To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (‘biorientation’) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.DOI: http://dx.doi.org/10.7554/eLife.01374.001

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.chromatin | histone | proteasome | protein dynamics | turnover P roteins are dynamic molecules. Their abundance is controlled by synthesis and degradation and they can be subject to posttranslational processing, modification, and demodification. In addition, most proteins are very mobile and undergo interactions with multiple other protein partners (1-4). However, little is known about the dynamics of proteins within macromolecular complexes in vivo (2, 4). Studying time-dependent changes in physical properties of proteins or protein turnover requires methods to distinguish resident (old) proteins from new proteins. Current methods that do so are usually based on fluorescent reporters or differential chemical labeling. For example, fluorescence recovery after photo bleaching relies on exchange of the old bleached protein by nonbleached proteins (1, 3, 4). Alternative methods involve time-dependent changes in fluorescence, nonspecific pulse-chase labeling of proteins with labeled amino acids, or labeling with chemical dyes that specifically bind to short tags (5-7). Although suitable for detection of proteins by microscopy or mass spectrometry, a limitation of these methods is that they do not provide a handle for biochemical analysis of old and new proteins and their complexes. To solve this problem and to eliminate the requirement for chemical labels or UV light we developed recombination-induced tag exchange (RITE), a method in which a genetic epitope tag is switched by transient induction of a site-specific recombinase. As a consequence, old and newly synthesized proteins are differentially tagged, which enables monitoring of protein dynamics by multiple techniques, as illustrated here. In contrast to inducible expression strategies (8-12), differential tagging by a time-controlled site-specific protease (13), or the labeling methods described above, RITE allows parallel detection and purification of old and new proteins under physiological conditions and over long periods of time.We used RITE to probe the stability of chromatin. Photobleaching experiments using histones tagged with fluorescent reporters suggest that chromatin is a static comp...

Tracking of ancestral histone proteins over multiple generations of genome replication in yeast reveals that old histones move along genes from 3′ toward 5′ over time, and that maternal histones move up to around 400 bp during genomic replication.

Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core. Keywords: cell cycle; chromatin; H3K79; H3K4; Set1 EMBO reports (2011) 12, 956-962.