Quantification and Correction of Systematic Errors Due to Detector Time-Averaging in Single-Molecule Tracking Experiments

Single-molecule tracking is a powerful way to look at the dynamic organization of plasma membranes. However, there are some limitations to its use. For example, it was recently observed, using numerical simulation, that time-averaging effects inherent to the exposure time of detectors are likely to bias the apparent motion of molecules confined in microdomains. Here, we solve this apparently limiting issue analytically. We explore this phenomenon by calculating its effects on the observed diffusion coefficients and domain sizes. We demonstrate that the real parameters can be easily recovered from the measured apparent ones. Interestingly, we find that single-molecule tracking can be used to explore events occurring at a timescale smaller than the exposure time.Comment: 3 pages (Letter); 1 figur

Dependence of DNA persistence length on ionic strength of solutions with monovalent and divalent salts: a joint theory-experiment study

Using high-throughput Tethered Particle Motion single molecule experiments, the double-stranded DNA persistence length, $L_p$, is measured in solutions with Na$^+$ and Mg$^{2+}$ ions of various ionic strengths, $I$. Several theoretical equations for $L_p(I)$ are fitted to the experimental data, but no decisive theory is found which fits all the $L_p$ values for the two ion valencies. Properly extracted from the particle trajectory using simulations, $L_p$ varies from 30~nm to 55~nm, and is compared to previous experimental results. For the Na$^+$ only case, $L_p$ is an increasing concave function of $I^{-1}$, well fitted by Manning's electrostatic stretching approach, but not by classical Odjik-Skolnick-Fixman theories with or without counter-ion condensation. With added Mg$^{2+}$ ions, $L_p$ shows a marked decrease at low $I$, interpreted as an ion-ion correlation effect, with an almost linear law in $I^{-1}$, fitted by a proposed variational approach

Comment to the Paper of Michael J. Saxton: "A Biological Interpretation of Transient Anomalous Subdiffusion. I. Qualitative Model"

In a recent paper, Michael J. Saxton proposes to interpret as anomalous diffusion the occurrence of apparent transient sub-diffusive regimes in mean-squared displacements (MSD) plots, calculated from experimental trajectories of molecules diffusing in living cells, acquired by Single Particle (or Molecule) Tracking techniques (SPT or SMT). In this Comment, without questioning the existence of sub-diffusive behaviors, which certainly play a key role in numbers of mechanisms in living systems, we point out that the data used by J.M. Saxton can as well be fitted by a simple law, resulting from confined diffusion at short times, with a slower free diffusion superimposed at larger times. When visualizing MSD plots, the transition from short-term diffusion confined in domains of size L, to slower, longer-term free diffusion, can be confused with anomalous diffusion over several orders of magnitude of time.Comment: To appear in Biophysical Journa

A model for the molecular organisation of the IS911 transpososome

Tight regulation of transposition activity is essential to limit damage transposons may cause by generating potentially lethal DNA rearrangements. Assembly of a bona fide protein-DNA complex, the transpososome, within which transposition is catalysed, is a crucial checkpoint in this regulation. In the case of IS911, a member of the large IS3 bacterial insertion sequence family, the transpososome (synaptic complex A; SCA) is composed of the right and left inverted repeated DNA sequences (IRR and IRL) bridged by the transposase, OrfAB (the IS911-encoded enzyme that catalyses transposition). To characterise further this important protein-DNA complex in vitro, we used different tagged and/or truncated transposase forms and analysed their interaction with IS911 ends using gel electrophoresis. Our results allow us to propose a model in which SCA is assembled with a dimeric form of the transposase. Furthermore, we present atomic force microscopy results showing that the terminal inverted repeat sequences are probably assembled in a parallel configuration within the SCA. These results represent the first step in the structural description of the IS911 transpososome, and are discussed in comparison with the very few other transpososome examples described in the literature

Probing DNA conformational changes with high temporal resolution by Tethered Particle Motion

The Tethered Particle Motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150~nm). Notably we demonstrate that, for a particle of radius 20~nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20~ms.Comment: Improved version, to appear in Physical Biology. 10 pages + 10 pages of supporting materia

Oligomeric and polymeric surfactants for the transfer of luminescent ZnO nanocrystals to water

International audienceThe water dispersion of luminescent nanocrystals (NCs) synthesized in organic solvent by encapsulation in a surfactant bilayer is a promising strategy for preserving the optical properties of NCs. The phase transfer of highly monodispersed ZnO NCs using the monomer, dimer, trimer and polymer of a series of alkyl ammonium surfactants is compared. Transfer yields over 60% could be obtained with the oligomers and the polymer. In contrast, we observed no measurable transfer using the single chain surfactant. NMR spectroscopy, including DOSY and NOESY, demonstrated that increasing the oligomerization number ameliorates the stability within the coating bilayer. The NCs exhibit a strong luminescence in water and show long term chemical and photo-chemical stability

A deep Chandra observation of the poor cluster AWM4 - II. The role of the radio jets in enriching the intra-cluster medium

We use a Chandra observation of the poor cluster AWM4 to map the temperature and abundance of the intra-cluster medium, so as to examine the influence of the central radio galaxy on its environment. While the cluster core is generally enriched to near-solar abundances, we find evidence of super-solar abundances correlated with the radio jets, extending ~35 kpc from the core of the central dominant galaxy NGC 6051 along its minor axis. We conclude that the enriched gas has been transported out of the central galaxy through the action of the radio source. We estimate the excess mass of iron in the entrained gas to be ~1.4x10^6 Msol, and find that this can be produced in the core of NGC 6051 within the timescale of the AGN outburst. The energy required to transport this gas to its current location is ~4.5x10^57 erg, a significant fraction of the estimated total mechanical energy output of the AGN, though this estimate is dependent on the degree of enrichment of the uplifted gas. The larger near-solar abundance region is also compatible with enrichment by metals mixed outward from NGC 6051 over a much longer timescale.Comment: Accepted for publication in MNRAS, 11 pages, 6 figure

TERMINAL FLOWER-1/CENTRORADIALIS inhibits tuberization via protein interaction with the tuberigen activation complex

This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Division as part of the Strategic Research Programme 2016-2021, by a GCRF Foundation Awards for Global Agricultural and Food Systems Research funded by the BBSRC project BB/P022553/1 and also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 835704. Research in Prat’s lab was funded by the Spanish Ministerio de Economía y Competitividad BIO2015-73019-EXP, and the aligned Japan EIG CONCERT (PIA102017-1) projects.Potato tuber formation is a secondary developmental program by which cells in the subapical stolon region divide and radially expand, to further differentiate into starch accumulating parenchyma. Whilst some details of the molecular pathway that signals tuberization are known, important gaps in our knowledge persist. Here the role of a member of the TERMINAL FLOWER 1/ CENTRORADIALIS gene family (termed StCEN ) in the negative control of tuberization is demonstrated for the first time. It is shown that reduced expression of StCEN accelerates tuber formation whereas transgenic lines over‐expressing this gene display delayed tuberization and reduced tuber yield. Protein‐protein interaction studies (yeast two hybrid and bimolecular fluorescence complementation) demonstrate that StCEN binds components of the recently described tuberigen activation complex. Using transient transactivation assays we show that the StSP6A tuberization signal is an activation target of the tuberigen activation complex, and that co‐expression of StCEN blocks StFD‐Like‐1 activation of the StSP6A gene. Transcriptomic analysis of transgenic lines mis‐expressing StCEN identify early transcriptional events in tuber formation. These results demonstrate that StCEN suppresses tuberization by directly antagonizing StSP6A function in stolons, identifying StCEN as a breeding marker to improve tuber initiation and yield, through the selection of genotypes with reduced StCEN expression.Publisher PDFPeer reviewe