For proper partitioning of chromosomes in mitosis, the chromosomal passenger complex (CPC) including Aurora B and survivin must be localized at the center of paired kinetochores, at the site called the inner centromere. It is largely unknown what defines the inner centromere and how the CPC is targeted to this site. Here, we show that the phosphorylation of histone H3-threonine 3 (H3-pT3) mediated by Haspin cooperates with Bub1-mediated histone 2A-serine 121 (H2A-S121) phosphorylation in targeting the CPC to the inner centromere in fission yeast and human cells. H3-pT3 promotes nucleosome binding of survivin, whereas phosphorylated H2A-S121 facilitates the binding of shugoshin, the centromeric CPC adaptor. Haspin colocalizes with cohesin by associating with Pds5, whereas Bub1 localizes at kinetochores. Thus, the inner centromere is defined by intersection of two histone kinases.

Bub1 is a multi-task protein kinase required for proper chromosome segregation in eukaryotes. Impairment of Bub1 in humans may lead to chromosomal instability (CIN) or tumorigenesis. Yet, the primary cellular substrate of Bub1 has remained elusive. Here, we show that Bub1 phosphorylates the conserved serine 121 of histone H2A in fission yeast Schizosaccharomyces pombe. The h2a-SA mutant, in which all cellular H2A-S121 is replaced by alanine, phenocopies the bub1 kinase-dead mutant (bub1-KD) in losing the centromeric localization of shugoshin proteins. Artificial tethering of shugoshin to centromeres largely restores the h2a-SA or bub1-KD-related CIN defects, a function that is evolutionally conserved. Thus, Bub1 kinase creates a mark for shugoshin localization and the correct partitioning of chromosomes.

In the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1-Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.

During development, sensory axons compete for limiting neurotrophic support, and local neurotrophin insufficiency triggers caspase-dependent axon degeneration. The signaling driving axon degeneration upon local deprivation is proposed to reside within axons. Our results instead support a model in which, despite the apoptotic machinery being present in axons, the cell body is an active participant in gating axonal caspase activation and axon degeneration. Loss of trophic support in axons initiates retrograde activation of a somatic pro-apoptotic pathway, which in turn is required for distal axon degeneration via an anterograde pro-degenerative factor. At a molecular level, the cell body is a convergence point of two signaling pathways whose integrated action drives upregulation of pro-apoptotic Puma, which, unexpectedly, is confined to the cell body. Puma then overcomes inhibition by pro-survival Bcl-xL and Bcl-w and initiates the anterograde pro-degenerative program, highlighting the role of the cell body as an arbiter of large-scale axon removal.

Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program.

Sister-chromatid cohesion is established by the cohesin complex in S phase and persists until metaphase, when sister chromatids are captured by microtubules emanating from opposite poles [1]. The Aurora-B-containing chromosome passenger complex (CPC) plays a crucial role in achieving chromosome bi-orientation by correcting erroneous microtubule attachment [2]. The centromeric localization of the CPC relies largely on histone H3-T3 phosphorylation (H3-pT3), which is mediated by the mitotic histone kinase Haspin/Hrk1 [3-5]. Hrk1 localization to centromeres depends largely on the cohesin subunit Pds5 in fission yeast [5]; however, it is unknown how Pds5 regulates Hrk1 localization. Here we identify a conserved Hrk1-interacting motif (HIM) in Pds5 and a Pds5-interacting motif (PIM) in Hrk1 in fission yeast. Mutations in either motif result in the displacement of Hrk1 from centromeres. We also show that the mechanism of Pds5-dependent Hrk1 recruitment is conserved in human cells. Notably, the PIM in Haspin/Hrk1 is reminiscent of the YSR motif found in the mammalian cohesin destabilizer Wapl and stabilizer Sororin, both of which bind PDS5 [6-12]. Similarly, and through the same motifs, fission yeast Pds5 binds to Wpl1/Wapl and acetyltransferase Eso1/Eco1, in addition to Hrk1. Thus, we have identified a protein-protein interaction module in Pds5 that serves as a chromatin platform for regulating sister-chromatid cohesion and chromosome bi-orientation.