Cell type‐specific nuclear pores: a case in point for context‐dependent stoichiometry of molecular machines

Ori, Alessandro; Banterle, Niccolò; Iskar, Murat; Andrés-Pons, Amparo; Escher, Claudia; Bui, Huy Khanh; Sparks, Lenore; Solis‐Mezarino, Victor; Rinner, Oliver; Bork, Peer; Lemke, Edward A.; Beck, Martin

doi:10.1038/msb.2013.4

Cited by 298 publications

(348 citation statements)

References 67 publications

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“…For example, blinking of PAFPs such as mEos2 has been shown to cause overcounting artifacts (25). Although previous studies devoted effort to calibrating these effects (25)(26)(27)(28), variation of photophysical properties with experimental conditions for compatible PAFPs complicates quantitative image reconstruction. In addition, undetected fluorescent proteins owing to problems such as misfolding lead to undercounting.…”

Section: Significancementioning

confidence: 99%

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Puchner

Walter

Kasper

et al. 2013

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

172

213

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Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.S ingle-cell analysis of protein abundance has revealed cell-tocell variation in the form of phenotypic (extrinsic) heterogeneity and intrinsic variation (1). Dynamic processes such as the cell cycle, endocytosis, and meiosis are regulated but also influenced by stochastic events involving small numbers of molecules (e.g., DNA transcription) (2, 3). Similarly, the size and molecular composition of small subcellular organelles are dynamically regulated but still subject to stochastic noise. Studying the biomolecular composition and size of organelles will illuminate the regulation and noise in these dynamically stable systems. A major challenge for such exploration, however, is the development of a combined approach for both resolving small structures below the optical diffraction limit and simultaneously counting biomolecules over several orders of magnitude (Fig. 1A).In the endocytic pathway, both vesicle morphology and biomolecular composition are dynamically regulated along a maturation path. Formation of vesicles, tethering, fusion, and maturation to endosomes are controlled by over 60 proteins in concert with conversion of phosphoinositides (PIs) (4, 5). Phosphatases and PI-kinases produce phosphatidylinositol 3-phosphate (PI3P) from plasma membrane phospholipids (6). PI3P is required for endocytosis (7) and membrane transport to early and late endosomes (8, 9). PI3P regulates the recruitment of proteins (10), such as Rab GTPases, which coordinate many aspects of vesicle identity (11, 12) and maturation to endosomes, including tethe...

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Section: Significancementioning

confidence: 99%

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Puchner

Walter

Kasper

et al. 2013

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

172

213

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“…The commonly used HeLa cells and the colon carcinoma‐derived cells (RKO) have drastically different morphology and nuclear size, due to a > 2‐fold smaller RKO nuclear size as compared to HeLa (average nuclear surface area 206.3 and 543.5 Å 2 for RKO and HeLa, respectively; Fig EV3). Proteome profiles from whole cell extract and isolated nuclei are available for both cell lines (Geiger et al , 2012; Ori et al , 2013). Indeed, when we analyzed whole cell data using a standard differential expression analysis tool [limma package (Ritchie et al , 2015; Phipson et al , 2016)], we observed that the majority (70%) of the nuclear proteins differentially regulated in isolated nuclei are classified as down‐regulated in RKO cells compared to HeLa cells, reflecting the smaller nuclear size of the former cell line.…”

Section: Resultsmentioning

confidence: 99%

“…Distributions, represented as boxplots, of nuclear surface area (Å 2 ) of 44 HeLa and 41 RKO cells (Ori et al , 2013). HeLa nuclei are significantly larger than RKO nuclei ( t ‐test P = 2.5 × 10 −15 ).…”

Section: Resultsmentioning

confidence: 99%

Quantifying compartment‐associated variations of protein abundance in proteomics data

Parca

Beck

Bork

et al. 2018

Molecular Systems Biology

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Quantitative mass spectrometry enables to monitor the abundance of thousands of proteins across biological conditions. Currently, most data analysis approaches rely on the assumption that the majority of the observed proteins remain unchanged across compared samples. Thus, gross morphological differences between cell states, deriving from, e.g., differences in size or number of organelles, are often not taken into account. Here, we analyzed multiple published datasets and frequently observed that proteins associated with a particular cellular compartment collectively increase or decrease in their abundance between conditions tested. We show that such effects, arising from underlying morphological differences, can skew the outcome of differential expression analysis. We propose a method to detect and normalize morphological effects underlying proteomics data. We demonstrate the applicability of our method to different datasets and biological questions including the analysis of sub‐cellular proteomes in the context of Caenorhabditis elegans aging. Our method provides a complementary perspective to classical differential expression analysis and enables to uncouple overall abundance changes from stoichiometric variations within defined group of proteins.

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“…For example, monitoring the functional assembly of the human spliceosomal hPrp19/CDC5L complex under various conditions 55, the F 1 F 0 ‐ATP synthase super‐assembly in H9c2 cardiomyoblasts undergoing cardiac‐like differentiation 56 or the determination of context‐dependent stoichiometry of the nuclear pore complex in various human cell lines, which showed unanticipated variability 57, 58.…”

Section: Biology Applicationsmentioning

confidence: 99%

Applications of targeted proteomics in systems biology and translational medicine

et al. 2015

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Biological systems are composed of numerous components of which proteins are of particularly high functional significance. Network models are useful abstractions for studying these components in context. Network representations display molecules as nodes and their interactions as edges. Because they are difficult to directly measure, functional edges are frequently inferred from suitably structured datasets consisting of the accurate and consistent quantification of network nodes under a multitude of perturbed conditions. For the precise quantification of a finite list of proteins across a wide range of samples, targeted proteomics exemplified by selected/multiple reaction monitoring (SRM, MRM) mass spectrometry has proven useful and has been applied to a variety of questions in systems biology and clinical studies. Here, we survey the literature of studies using SRM‐MS in systems biology and clinical proteomics. Systems biology studies frequently examine fundamental questions in network biology, whereas clinical studies frequently focus on biomarker discovery and validation in a variety of diseases including cardiovascular disease and cancer. Targeted proteomics promises to advance our understanding of biological networks and the phenotypic significance of specific network states and to advance biomarkers into clinical use.

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Cell type‐specific nuclear pores: a case in point for context‐dependent stoichiometry of molecular machines

Abstract: The stoichiometry of the human nuclear pore complex is revealed by targeted mass spectrometry and super-resolution microscopy. The analysis reveals that the composition of the nuclear pore and other nuclear protein complexes is remodeled as a function of the cell type.

Cited by 298 publications

References 67 publications

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Quantifying compartment‐associated variations of protein abundance in proteomics data

Applications of targeted proteomics in systems biology and translational medicine

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