Along-standing biological question is how a eukaryotic cell controls the size of its nucleus. We report here that in fission yeast, nuclear size is proportional to cell size over a 35-fold range, and use mutants to show that a 16-fold change in nuclear DNA content does not influence the relative size of the nucleus. Multi-nucleated cells with unevenly distributed nuclei reveal that nuclei surrounded by a greater volume of cytoplasm grow more rapidly. During interphase of the cell cycle nuclear growth is proportional to cell growth, and during mitosis there is a rapid expansion of the nuclear envelope. When the nuclear/cell (N/C) volume ratio is increased by centrifugation or genetic manipulation, nuclear growth is arrested while the cell continues to grow; in contrast, low N/C ratios are rapidly corrected by nuclear growth. We propose that there is a general cellular control linking nuclear growth to cell size.

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4 PAD ) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4 PAD -mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku-and Esc1-dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

Abstract-We present a new, robust, computational procedure for tracking fluorescent markers in time-lapse microscopy. The algorithm is optimized for finding the time-trajectory of single particles in very noisy dynamic (two-or three-dimensional) image sequences. It proceeds in three steps. First, the images are aligned to compensate for the movement of the biological structure under investigation. Second, the particle's signature is enhanced by applying a Mexican hat filter, which we show to be the optimal detector of a Gaussian-like spot in 1 2 noise. Finally, the optimal trajectory of the particle is extracted by applying a dynamic programming optimization procedure. We have used this software, which is implemented as a Java plug-in for the public-domain ImageJ software, to track the movement of chromosomal loci within nuclei of budding yeast cells. Besides reducing trajectory analysis time by several 100-fold, we achieve high reproducibility and accuracy of tracking. The application of the method to yeast chromatin dynamics reveals different classes of constraints on mobility of telomeres, reflecting differences in nuclear envelope association. The generic nature of the software allows application to a variety of similar biological imaging tasks that require the extraction and quantitation of a moving particle's trajectory.Index Terms-Dynamic programming (DP), fluorescence microscopy, image sequence analysis, living cell, particle tracking.

Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted. We found that local binding of the transcriptional activator VP16, but not of the Gal4 acidic domain, enhances chromatin mobility. The increase in movement did not correlate strictly with RNA polymerase II (PolII) elongation, but could be phenocopied by targeting the INO80 remodeler to the locus. Enhanced chromatin mobility required Ino80's ATPase activity. Consistently, the INO80-dependent remodeling of nucleosomes upon transcriptional activation of the endogenous PHO5 promoter enhanced chromatin movement locally. Finally, increased mobility at a double-strand break was also shown to depend in part on the INO80 complex. This correlated with increased rates of spontaneous gene conversion. We propose that local chromatin remodeling and nucleosome eviction increase large-scale chromatin movements by enhancing the flexibility of the chromatin fiber.[Keywords: chromatin remodeling; nuclear organization; transcription; VP16; Ino80; fluorescence microscopy; homologous recombination] Supplemental material is available for this article.Received August 15, 2011; revised version accepted January 13, 2012.DNA-based transactions such as transcription, replication, and repair take place in distinct nuclear subcompartments. Transcriptional silencing is frequently associated with the nuclear envelope or occurs near nucleoli (Towbin et al. 2009), whereas the activation of tissue-specific genes correlates with a shift of the relevant genes away from the nuclear periphery (Egecioglu and Brickner 2011). In contrast, genes activated under conditions of nutrient or temperature stress move to nuclear pores when they are induced (Taddei 2007). Finally, some types of damagenamely, irreparable DNA double-strand breaks (DSBs), collapsed replication forks, and eroded telomeres-relocate to nuclear pores to be processed for repair, unlike DSBs that can be repaired by recombination with a homologous template (for review, see Nagai et al. 2010). For these relocalization events to occur, whether at a promoter, a replication fork, or a DSB, chromatin must be mobile.Rapid time-lapse fluorescence microscopy of GFP-LacItagged genomic loci has shown that individual chromosomal domains move constantly in a near-random walk within a restrained volume of the nucleus (Hubner and Spector 2010). The measured radius of constraint for the movement of an average chromosomal locus (;0.6 mm) was roughly similar in every species investigated, although mobility was also shown to be influenced by local chromatin context (Marshall et al. 1997;Heun et al. 2001;Vazquez et al. 2001;Chubb et al. 2002;Gartenberg et al. 2004). For instance, lacO arrays inserted near budding yeast ce...

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.