scholarly journals Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

2020 ◽  
Author(s):  
Jack W Shepherd ◽  
Sarah Lecinski ◽  
Jasmine Wragg ◽  
Sviatlana Shashkova ◽  
Chris MacDonald ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Single Molecule ◽  
Copy Number ◽  
Yeast Cells ◽  
Molecular Crowding ◽  
Cell Analysis ◽  
Copy Numbers ◽  
Subcellular Processes ◽  
Elevated Glucose ◽  
Physical And Chemical

AbstractThe physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

scholarly journals Clinical Impact of Proximal Autosomal Imbalances

2012 ◽  
Vol 15 (2) ◽  
pp. 15-21 ◽  
Author(s):  
A.B. Hamid ◽  
A. Weise ◽  
M. Voigt ◽  
M. Bucksch ◽  
N. Kosyakova ◽  
...  
Keyword(s):  
Copy Number ◽  
Single Cell Analysis ◽  
Chromosomal Rearrangements ◽  
Marker Chromosome ◽  
Partial Trisomy ◽  
Clinical Impact ◽  
Cell Analysis ◽  
Small Supernumerary Marker Chromosome ◽  
Copy Numbers ◽  
Based Medicine

ABSTRACT Centromere-near gain of copy number can be induced by intra- or inter-chromosomal rearrangements or by the presence of a small supernumerary marker chromosome (sSMC). Interestingly, partial trisomy to hexasomy of euchromatic material may be present in clinically healthy or affected individuals, depending on origin and size of chromosomal material involved. Here we report the known minimal sizes of all centromere-near, i.e., proximal auto-somal regions in humans, which are tolerated; over 100 Mb of coding DNA are comprised in these regions. Additionally, we have summarized the typical symptoms for nine proximal autosomal regions including genes obviously sensitive to copy numbers. Overall, studying the carriers of specific chromosomal imbalances using genomics-based medicine, combined with single cell analysis can provide the genotype-phenotype correlations and can also give hints where copy-numbersensitive genes are located in the human genome.

scholarly journals Accucopy: accurate and fast inference of allele-specific copy number alterations from low-coverage low-purity tumor sequencing data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xinping Fan ◽  
Guanghao Luo ◽  
Yu S. Huang
Keyword(s):  
Copy Number ◽  
Bayesian Learning ◽  
Kernel Smoothing ◽  
Gaussian Mixture ◽  
Copy Number Alterations ◽  
Sequencing Data ◽  
Copy Numbers ◽  
Allele Specific ◽  
Tumor Sequencing ◽  
Low Coverage

Abstract Background Copy number alterations (CNAs), due to their large impact on the genome, have been an important contributing factor to oncogenesis and metastasis. Detecting genomic alterations from the shallow-sequencing data of a low-purity tumor sample remains a challenging task. Results We introduce Accucopy, a method to infer total copy numbers (TCNs) and allele-specific copy numbers (ASCNs) from challenging low-purity and low-coverage tumor samples. Accucopy adopts many robust statistical techniques such as kernel smoothing of coverage differentiation information to discern signals from noise and combines ideas from time-series analysis and the signal-processing field to derive a range of estimates for the period in a histogram of coverage differentiation information. Statistical learning models such as the tiered Gaussian mixture model, the expectation–maximization algorithm, and sparse Bayesian learning were customized and built into the model. Accucopy is implemented in C++ /Rust, packaged in a docker image, and supports non-human samples, more at http://www.yfish.org/software/. Conclusions We describe Accucopy, a method that can predict both TCNs and ASCNs from low-coverage low-purity tumor sequencing data. Through comparative analyses in both simulated and real-sequencing samples, we demonstrate that Accucopy is more accurate than Sclust, ABSOLUTE, and Sequenza.

scholarly journals Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

2018 ◽  
Vol 115 (17) ◽  
pp. E3969-E3977 ◽  
Author(s):  
Sasikumar Rajoo ◽  
Pascal Vallotton ◽  
Evgeny Onischenko ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Altered Expression ◽  
Copy Numbers ◽  
Quantitative Image ◽  
Multiple Copies ◽  
Almost All ◽  
High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

scholarly journals Improved chromatic and sensory characteristics of Plavac Mali wines – efficiency of maceration enzymes

2017 ◽  
Vol 35 (No. 3) ◽  
pp. 236-245 ◽  
Author(s):  
IVANA ALPEZA ◽  
KARIN KOVAČEVIĆ GANIĆ ◽  
ANDREJA VANZO ◽  
STANKA HERJAVEC
Keyword(s):  
Sensory Quality ◽  
Yeast Cells ◽  
Enzyme Preparations ◽  
Pectolytic Enzymes ◽  
Colour Parameters ◽  
The Moment ◽  
Red Grape ◽  
Different Characteristics ◽  
Physical And Chemical ◽  
Better Than

Two commercial enzyme preparations were used in the production of wine from the Croatian autochthonous red grape variety Plavac Mali in order to improve the extraction of polyphenolic components from grapes, chromatic parameters, and sensory quality. During two vintages, the conventional maceration without enzymes was compared with the maceration using products with different characteristics: pectinase with additional cellulase and hemicellulase activity and pectinase with inactive yeast cells. Both products affected polyphenolic extraction and colour parameters: intensity and hue, and ratio between the yellow, red, and blue colour in young wines (2 months after fermentation) and at the moment of bottling (9 months after fermentation). The correlation between anthocyanins and colour intensity was very strong. The expected reduction of quantitative chromatic parameters during aging was confirmed. Significantly better results were observed in wines produced with pectinase, in relation to all analysed physical and chemical parameters. The sensory analysis showed that wines produced with pure pectolytic enzymes were significantly better than those produced without the enzymes. A product of the combination of pectolytic enzymes and inactive yeast cells had a partial influence on the improvement of the phenolic and sensory quality. The overall quality was significantly more expressed in wines produced with pectolytic enzymes, especially in young wines.

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

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