Biology subjects

Dehapiot, B.

Publications and source records attributed to Dehapiot, B..

8 recordsLinked to original sources

bioRxiv2023

Theory of sarcomere assembly inferred from sequential ordering of myofibril components

Myofibrils in striated muscle cells are chains of regular cytoskeletal units termed sarcomeres, whose contractions drive voluntary movements of animals. Despite the well characterized order of the sarcomere components in mature sarcomeres, which explains the sarcomere contraction mechanism, the mechanism of molecular ordering during sarcomere assembly remains debated. Here, we put forward a theoretical framework for the self-assembly of sarcomeres. This theory is based on measurements of the sequential ordering of sarcomere components in developing Drosophila flight muscles, identified by applying a novel tracking-free algorithm: myosin, -actinin and the titin homologue Sallimus form periodic patterns before actin. Based on these results, we propose that myosin, Sallimus, and sarcomere Z-disc proteins including -actinin dynamically bind and unbind to an unordered bundle of actin filaments to establish an initial periodic pattern. As a consequence, periodicity of actin filaments is only established later. Our model proposes that non-local interactions between spatially extended myosin and titin/Sallimus containing complexes, and possibly tension-dependent feedback mediated by an -actinin catch-bond, drive this ordering process. We probe this hypothesis using mathematical models and derive predictive conditions for sarcomere pattern formation, guiding future experimental analysis.

biophysics↗

bioRxiv2023

Physical mechanisms of red blood cell splenic filtration

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 {micro}m RBCs pass through 0.3 {micro}m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with sub-micron wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 {micro}m-wide rigid slits at 37{degrees}C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28 % of RBCs in a 0.4 {micro}m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a -1 and -3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a 2D Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery. Significance StatementSplenic filtration of red blood cells through narrow interendothelial slits remains poorly understood despite its physiological significance as experiments and imaging of red cells passing through the slits are lacking. Here, we coupled live imaging, biomimetic submicron-fluidics, and multiscale modeling to quantify passage conditions. Remarkably, healthy 8-{micro}m cells can pass through 0.28-{micro}m slits at body temperature. This event is conditioned to cells being able to deform into two tether-connected equal spheres and, in limiting cases, to unfold their spectrin cytoskeleton. We showed that cells behave like a Newtonian fluid and that their dynamics is controlled by the inner fluid viscosity. We thus propose an in-vitro and in-silico approach to quantify splenic clearance of diseased cells and cells engineered for transfusion and drug delivery.

biophysics↗

bioRxiv2022

MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In mouse oocytes arrested at metaphase II, eccentric spindle positioning is associated with a chromatin-induced remodeling of the overlying cortex, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation was demonstrated, the role of ring myosin II, and its mechanism of activation, have remained elusive. Here, we show that ring myosin II activation requires Myotonic dystrophy kinase-Related Cdc42-binding Kinase (MRCK), downstream of polarized Cdc42. During anaphase-II, inhibition of MRCK resulted in spindle rotation defects and a decreased rate of polar body emission. Remarkably, some oocytes eventually achieved spindle rotation by disengaging one cluster of chromatids from the anaphase spindle. We show that the MRCK/myosin II pathway also regulates the flattening of the fertilization cone to initiate male pronucleus centration. These findings provide novel insights into mammalian oocyte polarization and the role of cortical myosin II in orchestrating asymmetric division.

cell biology↗

bioRxiv2021

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

developmental biology↗

bioRxiv2021

Cofilin regulates actin network homeostasis and microvilli length in mouse oocytes

How multiple actin networks coexist in a common cytoplasm, while competing for a shared pool of monomers, is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner at meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread microvilli elongation, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes, and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.

cell biology↗

bioRxiv2020

RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and two small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before the anaphase occurs. In metaphase II arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until the fertilization triggers the sister chromatids segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable the cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that the spindle rotation results from two antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling and second, an outward attraction exerted on both lots of chromatids by a RanGTP dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modelling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the lot of chromatids that eventually gets discarded are biologically predetermined.

developmental biology↗

bioRxiv2019

Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Tissue remodeling during embryogenesis is driven by the apical contractility of the epithelial cell cortex. This behavior arises notably from Rho1/Rok induced transient accumulation of non-muscle myosin II (MyoII pulses) pulling on actin filaments (F-Actin) of the medio-apical cortex. While recent studies begin to highlight the mechanisms governing the emergence of Rho1/Rok/MyoII pulsatility in different organisms, little is known about how the F-Actin organization influences this process. Focusing on Drosophila ectodermal cells during germband extension and amnioserosa cells during dorsal closure, we show that the medio-apical actomyosin cortex consists of two entangled F-Actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1 dependent manner. The other forms a persistent and homogeneous network independent of Rho1. We identify the Frl/Fmnl formin as a critical nucleator of the persistent network since modulating its level, in mutants or by overexpression, decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue scale morphogenetic defects. Our work sheds new lights on how the F-Actin cortex offers multiple levels of regulation to affect epithelial cells dynamics.

developmental biology↗

bioRxiv2017

Viscoelastic dissipation stabilizes cell shape changes during tissue morphogenesis

Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here we use Myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from Myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by Myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Our results argue that active junctional deformation is stabilized by dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.

developmental biology↗