Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Spatial confinement: A spur for axonal growth.

The concept of spatial confinement is the basis of cell positioning and guidance in in vitro studies. In vivo, it reflects many situations faced during embryonic development. In vitro, spatial confinement of neurons is achieved using different technological approaches: adhesive patterning, topographical structuring, microfluidics and the use of hydrogels. The notion of chemical or physical frontiers is particularly central to the behaviors of growth cones and neuronal processes under confinement. They encompass phenomena of cell spreading, boundary crossing, and path finding on surfaces with different adhesive properties. However, the most universal phenomenon related to confinement, regardless of how it is implemented, is the acceleration of neuronal growth. Overall, a bi-directional causal link emerges between the shape of the growth cone and neuronal elongation dynamics, both in vivo and in vitro. The sensing of adhesion discontinuities by filopodia and the subsequent spatial redistribution and size adaptation of these actin-rich filaments seem critical for the growth rate in conditions in which adhesive contacts and actin-associated clutching forces dominate. On the other hand, the involvement of microtubules, specifically demonstrated in 3D hydrogel environments and leading to ameboid-like locomotion, could be relevant in a wider range of growth situations. This review brings together a literature collected in distinct scientific fields such as development, mechanobiology and bioengineering that highlight the consequences of confinement and raise new questions at different cellular scales. Its ambition is to stimulate new research that could lead to a better understanding of what gives neurons their ability to establish and regulate their exceptional size.

Mitochondria: At the crossroads between mechanobiology and cell metabolism.

Metabolism and mechanics are two key facets of structural and functional processes in cells, such as growth, proliferation, homeostasis and regeneration. Their reciprocal regulation has been increasingly acknowledged in recent years: external physical and mechanical cues entail metabolic changes, which in return regulate cell mechanosensing and mechanotransduction. Since mitochondria are pivotal regulators of metabolism, we review here the reciprocal links between mitochondrial morphodynamics, mechanics and metabolism. Mitochondria are highly dynamic organelles which sense and integrate mechanical, physical and metabolic cues to adapt their morphology, the organization of their network and their metabolic functions. While some of the links between mitochondrial morphodynamics, mechanics and metabolism are already well established, others are still poorly documented and open new fields of research. First, cell metabolism is known to correlate with mitochondrial morphodynamics. For instance, mitochondrial fission, fusion and cristae remodeling allow the cell to fine-tune its energy production through the contribution of mitochondrial oxidative phosphorylation and cytosolic glycolysis. Second, mechanical cues and alterations in mitochondrial mechanical properties reshape and reorganize the mitochondrial network. Mitochondrial membrane tension emerges as a decisive physical property which regulates mitochondrial morphodynamics. However, the converse link hypothesizing a contribution of morphodynamics to mitochondria mechanics and/or mechanosensitivity has not yet been demonstrated. Third, we highlight that mitochondrial mechanics and metabolism are reciprocally regulated, although little is known about the mechanical adaptation of mitochondria in response to metabolic cues. Deciphering the links between mitochondrial morphodynamics, mechanics and metabolism still presents significant technical and conceptual challenges but is crucial both for a better understanding of mechanobiology and for potential novel therapeutic approaches in diseases such as cancer.

Synapses do not facilitate prion-like transfer of alpha-synuclein: a quantitative study in reconstructed unidirectional neural networks.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Neuronal growth from a volume perspective.

Microfluidic-based fluorescent exclusion method allows to tackle the issue of neuronal growth from a volume perspective. Based on this technology, we studied the two main actin-rich structures accompanying the early stages of neuron development, i.e. growth cones, located at the tip of growing neuronal processes, and propagative actin waves. Our work reveals that growth cones tend to loose volume during their forward motion, as do actin waves during their journey from the cell body to the tip of neuronal processes, before the total transfer of their remaining volume to the growth cone. Actin waves seem thus to supply material to increasingly distant growth cones as neurons develop. In addition, our work may suggest the existence of a membrane recycling phenomena associated to actin waves as a pulsatile anterograde source of material and by a continuous retrograde transport.

Parallelized Manipulation of Adherent Living Cells by Magnetic Nanoparticles-Mediated Forces.

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.

Nanoscale surface topography reshapes neuronal growth in culture.

Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nanopillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nanopillars with a typical distance between adhesion points of about 500 nm. These neurons produce fewer neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.

Tuning the adhesive geometry of neurons: length and polarity control.

Neurons acquire their functional and morphological axo-dendritic polarity by extending, from competing minor processes (neurites), one long axon among numerous dendrites. We employed complementary sets of micropatterns built from 2 and 6 µm wide stripes of various lengths to constrain hippocampal neuron shapes. Using these geometries, we have (i) limited the number of neuronal extensions to obtain a minimal in vitro system of bipolar neurons and (ii) controlled the neurite width during growth by the generation of a progressive cell shape asymmetry on either side of the cellular body. From this geometrical approach, we gained a high level of control of each neurite length and of the localization of axonal specification. To analyze these results, we developed a model based on a width and polarization dependent neurite elongation rate and on the existence of a critical neurite length that sets the axonal fate. Our data on the four series of micro-patterns developed for this study are described by a single set of growth parameters, well supported by experiments. The control of neuronal shapes by adhesive micro-patterns thereby offers a novel paradigm to follow the dynamical process of neurite lengthening and competition through the process of axonal polarization.

Surface Tension and Neuronal Sorting in Magnetically Engineered Brain-Like Tissue.

Engineered 3D brain-like models have advanced the understanding of neurological mechanisms and disease, yet their mechanical signature, while fundamental for brain function, remains understudied. The surface tension for instance controls brain development and is a marker of cell-cell interactions. Here, 3D magnetic brain-like tissue spheroids composed of intermixed primary glial and neuronal cells at different ratios are engineered. Remarkably, the two cell types self-assemble into a functional tissue, with the sorting of the neuronal cells toward the periphery of the spheroids, whereas the glial cells constitute the core. The magnetic fingerprint of the spheroids then allows their deformation when placed under a magnetic field gradient, at a force equivalent to a 70 g increased gravity at the spheroid level. The tissue surface tension and elasticity can be directly inferred from the resulting deformation, revealing a transitional dependence on the glia/neuron ratio, with the surface tension of neuronal tissue being much lower. The results suggest an underlying mechanical contribution to the exclusion of the neurons toward the outer spheroid region, and depict the glia/neuron organization as a sophisticated mechanism that should in turn influence tissue development and homeostasis relevant in the neuroengineering field.

Neuronal architectures with axo-dendritic polarity above silicon nanowires.

An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.

Poly-l-lysine/Laminin Surface Coating Reverses Glial Cell Mechanosensitivity on Stiffness-Patterned Hydrogels.

Brain tissues demonstrate heterogeneous mechanical properties, which evolve with aging and pathologies. The observation in these tissues of smooth to sharp rigidity gradients raises the question of brain cell responses to both different values of rigidity and their spatial variations, in dependence on the surface chemistry they are exposed to. Here, we used recent techniques of hydrogel photopolymerization to achieve stiffness texturing down to micrometer resolution in polyacrylamide hydrogels. We investigated primary neuron adhesion and orientation as well as glial cell proliferative properties on these rigidity-textured hydrogels for two adhesive coatings: fibronectin or poly-l-lysine/laminin. Our main observation is that glial cell adhesion and proliferation is favored on the stiffer regions when the adhesive coating is fibronectin and on the softer ones when it consists of poly-l-lysine/laminin. This behavior was unchanged by the presence or the absence of neuronal cells. In addition, glial cells were not confined by sharp, micron-scaled gradients of rigidity. Our observations suggest that rigidity sensing could involve adhesion-related pathways that profoundly depend on surface chemistry.

Bending stiffness of Candida albicans hyphae as a proxy of cell wall properties.

The cell wall is a key component of fungi. It constitutes a highly regulated viscoelastic shell which counteracts internal cell turgor pressure. Its mechanical properties thus contribute to define cell morphology. Measurements of the elastic moduli of the fungal cell wall have been carried out in many species including Candida albicans, a major human opportunistic pathogen. They mainly relied on atomic force microscopy, and mostly considered the yeast form. We developed a parallelized pressure-actuated microfluidic device to measure the bending stiffness of hyphae. We found that the cell wall stiffness lies in the MPa range. We then used three different ways to disrupt cell wall physiology: inhibition of beta-glucan synthesis, a key component of the inner cell wall; application of a hyperosmotic shock triggering a sudden decrease of the hyphal diameter; deletion of two genes encoding GPI-modified cell wall proteins resulting in reduced cell wall thickness. The bending stiffness values were affected to different extents by these environmental stresses or genetic modifications. Overall, our results support the elastic nature of the cell wall and its ability to remodel at the scale of the entire hypha over minutes.

Living cell imaging by far-field fibered interference scanning optical microscopy.

We report on the imaging of biological cells including living neurons by a dedicated fibered interferometric scanning optical microscope. The topography and surface roughness of mouse fibroblasts and hippocampal neurons are clearly revealed. This straightforward far-field technique allows fast, high resolution observation of samples in liquids without lengthy alignment procedures or costly components.

Cellular and Subcellular Contact Guidance on Microfabricated Substrates.

Topography of the extracellular environment is now recognized as a major biophysical regulator of cell behavior and function. The study of the influence of patterned substrates on cells, named contact guidance, has greatly benefited from the development of micro and nano-fabrication techniques, allowing the emergence of increasingly diverse and elaborate engineered platforms. The purpose of this review is to provide a comprehensive view of the process of contact guidance from cellular to subcellular scales. We first classify and illustrate the large diversity of topographies reported in the literature by focusing on generic cellular responses to diverse topographical cues. Subsequently, and in a complementary fashion, we adopt the opposite approach and highlight cell type-specific responses to classically used topographies (arrays of pillars or grooves). Finally, we discuss recent advances on the key subcellular and molecular players involved in topographical sensing. Throughout the review, we focus particularly on neuronal cells, whose unique morphology and behavior have inspired a large body of studies in the field of topographical sensing and revealed fascinating cellular mechanisms. We conclude by using the current understanding of the cell-topography interactions at different scales as a springboard for identifying future challenges in the field of contact guidance.

Fluid shear stress coupled with narrow constrictions induce cell type-dependent morphological and molecular changes in SK-BR-3 and MDA-MB-231 cells.

Cancer mortality mainly arises from metastases, due to cells that escape from a primary tumor, circulate in the blood as circulating tumor cells (CTCs), permeate across blood vessels and nest in distant organs. It is still unclear how CTCs overcome the harsh conditions of fluid shear stress and mechanical constraints within the microcirculation. Here, a minimal model of the blood microcirculation was established through the fabrication of microfluidic channels comprising constrictions. Metastatic breast cancer cells of epithelial-like and mesenchymal-like phenotypes were flowed into the microfluidic device. These cells were visualized during circulation and analyzed for their dynamical behavior, revealing long-lived plastic deformations and significant differences in biomechanics between cell types. γ-H2AX staining of cells retrieved post-circulation showed significant increase of DNA damage response in epithelial-like SK-BR-3 cells, while gene expression analysis of key regulators of epithelial-to-mesenchymal transition revealed significant changes upon circulation. This work thus documents first results of the changes at the cellular, subcellular and molecular scales induced by the two main mechanical stimuli arising from circulatory conditions, and suggest a significant role of this still elusive step of the metastatic cascade in cancer cells heterogeneity and aggressiveness.

The expression level of alpha-synuclein in different neuronal populations is the primary determinant of its prion-like seeding.

Alpha-synuclein (aSyn)-rich aggregates propagate in neuronal networks and compromise cellular homeostasis leading to synucleinopathies such as Parkinson's disease. Aggregated aSyn spread follows a conserved spatio-temporal pattern that is not solely dependent on connectivity. Hence, the differential tropism of aSyn-rich aggregates to distinct brain regions, or their ability to amplify within those regions, must contribute to this process. To better understand what underlies aSyn-rich aggregates distribution within the brain, we generated primary neuronal cultures from various brain regions of wild-type mice and mice expressing a reduced level of aSyn, and exposed them to fibrillar aSyn. We then assessed exogenous fibrillar aSyn uptake, endogenous aSyn seeding, and endogenous aSyn physiological expression levels. Despite a similar uptake of exogenous fibrils by neuronal cells from distinct brain regions, the seeded aggregation of endogenous aSyn differed greatly from one neuronal population to another. The different susceptibility of neuronal populations was linked to their aSyn expression level. Our data establish that endogenous aSyn expression level plays a key role in fibrillar aSyn prion-like seeding, supporting that endogenous aSyn expression level participates in selective regional brain vulnerability.

Topographical cues control the morphology and dynamics of migrating cortical interneurons.

In mammalian embryos, cortical interneurons travel long distances among complex three-dimensional tissues before integrating into cortical circuits. Several molecular guiding cues involved in this migration process have been identified, but the influence of physical parameters remains poorly understood. In the present study, we have investigated in vitro the influence of the topography of the microenvironment on the migration of primary cortical interneurons released from mouse embryonic explants. We found that arrays of PDMS micro-pillars of 10 µm size and spacing, either round or square, influenced both the morphology and the migratory behavior of interneurons. Strikingly, most interneurons exhibited a single and long leading process oriented along the diagonals of the square pillared array, whereas leading processes of interneurons migrating in-between round pillars were shorter, often branched and oriented in all available directions. Accordingly, dynamic studies revealed that growth cone divisions were twice more frequent in round than in square pillars. Both soma and leading process tips presented forward directed movements within square pillars, contrasting with the erratic trajectories and more dynamic movements observed among round pillars. In support of these observations, long interneurons migrating in square pillars displayed tight bundles of stable microtubules aligned in the direction of migration. Overall, our results show that micron-sized topography provides global spatial constraints promoting the establishment of different morphological and migratory states. Remarkably, these different states belong to the natural range of migratory behaviors of cortical interneurons, highlighting the potential importance of topographical cues in the guidance of these embryonic neurons, and more generally in brain development.

Redox-Triggered Control of Cell Adhesion and Deadhesion on Poly(lysine)-g-poly(ethylene oxide) Adlayers.

Spontaneous adsorption of poly(lysine)-g-poly(ethylene glycol) comb-like copolymers (PLL-g-PEG) is a versatile mean to coat substrates with polymer layers that resist cell adhesion. We prepared redox cleavable PLL-g-PEG to switch adhesion on demand. Redox sensitivity was obtained by introducing disulfide linkers between the PLL backbone and PEG strands. This modification was done alone or in combination with an azide end on the PEG strands that enabled in situ conjugations of adhesion peptides or fluorescent labels (by a simple application of commercially available molecules for copper-free click chemistry compatible with cell survival). To balance the functional (adhesion-promoting) vs cell-repellent copolymers, mixed layers of adjusted compositions were obtained by coadsorption from mixed solutions of the cleavable copolymer with noncleavable and repellant PLL-g-PEG. The deposition of copolymers and quantitative cleavage as triggered by reductive conditions (application of solutions of tris(carboxyethyl)phosphine, dithiothreitol, or glutathione) were characterized by QCM-D, XPS, and fluorescence microscopy. In cell culture conditions, redox-triggered cleavage was obtained by a nontoxic application of TCEP for a few minutes, enabling either to release cell attachment points (i.e., cleavage of RGD-presenting areas) or to "open" nonspecific adherent areas (i.e., transition from PEG-presenting areas to adherent PLL-like coatings).

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices.

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Reconstruction of directed neuronal networks in a microfluidic device with asymmetric microchannels.

Microfluidic devices for controlling neuronal connectivity in vitro are extremely useful tools for deciphering pathological and physiological processes occurring in neuronal networks. These devices allow the connection between different neuronal populations located into separate culture chambers through axon-selective microchannels. In order to implement specific features of brain connectivity such as directionality, it is necessary to control axonal growth orientation in these devices. Among the various strategies proposed to achieve this goal, one of the most promising and easily reproducible is the use of asymmetric microchannels. We present here a general protocol and several guidelines for the design, production and testing of a new paradigm of asymmetric microchannels geometries based on a "return to sender" strategy. In this method, axons are either allowed to travel between the emitting and receiving chambers within straight microchannels (forward direction), or are rerouted toward their initial location through curved microchannels (reverse direction). We introduce variations of these "arches" microchannels and evaluate their respective axonal filtering capacities. Importantly, one of these variants presents an almost complete filtration of axonal growth in the non-permissive direction while allowing robust axonal invasion in the other one, with a selectivity ratio as high as 99.7%.