André F. Maia

The spindle assembly checkpoint: Preventing chromosome mis-segregation during mitosis and meiosis

Aneuploidy is a common feature of many cancers, suggesting that genomic stability is essential to prevent tumorigenesis. Also, during meiosis, chromosome non‐disjunction produces gamete imbalance and when fertilized result in developmental arrest or severe birth defects. The spindle assembly checkpoint prevents chromosome mis‐segregation during both mitosis and meiosis. In mitosis, this control system monitors kinetochore‐microtubule attachment while in meiosis its role is still unclear. Interestingly, recent data suggest that defects in the spindle assembly checkpoint are unlikely to cause cancer development but might facilitate tumour progression. However, in meiosis a weakened checkpoint could contribute to age‐related aneuploidy found in humans.

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.

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.

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 homologue, 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.

Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

In order to improve the efficacy of conventional radiotherapy, attention has been paid to immune cells, which not only modulate cancer cell response to therapy but are also highly recruited to tumours after irradiation. Particularly, the effect of ionizing radiation on macrophages, using therapeutically relevant doses, is not well understood. To evaluate how radiotherapy affects macrophage behaviour and macrophage-mediated cancer cell activity, human monocyte derived-macrophages were subjected, for a week, to cumulative ionizing radiation doses, as used during cancer treatment (2 Gy/fraction/day). Irradiated macrophages remained viable and metabolically active, despite DNA damage. NF-kappaB transcription activation and increased Bcl-xL expression evidenced the promotion of pro-survival activity. A significant increase of pro-inflammatory macrophage markers CD80, CD86 and HLA-DR, but not CCR7, TNF and IL1B was observed after 10 Gy cumulative doses, while anti-inflammatory markers CD163, MRC1, VCAN and IL-10 expression decreased, suggesting the modulation towards a more pro-inflammatory phenotype. Moreover, ionizing radiation induced macrophage morphological alterations and increased their phagocytic rate, without affecting matrix metalloproteases (MMP)2 and MMP9 activity. Importantly, irradiated macrophages promoted cancer cell-invasion and cancer cell-induced angiogenesis. Our work highlights macrophage ability to sustain cancer cell activities as a major concern that needs to be addressed to improve radiotherapy efficacy.

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.

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.

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. Dyneins 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 dyneins 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.