Cancer cells’ ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.

Calcium ion (Ca2+) is an essential second messenger involved in multiple cellular and subcellular processes. Ca2+ can be released and sensed globally or locally within cells, providing complex signals of variable amplitudes and time-scales. The key function of Ca2+ in the regulation of acto-myosin contractility has provided a simple explanation for its role in the regulation of immune cell migration. However, many questions remain, including the identity of the Ca2+ stores, channels and upstream signals involved in this process. Here, we focus on dendritic cells (DCs), because their immune sentinel function heavily relies on their capacity to migrate within tissues and later on between tissues and lymphoid organs. Deciphering the mechanisms by which cytoplasmic Ca2+ regulate DC migration should shed light on their role in initiating and tuning immune responses.

Dynamic guidance of living cells is achieved by fine-tuning and spatiotemporal modulation on artificial polymer layers enabling reversible peptide display. Adjustment of surface composition and interactions is obtained by coadsorption of mixed poly(lysine) derivatives, grafted with either repellent PEG, RGD adhesion peptides, or T-responsive poly(N-isopropylacrylamide) strands. Deposition of mixed adlayers provides a straightforward mean to optimize complex substrates, which is here implemented to achieve (1) thermal control of ligand accessibility and (2) adjustment of relative adhesiveness between adjacent micropatterns, while preserving cell attachment during thermal cycles. The reversible polarization of HeLa cells along orthogonal stripes mimics guidance along natural matrices.

FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis. [Cell. 2016]

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.

Sign epistasis is a central evolutionary constraint, but its causal factors remain difficult to predict. Here we use the notion of parameterised optima to explain epistasis within a signalling cascade, and test these predictions in Escherichia coli. We show that sign epistasis arises from the benefit of tuning phenotypic parameters of cascade genes with respect to each other, rather than from their complex and incompletely known genetic bases. Specifically, sign epistasis requires only that the optimal phenotypic parameters of one gene depend on the phenotypic parameters of another, independent of other details, such as activating or repressing nature, position within the cascade, intra-genic pleiotropy or genotype. Mutational effects change sign more readily in downstream genes, indicating that optimising downstream genes is more constrained. The findings show that sign epistasis results from the inherent upstream-downstream hierarchy between signalling cascade genes, and can be addressed without exhaustive genotypic mapping.

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.

An original setup combining a very stable loading stage, an atomic force microscope and an environmental chamber, allows to obtain very stable sub-critical fracture propagation in oxide glasses under controlled environment, and subsequently to finely characterize the nanometric roughness properties of the crack surfaces. The analysis of the surface roughness is conducted both in terms of the classical root mean square roughness to compare with the literature, and in terms of more physically adequate indicators related to the self-affine nature of the fracture surfaces. Due to the comparable nanometric scale of the surface roughness, the AFM tip size and the instrumental noise, a special care is devoted to the statistical evaluation of the metrologic properties. The 2 roughness amplitude of several oxide glasses was shown to decrease as a function of the stress intensity factor, to be quite insensitive to the relative humidity and to increase with the degree of heterogeneity of the glass. The results are discussed in terms of several modeling arguments concerning the coupling between crack propagation, material's heterogeneity, crack tip plastic deformation and water diffusion at the crack tip. A synthetic new model is presented combining the predictions of a model by Wiederhorn et al. [1] on the effect of the material's heterogeneity on the crack tip stresses with the self-affine nature of the fracture surfaces.

We analyze the evolution of the mechanical response of a colloidal suspension to an external tensile stress, from fracture to flow, as a function of the distance from the sol–gel transition. We cease to observe cracks at a finite distance from the transition. In an intermediate region where the phenomenon is clearly hysteretic, we observe the coexistence of both flow and fracture. Even when cracks are observed, the material in fact flows over a distance that increases in the vicinity of the transition.

Transient compartments have been recently shown to be able to maintain functional replicators in the context of prebiotic studies. Here, we show that a broad class of selection dynamics is able to achieve this goal. We identify two key parameters, the relative amplification of nonactive replicators (parasites) and the size of compartments. These parameters account for competition and diversity, and the results are relevant to similar multilevel selection problems, such as those found in virus-host ecology and trait group selection.

The concept of using stimuli-responsive hydrogels to actuatefluids in microfluidic devices is particularly attractive, but limitations,in terms of spatial resolution, speed, reliability and integration, have hindered its development during the past two decades. By patterning and grafting poly(N-isopropylacrylamide) PNIPAM hydrogel films on plane substrates with a 2μm horizontal resolution and closing the system afterward, we have succeeded in unblocking bottlenecks that thermo-sensitive hydrogel technology has
been challenged with until now. In this paper, we demonstrate, for thefi rst time with this technology, devices with up to 7800
actuated micro-cages that sequester and release solutes, along with valves actuated individually with closing and opening switching
times of 0.6 ± 0.1 and 0.25± 0.15 s, respectively. Two applications of this technology are illustrated in the domain of single cell
handling and the nuclear acid amplification test (NAAT) for the Human Synaptojanin 1 gene, which is suspected to be involved in several neurodegenerative diseases such as Parkinson’s disease. The performance of the temperature-responsive hydrogels we
demonstrate here suggests that in association with their moderate costs, hydrogels may represent an alternative to the actuation orhandling techniques currently used in microfluidics, that are, pressure actuated polydimethylsiloxane (PDMS) valves and droplets

In capillary-driven fluid dynamics, simple departures from equilibrium offer the chance to quantitatively model the resulting relaxations. These dynamics in turn provide insight on both practical and fundamental aspects of thin-film hydrodynamics. In this work, we describe a model trilayer dewetting experiment elucidating the effect of solid, no-slip confining boundaries on the bursting of a liquid film in a viscous environment. This experiment was inspired by an industrial polymer processing technique, multilayer coextrusion, in which thousands of alternating layers are stacked atop one another. When pushed to the nanoscale limit, the individual layers are found to break up on time scales shorter than the processing time. To gain insight on this dynamic problem, we here directly observe the growth rate of holes in the middle layer of the trilayer films described above, wherein the distance between the inner film and solid boundary can be orders of magnitude larger than its thickness. Under otherwise identical experimental conditions, thinner films break up faster than thicker ones. This observation is found to agree with a scaling model that balances capillary driving power and viscous dissipation with a no-slip boundary condition at the solid substrate/viscous environment boundary. In particular, even for the thinnest middle-layers, no finite-size effect related to the middle film is needed to explain the data. The dynamics of hole growth is captured by a single master curve over four orders of magnitude in the dimensionless hole radius and time, and is found to agree well with predictions including analytical expressions for the dissipation.

Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential
of generating electrical current. Despite their high value for sustainable energy, organic compound
production and bioremediation, a tool to probe the natural biodiversity and to select most efficient
microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but
the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we
develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes.
Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant
assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the
mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain
hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the
bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads
or the inner wall of the conductive capsules’ shell that function as an anode from which electrons
produced by the cells are collected.

The osmotic pressure of polyelectrolyte solutions as a function of concentration has been calculated by Monte Carlo simulations of a spherical cell model and by molecular dynamics simulations with periodic boundary conditions. The results for the coarse-grained polyelectrolyte model are in good agreement with experimental results for sodium polyacrylate and the cell model is validated by the bulk simulations. The cell model offers an alternative perspective on osmotic pressure and also forms a direct link to even simpler models in the form of the Poisson–Boltzmann approximation applied to cylindrical and spherical geometries. As a result, the non-monotonic behaviour of the osmotic coefficient seen in simulated salt-free solutions is shown not to rely on a transition between a dilute and semi-dilute regime, as is often suggested when the polyion is modelled as a linear flexible chain. The non-monotonic behaviour is better described as the combination of a finite-size effect and a double-layer effect. Parameters that represent the linear nature of the polyion, including an alternative to monomer concentration, make it possible to display a generalised behaviour of equivalent chains, at least at low concentrations. At high concentrations, local interactions become significant and the exact details of the model become important. The effects of added salt are also discussed and one conclusion is that the empirical additivity rule, treating the contributions from the polyelectrolyte and any salt separately, is a reasonable approximation, which justifies the study of salt-free solutions.

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.