Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

Vizeacoumar, Franco J.; Dyk, Nydia Van; Vizeacoumar, Frederick S.; Cheung, Vincent C. K.; Li, Jingjing; Sydorskyy, Yaroslav; Nicolle, Case,; Z, Li; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan J.; Bloom, Kerry; Boone, Charles; Andrews, Brenda

doi:10.1083/jcb.200909013

Cited by 99 publications

(104 citation statements)

References 69 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These future models will probably first succeed in providing a complete quantitative description of low-dimensional spindles reconstituted in cytoplasmic extracts, recent studies of which revealed important scaling laws (Gaetz et al, 2006;Dinarina et al, 2009), and 'one-dimensional' yeast spindles in which individual elements can be more readily observed Vizeacoumar et al, 2010). Then, to put together comprehensive models of three-dimensional and complex spindles in animal cells, besides the MT-, KT-and chromosome-tracking data, we will need to understand collective motor action (Ally et al, 2009), ways in which stochastic gradients of regulatory molecules affect MTmotor dynamics (Athale et al, 2008), and to measure force maps in the spindle (Ke et al, 2009).…”

Section: Perspectivesmentioning

confidence: 99%

Towards a quantitative understanding of mitotic spindle assembly and mechanics

Mogilner

Craig

2010

Journal of Cell Science

View full text Add to dashboard Cite

SummaryThe 'simple' view of the mitotic spindle is that it self-assembles as a result of microtubules (MTs) randomly searching for chromosomes, after which the spindle length is maintained by a balance of outward tension exerted by molecular motors on the MTs connecting centrosomes and chromosomes, and compression generated by other motors on the MTs connecting the spindle poles. This picture is being challenged now by mounting evidence indicating that spindle assembly and maintenance rely on much more complex interconnected networks of microtubules, molecular motors, chromosomes and regulatory proteins. From an engineering point of view, three design principles of this molecular machine are especially important: the spindle assembles quickly, it assembles accurately, and it is mechanically robust -yet malleable. How is this design achieved with randomly interacting and impermanent molecular parts? Here, we review recent interdisciplinary studies that have started to shed light on this question. We discuss cooperative mechanisms of spindle self-assembly, error correction and maintenance of its mechanical properties, speculate on analogy between spindle and lamellipodial dynamics, and highlight the role of quantitative approaches in understanding the mitotic spindle design.

show abstract

Section: Perspectivesmentioning

confidence: 99%

Towards a quantitative understanding of mitotic spindle assembly and mechanics

Mogilner

Craig

2010

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…For example, in order to investigate yeast spindle morphogenesis, GFP-tagged tubulin was inserted into a yeast deletion library (Vizeacoumar et al, 2010). Another example, in human cells, is the use of a GFP-tagged version of core histone 2B, which acts as a marker for chromosomes in all cell cycle stages, to uncover genes involved in chromosome segregation during cell division using a high-throughput RNAi time-lapse screen (Neumann et al, 2006).…”

Section: Box 1 the Repertoire Of Available High-throughput Platformsmentioning

confidence: 99%

“…For example, it is now feasible to perform functional genomics screens by capturing a microscopic phenotype of cells in which each gene has been knocked out or knocked down systematically. This can be done, for instance, by RNA interference (RNAi) in cell culture Krishnan et al, 2008;Moffat et al, 2006;Neumann et al, 2006;Prudencio et al, 2008) or by using a deletion library, as has been performed in budding yeast (Vizeacoumar et al, 2010). Additionally, deletion libraries have been created for fission yeast (Kim et al, 2010) and for bacteria (e.g.…”

mentioning

confidence: 99%

Getting the whole picture: combining throughput with content in microscopy

Rimon

Schuldiner

2011

Journal of Cell Science

View full text Add to dashboard Cite

Commentary 3743Introduction Technological developments in the past years have led to the emergence of high-throughput methods in cell biology (Box 1), where multiple perturbations of a system are automatically repeated in a controlled and identical fashion. These capabilities allow the acquisition of vast amounts of data on a biological system. Evaluating all aspects of such data using computational and mathematical tools can then lead to an unbiased systematic representation of the process studied (Ahn et al., 2006). Technical breakthroughs, often driven by the pharmaceutical industry, have pushed this field forwards and have led to major advances in drug screening (reviewed by Zanella et al., 2010) and numerous discoveries in basic biology.The need for systematic and unbiased approaches has always been at the core of scientific efforts. However, the technology that is required for such efforts often displays an inverse relationship between throughput (speed) and content (biological information). Sydney Brenner referred to this phenomenon by saying that highthroughput experiments are in danger of creating "low-input, high-throughput, no-output biology" (Brenner, 2008). Therefore, it remains a major goal to develop high-throughput science that will give all the advantages of being systematic, accurate, fast and unbiased without giving up the requirement to provide profound and highly informative data.Among the variety of high-throughput approaches available to date (Box 1), one of the methods that holds the promise to bridge the gap between these expectations is high-throughput microscopy, often also referred to as high-content screening (HCS). This is mainly owing to the ability of microscopy methods to focus on single cells at a subcellular resolution, in a time-dependent manner and to measure a large number of parameters in each frame. In this Commentary, we focus on the current frontiers of HCS by presenting the wide range of tools that are utilized and the biological questions that can be tackled using such approaches. We also discuss possible ways to increase content while maintaining throughput at all levels of the screen -from sample design and preparation through to the acquisition and analysis capabilities of the high-content imager system. The power of high-throughput microscopyHigh-throughput microscopy can be employed to address a wide spectrum of biological questions. At the basis of this capability lies the ever-growing variety of labeling methods (discussed below) that allow visualization of cellular architecture and function, as well as developmental or behavioral processes (Fig. 1). In addition, the technological advance of biological tools and microscopic platforms now enables screens to be performed in an array of genetic backgrounds, under different growth conditions and at various time points, as well as allowing the comparison of multiple tissues, cell lines or organisms. Combining the richness of visualization approaches with various experimental strategies creates nearly endless options ...

show abstract

“…4C). Similarly, a high-throughput screen to identify mutants with microtubule defects uncovered the hooked microtubule phenotype in multiple nonessential NuA4 mutants (47). In agreement Boldface K represents acetyl lysine residues identified in S. cerevisiae acetylome study (4).…”

Section: Inverse Application Of Mchip-kat-ms Provides An Alternative mentioning

confidence: 70%

mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

Mitchell

Huard

Cotrut

et al. 2013

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

View full text Add to dashboard Cite

Significance Recent proteomic studies have revealed that lysine acetylation is a global and ubiquitous posttranslational modification. However, in the vast majority of cases the lysine acetyltransferases (KATs) responsible for individual modifications remain unknown. Here we present a unique methodology that connects KATs to their substrates. To validate the methodology, we use the yeast KAT nucleosome acetyltransferase of histone H4 (NuA4) and identify both protein interactions and acetylation targets. Importantly, this methodology can be applied to any KAT and should aid in the linking of KATs to their cellular targets.

show abstract

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

Abstract: A combination of yeast genetics, synthetic genetic array analysis, and high-throughput screening reveals that sumoylation of Mcm21p promotes disassembly of the mitotic spindle.

Cited by 99 publications

References 69 publications

Towards a quantitative understanding of mitotic spindle assembly and mechanics

Towards a quantitative understanding of mitotic spindle assembly and mechanics

Getting the whole picture: combining throughput with content in microscopy

mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

Contact Info

Product

Resources

About