Counting Cytokinesis Proteins Globally and Locally in Fission Yeast

Wu, Jian-Qiu; Pollard, Thomas D.

doi:10.1126/science.1113230

Cited by 545 publications

(909 citation statements)

References 40 publications

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“…In parallel, EGFP‐tagging of endogenous proteins through genetic engineering offers opportunities for quantifying fluorescence signals at specific subcellular locations (e.g. Wu & Pollard, 2005; Bodor et al , 2014). Here, we have used these methodologies for a quantitative analysis of key proteins implicated in the duplication and function of human centrosomes.…”

Section: Introductionmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

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Section: Introductionmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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“…While each of these approaches have provided essential information about the distribution and dynamics of actin in living cells, each has one or more important disadvantages. Fluorescent G-actin, which is generated either via attachment of a chemical fluorophore or by fluorescent protein fusions, can alter actin assembly and processes dependent on actin assembly [Hird, 1996;Aizawa et al, 1997;Wu and Pollard, 2005] and, moreover, may not incorporate into all pools of F-actin [Kovar et al, 2006]. Further, because only a fraction of actin is in polymer form at any given time [Korn, 1982], imaging with fluorescent G-actin entails high background signal, unless specialized techniques such as speckling are employed [Waterman-Storer et al, 1998].…”

Section: Introductionmentioning

confidence: 99%

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

Burkel

Dassow

Bement

2007

Cell Motil. Cytoskeleton

452

485

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Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.

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“…Image processing methods are required to achieve the automated segmentation of gene expression domains and the quantification of gene products to allow for example the description of expression domain borders. A simple quantification strategy is based on the assumption of a linear relationship between fluorescence intensity and gene expression level (Wu and Pollard, 2005;Frise et al, 2010). The obtained measurements are often normalized with respect to the nuclei channel fluorescence , which is considered to be constant, to compensate for thickness-dependent signal detection.…”

Section: Data Typesmentioning

confidence: 99%

Assembling models of embryo development: Image analysis and the construction of digital atlases

Castro-González

Ledesma-Carbayo

Peyriéras

et al. 2012

Birth Defects Research Pt C

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Digital atlases of animal development provide a quantitative description of morphogenesis, opening the path toward processes modeling. Prototype atlases offer a data integration framework where to gather information from cohorts of individuals with phenotypic variability. Relevant information for further theoretical reconstruction includes measurements in time and space for cell behaviors and gene expression. The latter as well as data integration in a prototypic model, rely on image processing strategies. Developing the tools to integrate and analyze biological multidimensional data are highly relevant for assessing chemical toxicity or performing drugs preclinical testing. This article surveys some of the most prominent efforts to assemble these prototypes, categorizes them according to salient criteria and discusses the key questions in the field and the future challenges toward the reconstruction of multiscale dynamics in model organisms.

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Counting Cytokinesis Proteins Globally and Locally in Fission Yeast

Cited by 545 publications

References 40 publications

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

Assembling models of embryo development: Image analysis and the construction of digital atlases

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