An Algorithm to Automate Yeast Segmentation and Tracking

Doncic, Andreas; Eser, Umut; Atay, Oguzhan

doi:10.1371/journal.pone.0057970

Cited by 67 publications

(101 citation statements)

References 32 publications

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“…Images were taken for at most 20 positions every 3 min. Image analysis was performed as previously described (62). Gene activation time was calculated as in Skotheim et al (63) relative to the time Whi5 concentration in the nucleus is half-maximum, which corresponds to the point of commitment to cell division (37).…”

Section: Methodsmentioning

confidence: 99%

Form and function of topologically associating genomic domains in budding yeast

Eser

Chandler-Brown

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the longknown effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins. A n important distinction between eukaryotic and prokaryotic cells is the presence of the eukaryotic nucleus, which compartmentalizes the cell. It is becoming increasingly clear that the eukaryotic nuclear compartment contains additional layers of spatial organization, including the nucleolus, splicing bodies, transcriptional foci, and the peripheral localization of telomeres (1, 2). In addition, high-throughput chromosome conformation capture (Hi-C) technologies have recently revealed the spatial organization of chromatin into topologically associating domains (TADs) on the 100-kb to 1-Mb scale for mammals (3, 4), as well as the fly Drosophila melanogaster (5), the worm Caenorhabditis elegans (6), and the fission yeast Schizosaccharomyces pombe (7). Loci within a TAD are much more likely to interact with one another than with loci outside the domain (5,8,9).In metazoans, topological domains play important roles in coordinating the DNA-templated processes of replication and transcription (10-12). Chromatin within a TAD tends to have similar histone modifications, and consequently euchromatic or heterochromatic state, so that the genome is organized into self-associated globules that are either permissive or repressive of transcription. Repressive TADs are likely to be associated with the nuclear periphery (8). In addition to coordinating transcription, TADs also coordinate replication so that replication origins within a domain activate synchronously.That TAD nuclear organization is important for transcription and replication has motivated much recent work on the molecular mechanisms underlying TAD formation. The...

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Section: Methodsmentioning

confidence: 99%

Form and function of topologically associating genomic domains in budding yeast

Eser

Chandler-Brown

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Images were acquired every 3 min., and multiple fields were followed simultaneously. Fluorescence data were analyzed via automated algorithms [55] to determine midpoint times of Whi5-mCherry nuclear exit and Sic1-GFP degradation. Phase-contrast images were inspected manually to determine the time of bud emergence and the cell diameters at Whi5 exit and budding onset.…”

Section: Methodsmentioning

confidence: 99%

A Docking Interface in the Cyclin Cln2 Promotes Multi-site Phosphorylation of Substrates and Timely Cell-Cycle Entry

et al. 2015

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Summary Background Eukaryotic cell division is driven by cyclin-dependent kinases (CDKs). Distinct cyclin-CDK complexes are specialized to drive different cell cycle events, though the molecular foundations for these specializations are only partly understood. In budding yeast, the decision to begin a new cell cycle is regulated by three G1 cyclins (Cln1–Cln3). Recent studies revealed that some CDK substrates contain a novel docking motif that is specifically recognized by Cln1 and Cln2, and not by Cln3 or later S- or M-phase cyclins, but the responsible cyclin interface was unknown. Results Here, to explore the role of this new docking mechanism in the cell cycle, we first show that it is conserved in a distinct cyclin subtype (Ccn1). Then, we exploit phylogenetic variation to identify cyclin mutations that disrupt docking. These mutations disrupt binding to multiple substrates as well as the ability to use docking sites to promote efficient, multi-site phosphorylation of substrates in vitro. In cells where the Cln2 docking function is blocked, we observed reductions in the polarized morphogenesis of daughter buds and reduced ability to fully phosphorylate the G1/S transcriptional repressor Whi5. Furthermore, disruption of Cln2 docking perturbs the coordination between cell size and division, such that the G1/S transition is delayed. Conclusions The findings point to a novel substrate interaction interface on cyclins, with patterns of conservation and divergence that relate to functional distinctions among cyclin subtypes. Furthermore, this docking function helps ensure full phosphorylation of substrates with multiple phosphorylation sites, and this contributes to punctual cell cycle entry.

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“…Automatic hardware-based focusing was performed by Definite Focus. The blue fluorescence (480/40 nm) and yellow fluorescence (535/30 nm) on excitation at 436 ± 25 nm, as well as phase images, were recorded every 30 s. Images were analyzed using custom Matlab software [22] that automatically tracks cells and quantifies fluorescence intensities. Mean Venus/eCFP pixel intensities for each cell were determined from 5 min before until 10 min after the step increase in glucose.…”

Section: Methodsmentioning

confidence: 99%

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics

Peroza

Ewald

Parakkal

et al. 2015

Analytical Biochemistry

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Trehalose-6-phosphate is a pivotal regulator of sugar metabolism, growth, and osmotic equilibrium in bacteria, yeasts, and plants. To directly visualize the intracellular levels of intracellular trehalose-6-phosphate, we developed a series of specific Förster resonance energy transfer (FRET) sensors for in vivo microscopy. We demonstrated real-time monitoring of regulation in the trehalose pathway of Escherichia coli. In Saccharomyces cerevisiae, we could show that the concentration of free trehalose-6-phosphate during growth on glucose is in a range sufficient for inhibition of hexokinase. These findings support the hypothesis of trehalose-6-phosphate as the effector of a negative feedback system, similar to the inhibition of hexokinase by glucose-6-phosphate in mammalian cells and controlling glycolytic flux.

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An Algorithm to Automate Yeast Segmentation and Tracking

Cited by 67 publications

References 32 publications

Form and function of topologically associating genomic domains in budding yeast

Form and function of topologically associating genomic domains in budding yeast

A Docking Interface in the Cyclin Cln2 Promotes Multi-site Phosphorylation of Substrates and Timely Cell-Cycle Entry

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics

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