononucleated cells, rather than the RS/H cells, are the main drivers of cHL.

Genome-wide RNAi screen for synthetic lethal interactions with the C. elegans kinesin-5 homolog BMK-1.

Kinesins are a superfamily of microtubule-based molecular motors that perform various transport needs and have essential roles in cell division. Among these, the kinesin-5 family has been shown to play a major role in the formation and maintenance of the bipolar mitotic spindle. Moreover, recent work suggests that kinesin-5 motors may have additional roles. In contrast to most model organisms, the sole kinesin-5 gene in Caenorhabditis elegans, bmk-1, is not required for successful mitosis and animals lacking bmk-1 are viable and fertile. To gain insight into factors that may act redundantly with BMK-1 in spindle assembly and to identify possible additional cellular pathways involving BMK-1, we performed a synthetic lethal screen using the bmk-1 deletion allele ok391. We successfully knocked down 82% of the C. elegans genome using RNAi and assayed viability in bmk-1(ok391) and wild type strains using an automated high-throughput approach based on fluorescence microscopy. The dataset includes a final list of 37 synthetic lethal interactions whose further study is likely to provide insight into kinesin-5 function.

Stability of kinetochore-microtubule attachment and the role of different KMN network components in Drosophila.

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability.

Aurora B kinase cooperates with CENP-E to promote timely anaphase onset.

Error-free chromosome segregation requires that all chromosomes biorient on the mitotic spindle. The motor protein Centromere-associated protein E (CENP-E) facilitates chromosome congression by mediating the lateral sliding of sister chromatids along existing K-fibers, while the mitotic kinase Aurora B detaches kinetochore-microtubule interactions that are not bioriented. Whether these activities cooperate to promote efficient chromosome biorientation and timely anaphase onset is not known. We here show that the chromosomes that fail to congress after CENP-E depletion displayed high centromeric Aurora B kinase activity. This activity destabilized spindle pole proximal kinetochore-microtubule interactions resulting in a checkpoint-dependent mitotic delay that allowed CENP-E-independent chromosome congression, thus reducing chromosome segregation errors. This shows that Aurora B keeps the mitotic checkpoint active by destabilizing kinetochore fibers of polar chromosomes to permit chromosome congression in CENP-E-compromised cells and implies that this kinase normally prevents pole proximal syntelic attachments to allow CENP-E-mediated congression of mono-oriented chromosomes.

RAMA1 is a novel kinetochore protein involved in kinetochore-microtubule attachment.

During mitosis, kinetochores need to attach to microtubules emanating from spindle poles. Several protein complexes have been shown to mediate the kinetochore-microtubule interaction. However, with the continually growing number of newly identified kinetochore proteins, it is unclear whether all major components of the kinetochore-microtubule interface have been identified. We therefore performed a high-throughput RNAi screen to identify additional factors involved in kinetochore-microtubule attachment, and identified RAMA1 as a novel regulator of this process. Depletion of RAMA1 results in severe chromosome alignment defects and a checkpoint-dependent mitotic arrest. We show that this is due to reduced kinetochore-microtubule attachments. RAMA1 localizes to the spindle and to outer kinetochores throughout all phases of mitosis and is recruited to kinetochores by the core kinetochore-microtubule attachment factor Hec1. Interestingly, unlike Hec1, the association of RAMA1 with kinetochores is highly dynamic, suggesting that it is not a structural component of the kinetochore. Consistent with this, all other kinetochore proteins tested do not require RAMA1 for their kinetochore localization. Taken together, these results identify RAMA1 as a novel kinetochore protein and suggest that RAMA1 may have a direct role in mediating kinetochore-microtubule interactions.

Sgt1, a co-chaperone of Hsp90 stabilizes Polo and is required for centrosome organization.

Sgt1 was described previously in yeast and humans to be a Hsp90 co-chaperone and required for kinetochore assembly. We have identified a mutant allele of Sgt1 in Drosophila and characterized its function. Mutations in sgt1 do not affect overall kinetochore assembly or spindle assembly checkpoint. sgt1 mutant cells enter less frequently into mitosis and arrest in a prometaphase-like state. Mutations in sgt1 severely compromise the organization and function of the mitotic apparatus. In these cells, centrioles replicate but centrosomes fail to mature, and pericentriolar material components do not localize normally resulting in highly abnormal spindles. Interestingly, a similar phenotype was described previously in Hsp90 mutant cells and correlated with a decrease in Polo protein levels. In sgt1 mutant neuroblasts, we also observe a decrease in overall levels of Polo. Overexpression of the kinase results in a substantial rescue of the centrosome defects; most cells form normal bipolar spindles and progress through mitosis normally. Taken together, these findings suggest that Sgt1 is involved in the stabilization of Polo allowing normal centrosome maturation, entry and progression though mitosis.

The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond.

During mitosis, correct bipolar chromosome attachment to the mitotic spindle is an essential prerequisite for the equal segregation of chromosomes. The spindle assembly checkpoint can prevent chromosome segregation as long as not all chromosome pairs have obtained bipolar attachment to the spindle. The chromosomal passenger complex plays a crucial role during chromosome alignment by correcting faulty chromosome-spindle interactions (e.g. attachments that do not generate tension). In the process of doing so, the chromosomal passenger complex generates unattached chromosomes, a specific situation that is known to promote checkpoint activity. However, several studies have implicated an additional, more direct role for the chromosomal passenger complex in enforcing the mitotic arrest imposed by the spindle assembly checkpoint. In this review, we discuss the different roles played by the chromosomal passenger complex in ensuring proper mitotic checkpoint function. Additionally, we discuss the possibility that besides monitoring the presence of unattached kinetochores, the spindle assembly checkpoint may also be capable of responding to chromosome-microtubule interactions that do not generate tension and we propose experimental set-ups to study this.

BubR1 and CENP-E have antagonistic effects upon the stability of microtubule-kinetochore attachments in Drosophila S2 cell mitosis.

The spindle assembly checkpoint ensures the fidelity of chromosome segregation at each cell division cycle. Previous reports have indicated that in higher eukaryotes checkpoint proteins, such as BubR1, are also implicated in chromosome congression, more specifically that BubR1 regulates chromosome-spindle attachments. Also, several studies have shown that BubR1 interacts with the microtubule motor protein CENP-E. Whether this association contributes to the regulation of chromosome-spindle attachments is not yet known. Accordingly, we performed a detailed analysis of microtubule-kinetochore interactions after depletion of BubR1 and the Drosophila CENP-E homolog, CENP-meta by RNAi. We find that depletion of BubR1 affects mitosis very differently from depletion of CENP-meta. While BubR1-depleted cells exit mitosis prematurely due to loss of SAC activity, CENP-meta-depleted cells accumulate in prometaphase and do not exit mitosis after spindle damage. Also, in contrast to cells depleted for CENP-meta, cells depleted for BubR1 very rarely reach full metaphase alignment even if arrested in mitosis with the proteasome inhibitor MG132. More importantly, we show for the first time that BubR1-depleted cells contain a high frequency of either monoriented or fully unattached chromosomes while most CENP-meta dsRNAi-treated cells have chromosomes attached to spindle microtubules. Moreover, simultaneous depletion of both proteins reveals that absence of CENP-meta is able to partially rescue the unattached chromosome phenotype observed after BubR1 depletion. These results strongly suggest that while BubR1 is required to promote stable microtubule kinetochore attachment, CENP-E appears to be required to destabilize kinetochore attachment. Overall our results suggest that activation of the mechanism that corrects inappropriate kinetochore attachment requires the antagonistic effects of BubR1 and CENP-E.