Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule diversity by means of tubulin post-translational modifications. This so-called “tubulin code” might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed.

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.DOI: http://dx.doi.org/10.7554/eLife.05068.001

© F e r r a t a S t o r t i F o u n d a t i o npermeabilized in ice-cold PERM buffer III, washed in staining buffer and stained with the following antibodies: CD79a-APC; Akt-Alexa Fluor 488, PTEN-PE, phospho-Akt (S473)-Alexa Fluor 488, phospho-Akt (T308)-PE, and phospho-STAT5 (Y694)-Alexa Fluor 488. Samples were analyzed on a FACSAria or LSRFortessa using the gating strategy indicated in the Online Supplementary Appendix (Online Supplementary Figure S1). Endogenous PTEN in vitro lipid phosphatase assayPTEN phosphatase activity was measured in vitro as previously described. 13Endogenous CK2 in vitro kinase assay CK2 activity was measured in vitro as previously described. 15 Treatment with signaling inhibitorsCells were cultured in control medium or in the presence of CX-4945 or LY294002 for the indicated time points and used for protein and viability analysis. ImmunoblottingCell lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the antibodies against actin, phospho-PTEN (S380), PTEN, CK2α and CK2α'. Analysis of cell viability and apoptosisCell viability was determined by double-staining with APC or FITC-conjugated Annexin V and propidium iodide (PI) and flow cytometry analysis, as previously described. 16 Statistical analysisDifferences between populations were calculated using unpaired two-tailed Students's t-test or Mann-Whitney test, as appropriate. Correlations were analyzed using the Pearson correlation coefficient. P<0.05 was considered significant. Results JAK/STAT and PI3K/Akt pathways are hyperactivated in adult B-ALL cellsHyperactivation of signaling pathways involved in promotion of proliferation and survival is commonly associated with cancer progression. Previous studies have shown that one of these signaling cascades, the JAK/STAT pathway, is constitutively activated in B-ALL patients displaying the BCR-ABL fusion (also known as Philadelphia chromosome (Ph)-positive cases) or CRFL2 overexpression in combination or not with activating mutations in JAK1 and JAK2. 3,17 Using phospho-specific flow cytometry and a gating strategy that enabled us to focus on blast cells (Online Supplementary Figure S1) to compare primary bone marrow cells from healthy donors with B-ALL blasts collected from leukemia patients at diagnosis (Table 1), we con-PI3K/Akt pathway activation in adult ALL haematologica | 2014; 99(6) 1063 TdT+, cCD79a+,CD19+, TdT+,CD10+, cCD79a+,CD19+, TdT+,cyIgM+, © F e r r a t a S t o r t i F o u n d a t i o n firmed that adult leukemia cases displayed constitutive hyperactivation of the JAK/STAT pathway ( Figure 1A; Online Supplementary Figures S1 and S2). Moreover, we found a discrete subgroup of samples that presented very high levels of STAT5 phosphorylation. In line with the knowledge that BCR-ABL drives STAT5 activation, 17,18 this group was enriched in Ph-positive cases ( Figure 1A, red labels).In contrast to JAK/STAT pathway, the activation status of PI3K/Akt signaling pathway and its potential role in adult ALL remain less well...

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