JC-1 was used to dye NB4 cells to visually detect early apoptosis

g detection of centromere LY3039478 cohesion defects in metaphase spreads. In our hands, this phenotype is observed not only in Pds5B knockout MEFs but also in hepatocytes from Pds5B knockout embryos. Also in HeLa cells, downregulation of Pds5B levels results in centromeric cohesion defects. Taken together with previous analysis of SA1 and SA2 in mouse and human cells, it is likely that cohesin-SA1Pds5A and cohesin-SA1-Pds5B act at telomeres, cohesin-SA2Pds5B functions preferentially at centromeres, and these three complexes as well as cohesin-SA2-Pds5A perform cohesion along chromosome arms. The molecular determinants that direct these specificities remain to be identified. It is also unclear why telomeric and centromeric cohesin complexes should be different. We speculate that cohesion serves different purposes at different chromosomal regions. While telomere cohesion ensures telomere replication, centromere cohesion promotes chromosome biorientation by providing tension and proper localization of the chromosomal passenger complex. Centromeric cohesion is viewed as being the most critical for proper chromosome segregation and is the likely reason for cohesin accumulation at the centromeres of mitotic chromosomes. Pds5B null cells have major defects in centromeric cohesion as judged by metaphase chromosome spreads, but nevertheless are capable of forming a metaphase plate and initiating anaphase with only a short delay compared with wild-type cells. We envision that cohesion mediated by cohesin bound to Pds5A along chromosome arms, as well as DNA catenation, provides sufficient linkages between the sister chromatids to allow chromosome alignment in Pds5B null cells. Even though interkinetochore stretch may be reduced in the absence of cohesin-mediated centromere cohesion, the residual intrakinetochore stretch may be sufficient to satisfy the SAC. However, we suspect that Pds5B null cells begin anaphase without having achieved proper biorientation, which leads to chromosome segregation defects and, eventually, to aneuploidy. We have shown that the accumulation of Aurora B at the inner centromere is defective in Pds5B null cells, possibly through the impaired recruitment of Haspin, which may further prevent the correction of improper attachments and a cell-cycle delay. In S. pombe, Pds5 is also required for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811088 proper localization of Haspin and a physical interaction between the two proteins has been detected. Results from different model organisms have suggested that Pds5 proteins act as both positive and negative regulators of cohesion, functioning for the establishment and maintenance of cohesion, but also for its destabilization. Our analysis indicates that Pds5A and Pds5B are indeed critical for cohesion establishment and maintenance in vertebrates, as these proteins are required for full Smc3 Pds5B is required for cohesion establishment and Aurora B accumulation M Carretero et al when the two Pds5 proteins are absent. However, a cross-talk must exist between the CoATs and Pds5 proteins, which is essential for Smc3 acetylation and cohesion establishment and maintenance. In particular, we observe the reduced presence of Esco2 at replicating PCH foci in Pds5B null cells, which correlates with reduced Sororin at PCH and centromere cohesion defects. A lack of centromeric cohesion has been proposed to underlie the pathology of the Roberts syndrome. From the results presented here, one could expect Pds5B mutations to be causative of this disease. Howev