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1.
Proc Natl Acad Sci U S A ; 120(44): e2300095120, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37874856

RESUMO

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 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]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 submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °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 [Formula: see text]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 [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional 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.


Assuntos
Eritrócitos , Baço , Eritrócitos/metabolismo , Citoesqueleto , Microfluídica , Espectrina/metabolismo
2.
PLoS Biol ; 19(9): e3001376, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34491981

RESUMO

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II-arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable 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 spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, 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 set of chromatids that eventually gets discarded are biologically predetermined.


Assuntos
Segregação de Cromossomos , Meiose , Oócitos/citologia , Fuso Acromático , Actinas/metabolismo , Animais , Feminino , Camundongos , Proteína cdc42 de Ligação ao GTP , Proteína rhoA de Ligação ao GTP
3.
Brain ; 146(10): 4247-4261, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37082944

RESUMO

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.


Assuntos
Bumetanida , Inibidores de Simportadores de Cloreto de Sódio e Potássio , Camundongos , Animais , Bumetanida/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Microglia/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Parvalbuminas/metabolismo , Parvalbuminas/farmacologia , Membro 2 da Família 12 de Carreador de Soluto , Interneurônios/metabolismo , Neurogênese
4.
J Cell Sci ; 134(24)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34841429

RESUMO

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 upon meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread elongation of microvilli, 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.


Assuntos
Fatores de Despolimerização de Actina , Actinas , Fatores de Despolimerização de Actina/genética , Animais , Homeostase , Meiose , Camundongos , Microvilosidades , Oócitos , Fuso Acromático
5.
Proc Natl Acad Sci U S A ; 109(29): 11705-10, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22753521

RESUMO

In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells.


Assuntos
Actinas/metabolismo , Citoplasma/metabolismo , Meiose/fisiologia , Xenopus/fisiologia , Animais , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Cinética , Microscopia de Fluorescência , Xenopus/metabolismo
6.
Dev Biol ; 377(1): 202-12, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23384564

RESUMO

Asymmetric meiotic divisions in mammalian oocytes rely on the eccentric positioning of the spindle and the remodeling of the overlying cortex, resulting in the formation of small polar bodies. The mechanism of this cortical polarization, exemplified by the formation of a thick F-actin cap, is poorly understood. Cdc42 is a major player in cell polarization in many systems; however, the spatio-temporal dynamics of Cdc42 activation during oocyte meiosis, and its contribution to mammalian oocyte polarization, have remained elusive. In this study, we investigated Cdc42 activation (Cdc42-GTP), dynamics and role during mouse oocyte meiotic divisions. We show that Cdc42-GTP accumulates in restricted cortical regions overlying meiotic chromosomes or chromatids, in a Ran-GTP-dependent manner. This polarized activation of Cdc42 is required for the recruitment of N-WASP and the formation of F-actin-rich protrusions during polar body formation. Cdc42 inhibition in MII oocytes resulted in the release of N-WASP into the cytosol, a loss of the polarized F-actin cap, and a failure to protrude the second polar body. Cdc42 inhibition also resulted in central spindle defects in activated MII oocytes. In contrast, emission of the first polar body during oocyte maturation could occur in the absence of a functional Cdc42/N-WASP pathway. Therefore, Cdc42 is a new protagonist in chromatin-induced cortical polarization in mammalian oocytes, with an essential role in meiosis II completion, through the recruitment and activation of N-WASP, downstream of the chromatin-centered Ran-GTP gradient.


Assuntos
Divisão Celular Assimétrica , Oócitos/citologia , Oócitos/enzimologia , Corpos Polares/citologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Cromossomos de Mamíferos/metabolismo , Citocinese , Ativação Enzimática , Feminino , Guanosina Trifosfato/metabolismo , Meiose , Camundongos , Transporte Proteico , Transdução de Sinais , Fuso Acromático/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/metabolismo
7.
Dev Cell ; 59(1): 156-172.e7, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38103554

RESUMO

During morphogenesis, mechanical forces induce large-scale deformations; yet, how forces emerge from cellular contractility and adhesion is unclear. In Drosophila embryos, a tissue-scale wave of actomyosin contractility coupled with adhesion to the surrounding vitelline membrane drives polarized tissue invagination. We show that this process emerges subcellularly from the mechanical coupling between myosin II activation and sequential adhesion/de-adhesion to the vitelline membrane. At the wavefront, integrin clusters anchor the actin cortex to the vitelline membrane and promote activation of myosin II, which in turn enhances adhesion in a positive feedback. Following cell detachment, cortex contraction and advective flow amplify myosin II. Prolonged contact with the vitelline membrane prolongs the integrin-myosin II feedback, increases integrin adhesion, and thus slows down cell detachment and wave propagation. The angle of cell detachment depends on adhesion strength and sets the tensile forces required for detachment. Thus, we document how the interplay between subcellular mechanochemical feedback and geometry drives tissue morphogenesis.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Integrinas/metabolismo , Morfogênese/fisiologia
8.
J Cell Biol ; 222(11)2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37651121

RESUMO

Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, 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 is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.


Assuntos
Actinas , Miosina Tipo II , Oócitos , Proteínas Serina-Treonina Quinases , Polos do Fuso , Animais , Masculino , Camundongos , Citoesqueleto de Actina , Proteínas do Citoesqueleto , Miosina Tipo II/metabolismo , Rotação , Feminino , Proteínas Serina-Treonina Quinases/metabolismo , Polos do Fuso/metabolismo , Anáfase
9.
Elife ; 122023 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-36913486

RESUMO

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term 'meisosomes,' in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.


Assuntos
Proteínas de Caenorhabditis elegans , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epiderme/metabolismo , Células Epidérmicas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Matriz Extracelular/metabolismo
10.
Elife ; 112022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35920628

RESUMO

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. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and 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.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Miofibrilas/fisiologia , Sarcômeros
11.
Nat Cell Biol ; 22(7): 791-802, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483386

RESUMO

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. Here, we show that the medioapical 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 formin Frl (also known as Fmnl) 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.


Assuntos
Citoesqueleto de Actina/fisiologia , Drosophila melanogaster/metabolismo , Células Epiteliais/patologia , Forminas/fisiologia , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Polaridade Celular , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Morfogênese , Miosina Tipo II/genética , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética
12.
Curr Biol ; 27(20): 3132-3142.e4, 2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-28988857

RESUMO

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. Finally, we show that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. Our results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Animais , Fenômenos Biomecânicos , Forma Celular , Drosophila melanogaster/citologia
13.
Cell Cycle ; 12(11): 1672-8, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23656777

RESUMO

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Cromatina/metabolismo , Meiose , Camundongos , Mutação , Oócitos/metabolismo , Fosforilação , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
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