Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions.

The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repli-seq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.

Evidence for DNA Sequence Encoding of an Accessible Nucleosomal Array across Vertebrates.

Nucleosome-depleted regions around which nucleosomes order following the "statistical" positioning scenario were recently shown to be encoded in the DNA sequence in human. This intrinsic nucleosomal ordering strongly correlates with oscillations in the local GC content as well as with the interspecies and intraspecies mutation profiles, revealing the existence of both positive and negative selection. In this letter, we show that these predicted nucleosome inhibitory energy barriers (NIEBs) with compacted neighboring nucleosomes are indeed ubiquitous to all vertebrates tested. These 1 kb-sized chromatin patterns are widely distributed along vertebrate chromosomes, overall covering more than a third of the genome. We have previously observed in human deviations from neutral evolution at these genome-wide distributed regions, which we interpreted as a possible indication of the selection of an open, accessible, and dynamic nucleosomal array to constitutively facilitate the epigenetic regulation of nuclear functions in a cell-type-specific manner. As a first, very appealing observation supporting this hypothesis, we report evidence of a strong association between NIEB borders and the poly(A) tails of Alu sequences in human. These results suggest that NIEBs provide adequate chromatin patterns favorable to the integration of Alu retrotransposons and, more generally to various transposable elements in the genomes of primates and other vertebrates.

Evidence of selection for an accessible nucleosomal array in human.

BACKGROUND: Recently, a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix has been used to reveal some enrichment of nucleosome-inhibiting energy barriers (NIEBs) nearby ubiquitous human "master" replication origins. Here we use this model to predict the existence of about 1.6 millions NIEBs over the 22 human autosomes. RESULTS: We show that these high energy barriers of mean size 153 bp correspond to nucleosome-depleted regions (NDRs) in vitro, as expected, but also in vivo. On either side of these NIEBs, we observe, in vivo and in vitro, a similar compacted nucleosome ordering, suggesting an absence of chromatin remodeling. This nucleosomal ordering strongly correlates with oscillations of the GC content as well as with the interspecies and intraspecies mutation profiles along these regions. Comparison of these divergence rates reveals the existence of both positive and negative selections linked to nucleosome positioning around these intrinsic NDRs. Overall, these NIEBs and neighboring nucleosomes cover 37.5 % of the human genome where nucleosome occupancy is stably encoded in the DNA sequence. These 1 kb-sized regions of intrinsic nucleosome positioning are equally found in GC-rich and GC-poor isochores, in early and late replicating regions, in intergenic and genic regions but not at gene promoters. CONCLUSION: The source of selection pressure on the NIEBs has yet to be resolved in future work. One possible scenario is that these widely distributed chromatin patterns have been selected in human to impair the condensation of the nucleosomal array into the 30 nm chromatin fiber, so as to facilitate the epigenetic regulation of nuclear functions in a cell-type-specific manner.

Time-lapse scanning surface plasmon microscopy of living adherent cells with a radially polarized beam.

We report on a fibered high-resolution scanning surface plasmon microscope for long term imaging of living adherent cells. The coupling of a high numerical aperture objective lens and a fibered heterodyne interferometer enhances both the sensitivity and the long term stability of this microscope, allowing for time-lapse recording over several days. The diffraction limit is reached with a radially polarized illumination beam. Adherence and motility of living C2C12 myoblast cells are followed for 50 h, revealing that the dynamics of these cells change after 10 h. This plasmon enhanced evanescent wave microscopy is particularly suited for investigating cell adhesion, since it can not only be performed without staining of the sample but it can also capture in real time the exchange of extracellular matrix elements between the substrate and the cells.

Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.

Plasmon-based tomographic microscopy.

The imaging principle of the scanning surface plasmon microscope (SSPM) springs from the high sensitivity of surface plasmons to modifications of material properties near the dielectric-metal interface. In this paper, we show that tomographic techniques can be applied to SSPM imaging of dielectric objects to reach resolutions beyond the diffraction-limited half-wavelength scale. Furthermore, this high resolution is not limited to the multiple scattering regime. Finally, we conclude that SSPM is less sensitive to noise because it provides higher contrast ratio than other far-field microscopies.

The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA.

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.

TRF2 promotes, remodels and protects telomeric Holliday junctions.

The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks.

Guided wave microscopy: mastering the inverse problem.

Surface plasmon microscopy is widely recognized for its high sensitivity to nanoscale dielectric or metallic structures confined in a close neighborhood of a gold surface. Recently, its coupling to high-numerical-aperture objective lenses pushed its resolution down to the diffraction limit. Here, we show that the same microscope configuration can be used to excite standing guided waves in asymmetric slabs, which definitely extends the range of applications of this type of microscopy from nano- to microscale structure imaging. We demonstrate experimentally on PPMA films that the V(Z) response of a scanning surface plasmon microscope can be Fourier inverted in order to obtain the reflectivity curve R(ν). When the guided waves are excited, R(ν) shows a finite number of sharp peaks corresponding to quantified guiding modes from which one can extract both the refractive index (RI) and the thickness of the layer at the point focused by the microscope. This device can thus be used to reconstruct RI and thickness contours of dielectric samples with a high spatial resolution.

In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy.

Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.

Nucleosome positioning by genomic excluding-energy barriers.

Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.

Power-law and log-normal avalanche size statistics in random growth processes.

We study the avalanche statistics observed in a minimal random growth model. The growth is governed by a reproduction rate obeying a probability distribution with finite mean a[over ¯] and variance v_{a}. These two control parameters determine if the avalanche size tends to a stationary distribution (finite scale statistics with finite mean and variance, or power-law tailed statistics with exponent ∈(1,3]), or instead to a nonstationary regime with log-normal statistics. Numerical results and their statistical analysis are presented for a uniformly distributed growth rate, which are corroborated and generalized by mathematical results. The latter show that the numerically observed avalanche regimes exist for a wide family of growth rate distributions, and they provide a precise definition of the boundaries between the three regimes.

Modeling of the scanning surface plasmon microscope.

We propose a family of exact solutions of Maxwell's equations to model some aspects of the imaging process involved in the scanning surface plasmon microscope (SSPM). More precisely, we compute the SSPM response of a spherical nanoparticle immobilized close to a thin gold layer and illuminated by a tightly focused spot. We discuss the influence of parameters such as the defocus and the width of the gold layer on the image contrast. We show that this microscopy combines a subwavelength spatial resolution together with high sensitivity to small changes in dielectric properties on the nanoparticle.

Multifractal Desynchronization of the Cardiac Excitable Cell Network During Atrial Fibrillation. II. Modeling.

In a companion paper (I. Multifractal analysis of clinical data), we used a wavelet-based multiscale analysis to reveal and quantify the multifractal intermittent nature of the cardiac impulse energy in the low frequency range ≲ 2Hz during atrial fibrillation (AF). It demarcated two distinct areas within the coronary sinus (CS) with regionally stable multifractal spectra likely corresponding to different anatomical substrates. The electrical activity also showed no sign of the kind of temporal correlations typical of cascading processes across scales, thereby indicating that the multifractal scaling is carried by variations in the large amplitude oscillations of the recorded bipolar electric potential. In the present study, to account for these observations, we explore the role of the kinetics of gap junction channels (GJCs), in dynamically creating a new kind of imbalance between depolarizing and repolarizing currents. We propose a one-dimensional (1D) spatial model of a denervated myocardium, where the coupling of cardiac cells fails to synchronize the network of cardiac cells because of abnormal transjunctional capacitive charging of GJCs. We show that this non-ohmic nonlinear conduction 1D modeling accounts quantitatively well for the "multifractal random noise" dynamics of the electrical activity experimentally recorded in the left atrial posterior wall area. We further demonstrate that the multifractal properties of the numerical impulse energy are robust to changes in the model parameters.

Prestressed cells are prone to cytoskeleton failures under localized shear strain: an experimental demonstration on muscle precursor cells.

We report on a wavelet based space-scale decomposition method for analyzing the response of living muscle precursor cells (C2C12 myoblasts and myotubes) upon sharp indentation with an AFM cantilever and quantifying their aptitude to sustain such a local shear strain. Beyond global mechanical parameters which are currently used as markers of cell contractility, we emphasize the necessity of characterizing more closely the local fluctuations of the shear relaxation modulus as they carry important clues about the mechanisms of cytoskeleton strain release. Rupture events encountered during fixed velocity shear strain are interpreted as local disruptions of the actin cytoskeleton structures, the strongest (brittle) ones being produced by the tighter and stiffer stress fibers or actin agglomerates. These local strain induced failures are important characteristics of the resilience of these cells, and their aptitude to maintain their shape via a quick recovery from local strains. This study focuses on the perinuclear region because it can be considered as a master mechanical organizing center of these muscle precursor cells. Using this wavelet-based method, we combine the global and local approaches for a comparative analysis of the mechanical parameters of normal myoblasts, myotubes and myoblasts treated with actomyosin cytoskeleton disruptive agents (ATP depletion, blebbistatin).

Enzymatic activity of immobilized yeast phosphoglycerate kinase.

This work reports the first evidence that recombinant yeast phosphoglycerate kinase (PGK) is still significantly active when immobilized on glass and muscovite mica. Using previous work to improve the sensitivity of the existing setup, Tapping Mode atomic force microscopy (AFM) was used in a liquid environment to determine the surface enzyme coverage of derivatized mica and glass slides. When associated to spectrophotometric measurements, the AFM data allows assessing the catalytic constant of surface enzymes and comparing it to bulk values. The validity of the Michaelis-Menten model for surface reactions is discussed, supported by spectroscopic measurements of the surface consumption of 1,3-bis-phosphoglycerate (1,3-BPG). Only a few percent of the enzyme material maintains its initial bulk activity. This value could constitute a guideline for biosensors made with the method used here whenever a rapid assessment of the remaining surface activity is needed.

Multifractal Desynchronization of the Cardiac Excitable Cell Network During Atrial Fibrillation. I. Multifractal Analysis of Clinical Data.

Atrial fibrillation (AF) is a cardiac arrhythmia characterized by rapid and irregular atrial electrical activity with a high clinical impact on stroke incidence. Best available therapeutic strategies combine pharmacological and surgical means. But when successful, they do not always prevent long-term relapses. Initial success becomes all the more tricky to achieve as the arrhythmia maintains itself and the pathology evolves into sustained or chronic AF. This raises the open crucial issue of deciphering the mechanisms that govern the onset of AF as well as its perpetuation. In this study, we develop a wavelet-based multi-scale strategy to analyze the electrical activity of human hearts recorded by catheter electrodes, positioned in the coronary sinus (CS), during episodes of AF. We compute the so-called multifractal spectra using two variants of the wavelet transform modulus maxima method, the moment (partition function) method and the magnitude cumulant method. Application of these methods to long time series recorded in a patient with chronic AF provides quantitative evidence of the multifractal intermittent nature of the electric energy of passing cardiac impulses at low frequencies, i.e., for times (≳0.5 s) longer than the mean interbeat (≃ 10-1 s). We also report the results of a two-point magnitude correlation analysis which infers the absence of a multiplicative time-scale structure underlying multifractal scaling. The electric energy dynamics looks like a "multifractal white noise" with quadratic (log-normal) multifractal spectra. These observations challenge concepts of functional reentrant circuits in mechanistic theories of AF, still leaving open the role of the autonomic nervous system (ANS). A transition is indeed observed in the computed multifractal spectra which group according to two distinct areas, consistently with the anatomical substrate binding to the CS, namely the left atrial posterior wall, and the ligament of Marshall which is innervated by the ANS. In a companion paper (II. Modeling), we propose a mathematical model of a denervated heart where the kinetics of gap junction conductance alone induces a desynchronization of the myocardial excitable cells, accounting for the multifractal spectra found experimentally in the left atrial posterior wall area.

Numerical Study of Novel Ratiometric Sensors Based on Plasmon-Exciton Coupling.

We numerically studied the optical properties of spherical nanostructures made of an emitter core coated by a silver shell through the generalized Mie theory. When there is a strong coupling between the localized surface plasmon in the metallic shell and the emitter exciton in the core, the extinction spectra exhibit two peaks. Upon adsorption of analytes on these core-shell nanostructures, the intensities of the two peaks change with opposite trends. This property makes them potential sensitive ratiometric sensors. Molecule adsorption on these nanostructures can be quantified through a very simple optical configuration likely resulting in a much faster acquisition time compared with systems based on the traditional metal nanoparticle surface plasmon resonance (SPR) biosensors.