80 research outputs found
Stochastic phenotype transition of a single cell in an intermediate region of gene-state switching
Multiple phenotypic states often arise in a single cell with different
gene-expression states that undergo transcription regulation with positive
feedback. Recent experiments have shown that at least in E. coli, the gene
state switching can be neither extremely slow nor exceedingly rapid as many
previous theoretical treatments assumed. Rather it is in the intermediate
region which is difficult to handle mathematically.Under this condition, from a
full chemical-master-equation description we derive a model in which the
protein copy-number, for a given gene state, follow a deterministic mean-field
description while the protein synthesis rates fluctuate due to stochastic
gene-state switching. The simplified kinetics yields a nonequilibrium landscape
function, which, similar to the energy function for equilibrium fluctuation,
provides the leading orders of fluctuations around each phenotypic state, as
well as the transition rates between the two phenotypic states. This rate
formula is analogous to Kramers theory for chemical reactions. The resulting
behaviors are significantly different from the two limiting cases studied
previously.Comment: 6 pages,4 figure
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Central Dogma at the Single-Molecule Level in Living Cells
Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of gene expression and regulation, and the demonstration that a single-molecule event can determine the phenotype of a cell.Chemistry and Chemical Biolog
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Probing Dynein and Kinesin Stepping with Mechanical Manipulation in a Living Cell
Molecular motors: By combining optical tweezers and high-speed particle tracking, individual steps of microtubule motor proteins transporting organelles can be detected under known force loads in living mammalian cells (see figure).
We report a label-free assay for simultaneous optical manipulation and tracking of endogenous lipid droplets as actively transported cargoes in a living mammalian cell with sub-millisecond time resolution. Using an EM-CCD camera as a highly sensitive quadrant detector, we can detect steps of dynein- and kinesin-driven cargoes under known force loads. We can distinguish single and multiple motor-driven cargoes and show that the stall forces for inward and outward transported cargoes are similar. By combining the stall force observable with the ability to detect individual steps, we can characterize kinesin- and dynein-driven active transport in different force regimes.Chemistry and Chemical Biolog
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Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell
Kindred cells can have different genomes because of dynamic changes in DNA. Single cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome amplification bias, resulting in low genome coverage. Here we report a new amplification method: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that offer high uniformity across the genome. Sequencing MALBAC amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy number variations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to call individual single nucleotide variations (SNVs) with no false positives observed. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.Chemistry and Chemical Biolog
Genome-Wide Study of mRNA Degradation and Transcript Elongation in Escherichia coli
An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome‐wide RNA degradation study in Escherichia coli using RNA‐seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNA's 5′ end, and the exponential decay. This gives a more accurate RNA lifetime and RNA polymerase elongation rate simultaneously genome‐wide. Transcripts typically have a single RNA decay constant along all positions, which is distinct between different operons, indicating that RNA stability is unlikely determined by local sequences. These measurements allowed us to establish a model for RNA processing involving co‐transcriptional degradation, providing quantitative description of the macromolecular coordination in gene expression in bacteria on a system‐wide level.Chemistry and Chemical Biolog
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Raman Spectroscopy: Stimulated Raman Scattering Microscopy for Label-Free Chemical Imaging
Coherent anti-Stokes Raman techniques are increasing the utility of Raman scattering for chemical and biological diagnostics.Chemistry and Chemical Biolog
Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy
The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.Chemistry and Chemical Biolog
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A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell
By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.Chemistry and Chemical Biolog
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Digital RNA Sequencing Minimizes Sequence-Dependent Bias and Amplification Noise with Optimized Single-Molecule Barcodes
RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq.Chemistry and Chemical Biolog
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