Sylvain Gabriele

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. Our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. On the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

A Possible Role for Integrin Signaling in Diffuse Axonal Injury

Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury.

Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments.

Hierarchical wrinkling patterns

This paper reports a simple and flexible method for generating hierarchical patterns from wrinkling instability. Complex features with gradually changing topographies are generated by using the spontaneous wrinkling of a rigid membrane (titanium) on a soft foundation (polystyrene) compressed via the diffusion of a solvent. We show that the morphology of these unreported wrinkled patterns is directly related to the rheological properties of the polymer layer and the geometry of the diffusion front. Based on these ingredients, we rationalize the mechanism for the formation of hierarchical wrinkling patterns and quantify our experimental findings with a simple scaling theory. Finally, we illustrate the relevance of our structuration method by studying the mechanosensitivity of fibroblasts.

Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites

Increasing evidences show that the actin cytoskeleton is a key parameter of the nuclear remodeling process in response to the modifications of cellular morphology. However, detailed information on the interaction between the actin cytoskeleton and the nuclear lamina was still lacking. We addressed this question by constraining endothelial cells on rectangular fibronectin-coated micropatterns and then using Structured Illumination Microscopy (SIM) to observe the interactions between actin stress fibers, nuclear lamina and LINC complexes at a super-resolution scale. Our results show that tension in apical actin stress fibers leads to deep nuclear indentations that significantly deform the nuclear lamina. Interestingly, indented nuclear zones are characterized by a local enrichment of LINC complexes, which anchor apical actin fibers to the nuclear lamina. Moreover, our findings indicate that nuclear indentations induce the formation of segregated domains of condensed chromatin. However, nuclear indentations and condensed chromatin domains are not irreversible processes and both can relax in absence of tension in apical actin stress fibers.

Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury

Although pathological changes in axonal morphology have emerged as important features of traumatic brain injury (TBI), the mechanical vulnerability of the axonal microcompartment relative to the cell body is not well understood. We hypothesized that soma and neurite microcompartments exhibit distinct mechanical behaviors, rendering axons more sensitive to a mechanical injury. In order to test this assumption, we combined protein micropatterns with magnetic tweezer rheology to probe the viscoelastic properties of neuronal microcompartments. Creep experiments revealed two opposite rheological behaviors within cortical neurons: the cell body was soft and characterized by a solid-like response, whereas the neurite compartment was stiffer and viscous-like. By using pharmacological agents, we demonstrated that the nucleus is responsible for the solid-like behavior and the stress-stiffening response of the soma, whereas neurofilaments have a predominant contribution in the viscous behavior of the neurite. Furthermore, we found that the neurite is a mechanosensitive compartment that becomes softer and adopts a pronounced viscous state on soft matrices. Together, these findings highlight the importance of the regionalization of mechanical and rigidity-sensing properties within neuron microcompartments in the preferential damage of axons during traumatic brain injury and into potential mechanisms of axonal outgrowth after injury.

Cell confinement: putting the squeeze on the nucleus

The nucleus has long been considered as a passive compartment containing the genetic information. However, recent attention to its structure, mechanical properties and physical connections with other cellular compartments has shown that the nucleus changes dynamically its morphology and internal organization for important cellular processes, especially those associated with cellular confinement. In this paper, we review some recent progress in experimental investigations of nuclear squeezing that lead to a better understanding of the nuclear remodeling in response to various situations of cellular confinement. We will discuss compelling examples of original experiments performed with microsystems that have recently brought new insights into the close relationship between nuclear mechanics and cellular organization. We will show that the study of nuclear confinement with microsystems has opened up new experimental avenues that already offer promising clues for understanding diseases that are associated with defective nuclear mechanics.

Influence of Substrate Properties on the Dewetting Dynamics of Viscoelastic Polymer Films

We studied the dewetting process of thin polystyrene (PS) films on silicon substrates, coated with a thin, irreversibly adsorbed polydimethylsiloxane (PDMS) layer, by optical microscopy and atomic force microscopy. Besides demonstrating the exceptional potential of dewetting for a sensitive characterization of rheological properties of PS thin films, characterized by a stress-relaxation time, τ1, we focused on the influence of the frictional behaviour (energy dissipation mechanism) at the interface between the PDMS-coated silicon wafer and the PS film on the dewetting process. Our results show that the initial stages of dewetting depend sensitively on the thickness and the way the PDMS layer was adsorbed. The maximum width of the dewetting rim at τ1 increased with increasing PDMS layer thickness, which can be interpreted as an increase of the effective, velocity-dependent slippage length. Interestingly, τ1 was found to be almost independent of the substrate properties. Our results demonstrate that dewetting is a really powerful approach for rheological and frictional studies of thin polymer films.

Influence of chain interdiffusion between immiscible polymers on dewetting dynamics

From dewetting and neutron reflectivity experiments, we demonstrate the major influence of chain interdiffusion on the friction properties for a couple of immiscible polymers. The interface between two immiscible polymers, polystyrene (PS) and polydimethylsiloxane (PDMS), was studied by neutron reflectivity and dewetting by using free PS chains and PDMS brushes. Unexpectedly, we found that the PS chains diffuse in the PDMS brushes at temperatures well below the glass transition temperatures of PS, the dynamics being largely determined by the grafting density of the brush. These observations strongly suggest that the puzzling ageing of PS thin films observed from dewetting experiments can be directly related to modifications of the PS/PDMS interface.

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins

ABSTRACT The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the laminas major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.