30 research outputs found
Characterizing mRNA Interactions with RNA Granules during Translation Initiation Inhibition
When cells experience environmental stresses, global translational arrest is
often accompanied by the formation of stress granules (SG) and an increase in
the number of p-bodies (PBs), which are thought to play a crucial role in the
regulation of eukaryotic gene expression through the control of mRNA translation
and degradation. SGs and PBs have been extensively studied from the perspective
of their protein content and dynamics but, to date, there have not been
systematic studies on how they interact with native mRNA granules. Here, we
demonstrate the use of live-cell hybridization assays with multiply-labeled
tetravalent RNA imaging probes (MTRIPs) combined with immunofluorescence, as a
tool to characterize the polyA+ and β-actin mRNA distributions within
the cytoplasm of epithelial cell lines, and the changes in their colocalization
with native RNA granules including SGs, PBs and the RNA exosome during the
inhibition of translational initiation. Translation initiation inhibition was
achieved via the induction of oxidative stress using sodium arsenite, as well as
through the use of Pateamine A, puromycin and cycloheximide. This methodology
represents a valuable tool for future studies of mRNA trafficking and regulation
within living cells
Elementary simulation of tethered Brownian motion
We describe a simple numerical simulation, suitable for an undergraduate
project (or graduate problem set), of the Brownian motion of a particle in a
Hooke-law potential well. Understanding this physical situation is a practical
necessity in many experimental contexts, for instance in single molecule
biophysics; and its simulation helps the student to appreciate the dynamical
character of thermal equilibrium. We show that the simulation succeeds in
capturing behavior seen in experimental data on tethered particle motion.Comment: Submitted to American Journal of Physic
Tethered Particle Motion as a Diagnostic of DNA Tether Length
The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping
DNA Looping Kinetics Analyzed Using Diffusive Hidden Markov Model
Tethered particle experiments use light microscopy to measure the position of
a micrometer-sized bead tethered to a microscope slide via a ~micrometer length
polymer, in order to infer the behavior of the invisible polymer. Currently,
this method is used to measure rate constants of DNA loop formation and
breakdown mediated by repressor protein that binds to the DNA. We report a new
technique for measuring these rates using a modified hidden Markov analysis
that directly incorporates the diffusive motion of the bead, which is an
inherent complication of tethered particle motion because it occurs on a time
scale between the sampling frequency and the looping time. We compare looping
lifetimes found with our method, which are consistent over a range of sampling
frequencies, to those obtained via the traditional threshold-crossing analysis,
which vary depending on how the raw data are filtered in the time domain. Our
method does not involve such filtering, and so can detect short-lived looping
events and sudden changes in looping behavior.Comment: 3 page pdf including 3 figures corrections: 2nd page, 1st column,
values of diffusion coefficient, spring constant and the decay time were
typed incorrectly. No conlcusions were affecte
Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA
International audienceThe characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile , and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening