Biology subjects

Piel, M.

Publications and source records attributed to Piel, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Mechanisms of genetic instability in a single S-phase following whole genome doubling

Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

cell biology↗

bioRxiv2021

A mechano-osmotic feedback couples cell volume to the rate of cell deformation

Mechanics has been a central focus of physical biology in the past decade. In comparison, the osmotic and electric properties of cells are less understood. Here we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spread, migrate or are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechano-sensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.

biophysics↗

bioRxiv2021

Volume growth in animal cells is cell cycle dependent and shows additive fluctuations

The way proliferating animal cells coordinate the growth of their mass, volume, and other relevant size parameters is a long-standing question in biology. Studies focusing on cell mass have identified patterns of mass growth as a function of time and cell cycle phase, but little is known about volume growth. To address this question, we improved our fluorescence exclusion method of volume measurement (FXm) and obtained 1700 single-cell volume growth trajectories of HeLa cells. We find that, during most of the cell cycle, volume growth is close to exponential and proceeds at a higher rate in S-G2 than in G1. Comparing the data with a mathematical model, we establish that the cell-to-cell variability in volume growth arises from constant-amplitude fluctuations in volume steps, rather than fluctuations of the underlying specific growth rate. We hypothesize that such "additive noise" could emerge from the processes that regulate volume adaptation to biophysical cues, such as tension or osmotic pressure.

cell biology↗

bioRxiv2020

Calculation of the force field required for nucleus deformation during cell migration through constrictions

During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions [1]. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. We extract the nuclear shape from the boundary of the fluorescently stained region in each consecutive image over time. From this we calculate the deformation field between images and use our elastic models to calculate the traction force field. Our calculations therefore predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations. Author summaryMany cell types are able to migrate and squeeze through constrictions that are narrower than the cells resting radius. For example, both immune cells and metastatic cancer cells change their shape to migrate through small holes in the complex tissue media they move in. During migration the cell nucleus is more difficult to deform than the cell cytoplasm and therefore significant forces are required for a cell to pass through spaces that are smaller than the resting size of the nucleus. Experimental measurements of these forces are extremely challenging but experimental images of nuclear deformation are regularly obtained in many labs. Therefore we present a computational method to analyse experimental images of nuclear deformation to deduce the forces required to produce such deformations. A mechanical model of the nucleus is necessary for this analysis and here we present two different models. The first treats the nucleus as a homogeneous elastic solid and the second treats the nucleus as an elastic shell. Our computational tool enables us to obtain detailed information about forces causing deformation from microscopy images.

biophysics↗

bioRxiv2020

Pinching the cortex of live cells reveals thickness instabilities caused by Myosin II motors

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

biophysics↗

bioRxiv2020

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not physiologically relevant for their uncoupling activity.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocyte. In the presence of long-chain fatty acids (LCFA), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Several atomic models of UCP1 and UCP2 have been obtained by NMR in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiment on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of S. cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S and K56S/K269S mutants did not display any default in activation, which shows that the NMR experiments in DPC detergent are not relevant to understand UCP1 function.

biochemistry↗

bioRxiv2020

Cell clusters adopt a collective amoeboid mode of migration in confined non-adhesive environments

Cell migration is essential to most living organisms. Single cell migration involves two distinct mechanisms, either a focal adhesion- and traction-dependent mesenchymal motility or an adhesion-independent but contractility-driven propulsive amoeboid locomotion. Cohesive migration of a group of cells, also called collective cell migration, has been only described as an adhesion- and traction-dependent mode of locomotion where the driving forces are mostly exerted at the front by leader cells. Here, by studying primary cancer specimens and cell lines from colorectal cancer, we demonstrate the existence of a second mode of collective migration which does not require adhesion to the surroundings and relies on a polarised supracellular contractility. Cell clusters confined into non-adhesive microchannels migrate in a rounded morphology, independently of the formation of focal adhesions or protruding leader cells, and lacking internal flow of cells, ruling-out classical traction-driven collective migration. Like single cells migrating in an amoeboid fashion, the clusters display a supracellular actin cortex with myosin II enriched at the rear. Using pharmacological inhibitors and optogenetics, we show that this polarised actomyosin activity powers migration and propels the clusters. This new mode of migration, that we named collective amoeboid, could be enabled by intrinsic or extrinsic neoplasic features to enable the metastatic spread of cancers. One Sentence SummaryClusters organise as polarised and contractile super-cells to migrate without adhesion.

cell biology↗

bioRxiv2020

Compromised nuclear envelope integrity drives tumor cell invasion

While mutations leading to a fragile envelope of the cell nucleus are well known to cause diseases such as muscular dystrophies or accelerated aging, the pathophysiological consequences of the recently discovered mechanically induced nuclear envelope ruptures in cells harboring no mutation are less known. Here we show that repeated loss of nuclear envelope integrity in nuclei experiencing mechanical constraints promotes senescence in nontransformed cells, and induces an invasive phenotype including increased collagen degradation in human breast cancer cells, both in vitro and in a mouse xenograft model of breast cancer progression. We show that these phenotypic changes are due to the presence of chronic DNA damage and activation of the ATM kinase. In addition, we found that depletion of the cytoplasmic exonuclease TREX1 is sufficient to abolish the DNA damage in mechanically challenged nuclei and to suppress the phenotypes associated with the loss of nuclear envelope integrity. Our results also show that TREX1-dependent DNA damage induced by physical confinement of tumor cells inside the mammary duct drives the progression of in situ breast carcinoma to the invasive stage. 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.

cell biology↗

bioRxiv2020

Acto-myosin network geometry defines centrosome position

The centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin, in which microtubules appeared bent by compressive forces. The disassembly of the actin network away from the cell edges defines an inner zone where actin bundles were absent and microtubules were more radially organized. The production of dynein-based forces on microtubules places the centrosome at the center of this inner zone. Cell adhesion pattern and contractile forces define the shape and position of the inner zone in which the centrosome-microtubule network is centered.

cell biology↗

bioRxiv2019

The nucleus acts as a ruler tailoring cell responses to spatial constraints

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. Here, we found that cells measure the degree of spatial confinement using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, upregulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force enabling their passage through restrictive pores in complex three-dimensional (3D) environments, a process relevant to cancer cell invasion, immune responses and embryonic development. One Sentence SummaryNuclear envelope expansion above a threshold triggers a contractile cell response and thus acts as a ruler for the degree of cell deformation.

cell biology↗

bioRxiv2019

Adhesion-free cell migration by topography-based force transduction

Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force-coupling is usually mediated by transmembrane adhesion receptors, especially these of the integrin family, amoeboid cells like leukocytes can migrate extremely fast despite very low adhesive forces1. We show that leukocytes cannot only migrate under low adhesion but indeed can transduce forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographic features of the substrate to propel themselves. Here, the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating shear forces sufficient to drive deformations towards the back of the cell. Notably, adhesion dependent and adhesion independent migration are not exclusive but rather variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate simultaneously. As adhesion free migration is independent of the chemical composition of the environment it renders cells completely autonomous in their locomotive behavior.

cell biology↗

bioRxiv2019

Profilin and formin constitute a pacemaker system for robust actin filament growth

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

biochemistry↗

bioRxiv2019

Oncogenic signaling alters cell shape and mechanics to facilitate cell division under confinement.

When cells enter mitosis, they become spherical and mechanically stiffen. We used MCF10A cell lines as a model system in which to investigate the effect of induced oncogene expression on mitotic entry. We find that activation of oncogenic RasV12, for as little as five hours, changes the way cells divide. RasV12-dependent activation of the MEK-ERK signalling cascade alters acto-myosin contractility to enhance mitotic rounding. RasV12 also affects cell mechanics, so that RasV12 expressing cells are softer in interphase but stiffen more upon entry into mitosis. As a consequence, RasV12 expression augments the ability of cells to round up and divide faithfully when confined underneath a stiff hydrogel. Conversely, inhibition of the Ras-ERK pathway reduces mitotic rounding under confinement, resulting in chromosome segregation defects. These data suggest a novel mechanism by which oncogenic Ras-ERK signalling can aid division in stiff environments like those found in tumours.

cell biology↗

bioRxiv2019

Spatial Asymmetries of the Prophase Nucleus Regulate Dynein Recruitment to Ensure Mitotic Fidelity

During prophase, centrosomes need to separate and position to correctly assemble the mitotic spindle. This process occurs through the action of molecular motors, cytoskeletal networks and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. Here we show that during prophase the centrosomes-nucleus axis reorients, so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. This centrosomes-nucleus configuration depends on mechanical cues generated by mitotic chromosome condensation on the prophase nucleus. We further show these mechanosensitive cues act through SUN1/2 and NudE+NudEL to enable the polarized loading of Dynein on the NE. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation. We propose that chromosome segregation fidelity depends on the mechanical properties of the prophase nucleus that facilitate spindle assembly by regulating NE-Dynein localization.

cell biology↗

bioRxiv2018

Glioma cell migration in confined microchannels via a motor-clutch mechanism

Glioma tumor dispersion involves invading cells escaping the tumor bulk and migrating into the healthy brain parenchyma. Here, they encounter linearly aligned track-like tissue structures such as axon bundles and the perivascular space. These environments also contain micrometer-scale pores that impose mechanical confinement on invading cells. To study glioma cell migration in an in vitro system that reproduces some of these features, we used microfluidic devices with 60 m2 cross-sectional area channels that confine cells into one-dimensional (1D) tracks. Individual cell tracking revealed strongly persistent migration at a mean rate of 8.5 {+/-} 0.33 nm s-1. Notably, a 1D computational cell migration simulator predicts migration behaviors of glioma cells without significant adjustment of parameters estimated from previous experiments on two-dimensional (2D) substrates. Pharmacological inhibitors of integrin-mediated adhesions, myosin II activation, or drugs targeting F-actin assembly or microtubule dynamics influence migration consistent with simulations where relevant parameters are changed. These results suggest that cell parameters calibrated to a motor-clutch model on 2D substrates effectively predict 1D confined migration behaviors a priori. Our results outline a method for testing biophysical mechanisms of tumor cell migration in confined spaces and predicting the effects of anti-motility therapy.

biophysics↗

bioRxiv2018

Adhesion to nanofibers drives cell membrane remodeling through 1D wetting

The shape of cellular membranes is highly regulated by a set of conserved mechanisms. These mechanisms can be manipulated by bacterial pathogens to infect cells. Human endothelial cell plasma membrane remodeling by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unknown. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a novel physical mechanism we term \" onedimensional\" membrane wetting. We provide a theoretical model that gives the physical basis of 1D wetting and show that this mechanism occurs in model membranes interacting with model nanofibers, and in human cells interacting with model extracellular matrices. It is thus a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococcus during infection.

cell biology↗

bioRxiv2018

Macropinocytosis overcomes directional bias due to hydraulic resistance to enhance space exploration by dendritic cells

The migration of immune cells is guided by specific chemical signals, such as chemokine gradients. Their trajectories can also be diverted by physical cues and obstacles imposed by the cellular environment, such as topography, rigidity, adhesion, or hydraulic resistance. On the example of hydraulic resistance, it was shown that neutrophil preferentially follow paths of least resistance, a phenomenon referred to as barotaxis. We here combined quantitative imaging and physical modeling to show that barotaxis results from a force imbalance at the scale of the cell, which is amplified by the acto-myosin intrinsic polarization capacity. Strikingly, we found that macropinocytosis specifically confers to immature dendritic cells a unique capacity to overcome this physical bias by facilitating external fluid transport across the cell, thereby enhancing their space exploration capacity in vivo and promoting their tissue-patrolling function. Conversely, mature dendritic cells, which down-regulate macropinocytosis, were found to be sensitive to hydraulic resistance. Theoretical modeling suggested that barotaxis, which helps them avoid dead-ends, may accelerate their migration to lymph nodes, where they initiate adaptive immune responses. We conclude that the physical properties of the microenvironment of moving cells can introduce biases in their migratory behaviors but that specific active mechanisms such as macropinocytosis have emerged to diminish the influence of these biases, allowing motile cells to reach their final destination and efficiently fulfill their functions.

cell biology↗

bioRxiv2017

Polarization of Myosin II refines tissue material properties to buffer mechanical stress.

As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces, such as those required for morphogenetic movements, are instrumental to the development and sculpting of tissues. However, mechanical forces can also lead to accumulation of substantial tensile stress, which if maintained, can result in tissue damage and impair tissue function. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis, once shape is determined after development. Buffering forces to prevent cellular changes in response to fluctuations of mechanical stress is critical during the lifetime of an adult organism. Here, through the development of a novel tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of Myosin II across the tissue. Mechanistically, this process is independent of conserved Rho-kinase signaling but is mediated by force-induced linear actin polymerization and depolymerization via the formin Diaphanous and actin severing protein Cofilin, respectively. Importantly, these stretch-induced actomyosin cables stiffen the tissue to limit changes in cell shape and to protect the tissue from further mechanical damage prior to stress dissipation. This tissue rigidification prevents fractures in the tissue from propagating by confining the damage locally to the injured cells. Overall this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that rapidly protects tissues from physical damage to preserve tissue shape.

biophysics↗