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

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

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

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

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