Biology subjects

Coppey, M.

Publications and source records attributed to Coppey, M..

6 recordsLinked to original sources

bioRxiv2018

Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration

Directional cell motility during organism and tissue development, homeostasis and disease requires symmetry breaking. This process relies on the ability of single cells to establish a front-rear polarity, and can occur in absence of external cues. The initiation of migration has been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cytoskeletal forces arising from continuous mechanical cell-substrate interaction has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to drive directional cell motility in absence of pre-established cytoskeleton polarity or morphogen gradients. A theoretical model validates the crucial role of adhesion dynamics during spontaneous symmetry breaking, proposing that the examined phenomenon can emerge independently of a complex self-polarizing system.\n\nOne sentence summaryCells can autonomously break their symmetry through traction force oscillations (mechanical instabilities) that lead to stochastic detachment of adhesion patches on one side of the cell and the subsequent initiation of migration.

biophysics↗

bioRxiv2018

Optogenetic dissection of Rac1 and Cdc42 gradient shaping

During migration, cells present a polarized activity that is aligned with the direction of motion. This cell polarity is established by an internal molecular circuitry, without the requirement of extracellular cues. At the heart of this circuitry, Rho GTPases spontaneously form spatial gradients that define the front and back of migrating cells. At the front of the cell, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away from the cell tip. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Combining optogenetics and cell micopatterning, we show that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by advection or diffusion mechanisms. Cdc42 simply follows the distribution of GEFs thanks to a uniform GAP activity, whereas Rac1 shaping requires the activity of an additional GAP, {beta}2-chimaerin, which is sharply localized at the tip of the cell. We find that {beta}2-chimaerin recruitment depends on feedbacks from Cdc42 and Rac1. Functionally, the extent -neither the slope nor the amplitude- of RhoGTPases gradients governs cell migration. A Cdc42 gradient with a short spatial extent is required to maximize directionality during cell migration while an extended Rac1 gradient controls the speed of the cell.

cell biology↗

bioRxiv2018

Precision in a rush: trade-offs between positioning and steepness of the hunchback expression pattern

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-o between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the genes ability to read positional information along the embryos anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies.

biophysics↗

bioRxiv2018

3 minutes to precisely measure morphogen concentration

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

developmental biology↗

bioRxiv2017

Fast Activation Cycles Of Rac1 At The Lamellipodium Tip Trigger Membrane Protrusion

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.\n\nSignificanceRac1 and RhoA GTPases are molecular switches controlling the actin cytoskeletal during cell migration. WAVE, Rac1 effector during cell protrusion, is concentrated at the lamellipodium tip. But, recent biosensor imaging studies suggested that Rac1 activation occurs behind the lamellipodium, while RhoA activation is maximal at the cell edge. Using single-molecule imaging and optogentics Rac1 activation we solved this apparent contradiction. We revealed a strong correlation between Rac1 activation and transient immobilizations at the lamellipodium tip, unlike RhoA. Furthermore, we demonstrated that Rac1 must be activated at the lamellipodium tip and not away from it to stimulate protrusion. Thus, fast cycling between activation and inhibition at the proximity of Rac1 targets ensures a local and fast control over Rac1 actions.\n\nAbbreviations

cell biology↗

bioRxiv2017

Gradients Of Rac1 Nanoclusters Support Spatial Patterns Of Rac1 Signaling

The dynamics of the cytoskeleton and cell shape relies on the coordinated activation of RhoGTPase molecular switches. Among them, Rac1 participates to the orchestration in space and time of actin branching and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single molecule imaging and super-resolution microscopy, we reveal an additional supramolecular organization of Rac1. We find that, similarly to H-Ras, Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble due to the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs, GAPs, downstream effectors, and possibly other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Using optogenetics and micropatterning tools, we find that activation of Rac1 leads to its immobilization in nanoclusters and that the local level of Rac1 activity matches the local density of nanoclusters. Altogether, our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms. This finding implies that graded distributions of nanoclusters might encode spatial information.\n\nSignificance statementThe plasma membrane of eukaryotic cells is a highly organized surface where hundreds of incoming signals are transduced to the intracellular space. How cells encode faithfully this myriad of signals is a fundamental question. Here we show that Rac1, a critical membrane-bound protein involved in the regulation of cytoskeletal dynamics, forms small aggregates together with other regulating proteins. These supramolecular assemblies, called nanoclusters, are the \"quantal\" units of signaling. By increasing the local concentration, nanoclusters set thresholds for downstream signaling and ensure the fidelity of information transduction. We found that Rac1 nanoclusters are distributed as spatial gradients matching the patterns of Rac1 activity. We propose that cells can encode positional information through distributed signaling quanta, hereby ensuring spatial fidelity.

biophysics↗