Actin dynamics modulate mechanosensitive immobilization of E-cadherin at adherens junctions.

Mechanical stress is increasingly being shown to be a potent modulator of cell-cell junctional morphologies in developmental and homeostatic processes. Intercellular force sensing is thus expected to be an important regulator of cell signalling and tissue integrity. In particular, the interplay between myosin contractility, actin dynamics and E-cadherin recruitment largely remains to be uncovered. We devised a suspended cell doublet assay to quantitatively assess the correlation between myosin II activity and local E-cadherin recruitment. The single junction of the doublet exhibited a stereotypical morphology, with E-cadherin accumulating into clusters of varied concentrations at the rim of the circular contact. This local recruitment into clusters derived from the sequestration of E-cadherin through a myosin-II-driven modulation of actin turnover. We exemplify how the regulation of actin dynamics provides a mechanism for the mechanosensitive response of cell contacts.

Is the in vitro ejection of bacteriophage DNA quasistatic? A bulk to single virus study.

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection-binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.

DNA translocation and unzipping through a nanopore: some geometrical effects.

This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

Force fluctuations assist nanopore unzipping of DNA.

We experimentally study the statistical distributions and the voltage dependence of the unzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We then propose a quantitative theoretical description considering the nanopore unzipping process as a random walk of the opening fork through the DNA sequence energy landscape biased by a time-fluctuating force. To achieve quantitative agreement fluctuations need to be correlated over the millisecond range and have an amplitude of order k(B)T/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that the unzipping process is efficiently enhanced by noise in the kHz range. We further show that the unzipping time of short 15 base-pair hairpins does not always increase with the global stability of the double helix and we theoretically study the role of DNA elasticity on the conversion of the electrical bias into a mechanical unzipping force.

Theoretical study of sequence-dependent nanopore unzipping of DNA.

We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.

Encoding folding paths of RNA switches.

RNA co-transcriptional folding has long been suspected to play an active role in helping proper native folding of ribozymes and structured regulatory motifs in mRNA untranslated regions (UTRs). Yet, the underlying mechanisms and coding requirements for efficient co-transcriptional folding remain unclear. Traditional approaches have intrinsic limitations to dissect RNA folding paths, as they rely on sequence mutations or circular permutations that typically perturb both RNA folding paths and equilibrium structures. Here, we show that exploiting sequence symmetries instead of mutations can circumvent this problem by essentially decoupling folding paths from equilibrium structures of designed RNA sequences. Using bistable RNA switches with symmetrical helices conserved under sequence reversal, we demonstrate experimentally that native and transiently formed helices can guide efficient co-transcriptional folding into either long-lived structure of these RNA switches. Their folding path is controlled by the order of helix nucleations and subsequent exchanges during transcription, and may also be redirected by transient antisense interactions. Hence, transient intra- and inter-molecular base pair interactions can effectively regulate the folding of nascent RNA molecules into different native structures, provided limited coding requirements, as discussed from an information theory perspective. This constitutive coupling between RNA synthesis and RNA folding regulation may have enabled the early emergence of autonomous RNA-based regulation networks.

Unzipping DNA with optical tweezers: high sequence sensitivity and force flips.

Force measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system.