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Heterochromatin is a nuclear area that contains highly condensed and transcriptionally inactive chromatin. Alterations in the organization of heterochromatin are correlated with changes in gene expression and genome stability, which affect various aspects of plant life. Thus, studies of the molecular mechanisms that regulate heterochromatin organization are important for understanding the regulation of plant physiology. Microscopically, heterochromatin can be characterized as chromocenters that are intensely stained with DNA-binding fluorescent dyes. Arabidopsis thaliana exhibits distinctive chromocenters in interphase nuclei, and genetic studies combined with cytological analyses have identified a number of factors that are involved in heterochromatin assembly and organization. In this review, I will summarize the factors involved in the regulation of heterochromatin organization in plants.
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Fertility is declining worldwide and many couples are turning towards assisted reproductive technologies (ART) to conceive babies. Organisms that propagate via sexual reproduction often come from the fusion between two gametes, an oocyte and a sperm, whose qualities seem to be decreasing in the human species. Interestingly, while the sperm mostly transmits its haploid genome, the oocyte transmits not only its haploid set of chromosomes but also its huge cytoplasm to its progeny. This is what can be defined as the maternal inheritance composed of chromosomes, organelles, lipids, metabolites, proteins and RNAs. To decipher the decline in oocyte quality, it is essential to explore the nature of the maternal inheritance, and therefore study the last stages of murine oogenesis, namely the end of oocyte growth followed by the two meiotic divisions. These divisions are extremely asymmetric in terms of the size of the daughter cells, allowing to preserve the maternal inheritance accumulated during oocyte growth within these huge cells to support early embryo development. Studies performed in Marie-Hélène Verlhac's lab have allowed to discover the unprecedented impact of original acto-myosin based mechanisms in the constitution as well as the preservation of this maternal inheritance and the consequences when these processes go awry.
La fécondité diminue mondialement et de nombreux couples se tournent vers les techniques de procréation médicalement assistée (PMA) pour concevoir des bébés. Les organismes se propageant par reproduction sexuée sont souvent issus de la fusion de deux gamètes, un ovocyte et un spermatozoïde, dont les qualités semblent diminuer dans l'espèce humaine. Si le spermatozoïde transmet principalement son génome haploïde, l'ovocyte transmet à sa progéniture non seulement son lot haploïde de chromosomes, mais aussi son immense cytoplasme. C'est ce que l'on peut définir comme l'héritage maternel, composé de chromosomes, d'organelles, de lipides, de métabolites, de protéines et d'ARNs. Pour comprendre la baisse de qualité des ovocytes, il est essentiel d'explorer la nature de cet héritage maternel, et donc d'étudier les dernières étapes de l'ovogenèse murine, à savoir la fin de la croissance ovocytaire suivie des deux divisions méiotiques. Ces divisions sont extrêmement asymétriques par la taille des cellules filles engendrées, ce qui permet de préserver l'héritage maternel accumulé pendant la croissance de cette énorme cellule, l'ovocyte, pour soutenir le développement précoce de l'embryon. Les études menées dans le laboratoire de Marie-Hélène Verlhac ont permis de découvrir l'impact sans précédent de mécanismes originaux dépendant de l'acto-myosine dans la constitution et la préservation de cet héritage maternel, ainsi que les conséquences des erreurs dans ces processus.
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Herança Materna , Oócitos , Animais , Feminino , Humanos , Camundongos , Herança Materna/genética , Meiose , Oogênese/genéticaRESUMO
In the development of chronic liver disease, the hepatic stellate cell (HSC) plays a pivotal role in increasing intrahepatic vascular resistance (IHVR) and inducing portal hypertension (PH) in cirrhosis. Our research demonstrated that HSC contraction, prompted by angiotensin II (Ang II), significantly contributed to the elevation of type I collagen (COL1A1) expression. This increase was intimately associated with enhanced cell tension and YAP nuclear translocation, mediated through α-smooth muscle actin (α-SMA) expression, microfilaments (MF) polymerization, and stress fibers (SF) assembly. Further investigation revealed that the Rho/ROCK signaling pathway regulated MF polymerization and SF assembly by facilitating the phosphorylation of cofilin and MLC, while Ca2+ chiefly governed SF assembly via MLC. Inhibiting α-SMA-MF-SF assembly changed Ang II-induced cell contraction, YAP nuclear translocation, and COL1A1 expression, findings corroborated in cirrhotic mice models. Overall, our study offers insights into mitigating IHVR and PH through cell mechanics, heralding potential breakthroughs.
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Angiotensina II , Células Estreladas do Fígado , Hipertensão Portal , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Animais , Hipertensão Portal/metabolismo , Hipertensão Portal/patologia , Camundongos , Colágeno Tipo I/metabolismo , Actinas/metabolismo , Proteínas de Sinalização YAP/metabolismo , Masculino , Transdução de Sinais , Camundongos Endogâmicos C57BL , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.
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Miosinas , Células Vegetais , Miosinas/metabolismo , Células Vegetais/metabolismo , Bryopsida/metabolismo , Bryopsida/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/metabolismo , Marchantia/metabolismo , Marchantia/crescimento & desenvolvimento , Desenvolvimento Vegetal/fisiologiaRESUMO
Regenerative medicine aims to identify new research strategies for the repair and restoration of tissues damaged by pathological or accidental events. Mesenchymal stem cells (MSCs) play a key role in regenerative medicine approaches due to their specific properties, such as the high rate of proliferation, the ability to differentiate into several cell lineages, the immunomodulatory potential, and their easy isolation with minimal ethical issues. One of the main goals of regenerative medicine is to modulate, both in vitro and in vivo, the differentiation potential of MSCs to improve their use in the repair of damaged tissues. Over the years, much evidence has been collected about the ability of cytochalasins, a large family of 60 metabolites isolated mainly from fungi, to modulate multiple properties of stem cells (SCs), such as proliferation, migration, and differentiation, by altering the organization of the cyto- and the nucleo-skeleton. In this review, we discussed the ability of two different cytochalasins, cytochalasins D and B, to influence specific SC differentiation programs modulated by several agents (chemical or physical) or intra- and extra-cellular factors, with particular attention to human MSCs (hMSCs).
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Adipogenia , Células-Tronco Mesenquimais , Humanos , Citocalasinas/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Linhagem da CélulaRESUMO
In this epoch, the disposal of multipollutant wastewater inevitably compromises life on Earth. In this study, the inclusion of Bacterial cellulose microfilaments reinforced chitosan adorned with melamine 2D plates creates a unique 3D bead structure for anionic dye removal. The establishment of an imine network between melamine and chitosan, along with the quantity of inter- and intrahydrogen bonds, boosts the specific surface area to 106.68 m2.g-1. Removal efficiency and in-depth comprehension of synthesized adsorbent characteristics were assessed using batch adsorption experiments and characterization methods. Additionally, pH, adsorbent quantity, time, beginning concentration of solution, and temperature were analyzed and optimized as adsorption essential factors. Owing to the profusion of hydroxyl, amine, imine functional groups and aromatic rings, the synthesized adsorbent intimated an astonishing maximum adsorption capacity of 3168 mg.g-1 in Congo red dye removal at pH 5.5. Based on the kinetic evaluation, pseudo-second-order (R2 = 0.999), pseudo-first-order (R2 = 0.964), and Avrami (R2 = 0.986) models were well-fitted with the kinetic results among the seven investigated models. The isothermal study reveals that the adsorption mechanism predominantly follows the Redlich-Peterson (R2 = 0.996), Koble-Carrigan, and Hill isotherm models (R2 = 0.994). The developed semi-natural sorbent suggests high adsorption capacity, which results from its exceptional structure, presenting promising implications for wastewater treatment.
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Quitosana , Poluentes Químicos da Água , Vermelho Congo/química , Quitosana/química , Celulose/química , Adsorção , Cinética , Iminas , Poluentes Químicos da Água/química , Concentração de Íons de HidrogênioRESUMO
Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability. Stomata are gateways of transpiration and gas exchange, the swift adjustment of stomatal aperture has a strong influence on plant drought resistance. Despite intensive investigations of stomatal closure during drought stress in past decades, little is known about how sequential signals are integrated during complete processes. Here, we discovered that the rapid Ca2+ signaling and subsequent abscisic acid (ABA) signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana, while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1) is the molecular switch for this entire process. During the early stage of osmotic shock responses, swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3 (CPK3) and CPK6. The phosphorylation restrained the microfilament binding affinity of SCAB1, which bring about the F-actin disassembly and stomatal closure initiation. As the osmotic stress signal continued, both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly. We further found that ABA signaling is indispensable for these attenuations, which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure. Notably, the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure. Taken together, our results support a model in which SCAB1 works as a molecular switch, and directs the microfilament rearrangement through integrating the sequentially generated Ca2+ and ABA signals during osmotic stress induced stomatal closure.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Pressão Osmótica , Cálcio/metabolismo , Actinas/metabolismo , Ácido Abscísico/metabolismo , Estômatos de Plantas/metabolismo , Plantas/metabolismo , Sinalização do Cálcio , Proteínas dos MicrofilamentosRESUMO
BACKGROUND: With the large-scale production and application of amorphous silica nanoparticles (aSiNPs), its adverse health effects are more worthy of our attention. Our previous research has demonstrated for the first time that aSiNPs induced cytokinesis failure, which resulted in abnormally high incidences of multinucleation in vitro, but the underlying mechanisms remain unclear. Therefore, the purpose of this study was firstly to explore whether aSiNPs induced multinucleation in vivo, and secondly to investigate the underlying mechanism of how aSiNPs caused abnormal cytokinesis and multinucleation. METHODS: Male ICR mice with intratracheal instillation of aSiNPs were used as an experimental model in vivo. Human hepatic cell line (L-02) was introduced for further mechanism study in vitro. RESULTS: In vivo, histopathological results showed that the rate of multinucleation was significantly increased in the liver and lung tissue after aSiNPs treatment. In vitro, immunofluorescence results manifested that aSiNPs directly caused microfilaments aggregation. Following mechanism studies indicated that aSiNPs increased ROS levels. The accumulation of ROS further inhibited the PI3k 110ß/Aurora B pathway, leading to a decrease in the expression of centralspindlin subunits MKLP1 and CYK4 as well as downstream cytokines regulation related proteins Ect2, Cep55, CHMP2A and RhoA. Meanwhile, the particles caused abnormal co-localization of the key mitotic regulatory kinase Aurora B and the centralspindlin complex by inhibiting the PI3k 110ß/Aurora B pathway. PI3K activator IGF increased the phosphorylation level of Aurora B and improved the relative ratio of the centralspindlin cluster. And ROS inhibitors NAC reduced the ratio of multinucleation, alleviated the PI3k 110ß/Aurora B pathway inhibition, and then increased the expression of MKLP1, CYK4 and cytokinesis-related proteins, whilst NAC restored the clustering of the centralspindlin. CONCLUSION: This study demonstrated that aSiNPs led to multinucleation formation both in vivo and in vitro. ASiNPs exposure caused microfilaments aggregation and inhibited the PI3k 110ß/Aurora B pathway through excessive ROS, which then hindered the centralspindlin cluster as well as restrained the expression of centralspindlin subunits and cytokinesis-related proteins, which ultimately resulted in cytokinesis failure and the formation of multinucleation.
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Citocinese , Fígado , Camundongos , Humanos , Animais , Masculino , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio , Citoesqueleto de Actina , Proteínas de Ciclo Celular , CinesinasRESUMO
Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy caused by mutations in the dystrophin gene. We characterized which isoforms of dystrophin were expressed by human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts obtained from control and DMD patients. Distinct dystrophin isoforms were observed; however, highest molecular weight isoform was absent in DMD patients carrying exon deletions or mutations in the dystrophin gene. The loss of the full-length dystrophin isoform in hiPSC-derived cardiac fibroblasts from DMD patients resulted in deficient formation of actin microfilaments and a metabolic switch from mitochondrial oxidation to glycolysis. The DMD hiPSC-derived cardiac fibroblasts exhibited a dysregulated mitochondria network and reduced mitochondrial respiration, with enhanced compensatory glycolysis to sustain cellular ATP production. This metabolic remodeling was associated with an exacerbated myofibroblast phenotype and increased fibroblast activation in response to pro fibrotic challenges. As cardiac fibrosis is a critical pathological feature of the DMD heart, the myofibroblast phenotype induced by the absence of dystrophin may contribute to deterioration in cardiac function. Our study highlights the relationship between cytoskeletal dynamics, metabolism of the cell and myofibroblast differentiation and provides a new mechanism by which inactivation of dystrophin in non-cardiomyocyte cells may increase the severity of cardiopathy.
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Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofina/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Fenótipo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Fibroblastos/metabolismo , Fibrose , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Many structural birth defects occur due to failure of tissue movement and fusion events during embryogenesis. Examples of such birth defects include failure of closure of the neural tube, palate, and ventral body wall. Actomyosin forces play a pivotal role in these closure processes, making proteins that regulate actomyosin dynamics a priority when studying the etiology of structural birth defects. SPECC1L (sperm antigen with calponin homology and coiled-coil domains 1 like) cytoskeletal protein associates with microtubules, filamentous actin, non-muscle myosin II (NMII), as well as membrane-associated components of adherens junctions. Patients with SPECC1L mutations show a range of structural birth defects affecting craniofacial development (hypertelorism, cleft palate), ventral body wall (omphalocele), and internal organs (diaphragmatic hernia, bicornuate uterus). Characterization of mouse models indicates that these syndromic mutations utilize a gain-of-function mechanism to affect intra- and supra-cellular actin organization. Interestingly, SPECC1L deficiency appears to affect the efficiency of tissue dynamics, making it an important cytoskeletal regulator to study tissue movement and fusion events during embryonic development. Here we summarize the SPECC1L-related syndrome mutations, phenotypes of Specc1l mouse models, and cellular functions of SPECC1L that highlight how it may regulate embryonic tissue dynamics.
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Actinas , Actomiosina , Animais , Feminino , Camundongos , Masculino , Actinas/metabolismo , Actomiosina/metabolismo , Sêmen , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismoRESUMO
Functional thin fabric with highly efficient cold protection properties are attracting the great attention of long-term dressing in a cold environment. Herein, a tri-layered bicomponent microfilament composite fabric comprised of a hydrophobic layer of PET/PA@C6 F13 bicomponent microfilament webs, an adhesive layer of LPET/PET fibrous web, and a fluffy-soft layer of PET/Cellulous fibrous web is designed and also successfully been fabricated through a facile process of dipping, combined with thermal belt bonding. The prepared samples exhibit a large resistance to wetting of alcohol, a high hydrostatic pressure of 5530 Pa, and brilliant water slipping properties, owing to the presence of dense micropores ranging from 25.1 to 70.3 µm, as well as to the smooth surface with an arithmetic mean deviation of surface roughness (Sa) ranging from 5.112 to 4.369 µm. Besides, the prepared samples exhibited good water vapor permeability, and a tunable CLO value ranging from 0.569 to 0.920, in addition to the fact that it exhibited a very suitable working temperature range of -5 °C to 15 °C. Additionally, it also showed excellent clothing tailorability including high mechanical strength with a remarkably soft texture and lightweight foldability that suitable for cold outdoor clothing applications.
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Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.
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Sêmen , Transdução de Sinais , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Sêmen/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Espermatogênese/fisiologia , Citoesqueleto de Actina/metabolismo , Barreira Hematotesticular/metabolismo , Mamíferos/metabolismoRESUMO
Spermatogenesis is a complicated process that includes spermatogonia differentiation, spermatocytes meiosis, spermatids spermiogenesis and final release of spermatozoa. Actin-related protein 3 (Arp3) and epidermal growth factor receptor pathway substrate 8 (Eps8) are two actin binding proteins that regulate cell adhesion in seminiferous tubules during mammalian spermatogenesis. However, the functions of these two proteins during spermatogenesis in nonmammalian species, especially Crustacea, are still unknown. Here, we cloned es-Arp3 and es-Eps8 from the testis of Chinese mitten crab Eriocheir sinensis. es-Arp3 and es-Eps8 were located in spermatocytes, spermatids and spermatozoa. Knockdown of es-Arp3 and es-Eps8 in vivo caused morphological changes to seminiferous tubules including delayed spermatozoa release, shedding of germ cells and vacuoles. Filamentous-actin (F-actin) filaments network was disorganized due to deficiency of es-Arp3 and es-Eps8. Accompanying this, four junctional proteins (α-catenin, ß-catenin, pinin and ZO1) displayed abnormal expression levels as well as penetrating biotin signals in seminiferous tubules. We also used the Arp2/3 complex inhibitor CK666 to block es-Arp3 activity and supported es-Arp3 knockdown results. In summary, our study demonstrated for the first time that es-Arp3 and es-Eps8 are important for spermatogenesis via regulating microfilament-mediated cell adhesion in Eriocheir sinensis.
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Barreira Hematotesticular , Espermatogênese , Animais , Masculino , Proteína 3 Relacionada a Actina/metabolismo , Barreira Hematotesticular/metabolismo , Espermatogênese/fisiologia , Testículo , Espermátides , Túbulos Seminíferos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mamíferos/metabolismoRESUMO
Thymosin ß4 (Tß4) is the ß-thymosin (Tßs) with the highest expression level in human cells; it makes up roughly 70-80% of all Tßs in the human body. Combining the mechanism and activity studies of Tß4 in recent years, we provide an overview of the subtle molecular mechanism, pharmacological action, and clinical applications of Tß4. As a G-actin isolator, Tß4 inhibits the polymerization of G-actin by binding to the matching site of G-actin in a 1:1 ratio through conformational and spatial effects. Tß4 can control the threshold concentration of G-actin in the cytoplasm, influence the balance of depolymerization and polymerization of F-actin (also called Tread Milling of F-actin), and subsequently affect cell's various physiological activities, especially motility, development and differentiation. Based on this, Tß4 is known to have a wide range of effects, including regulation of inflammation and tumor metastasis, promotion of angiogenesis, wound healing, regeneration of hair follicles, promotion of the development of the nervous system, and improving bone formation and tooth growth. Tß4 therefore has extensive medicinal applications in many fields, and serves to preserve the kidney, liver, heart, brain, intestine, and other organs, as well as hair loss, skin trauma, cornea repairing, and other conditions. In this review, we focus on the mechanism of action and clinical application of Tß4 for its main biological functions.
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Actinas , Timosina , Humanos , Actinas/genética , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Timosina/farmacologia , Timosina/química , Timosina/metabolismo , CicatrizaçãoRESUMO
Textile scaffolds that are either 2D or 3D with tunable shapes and pore sizes can be made through textile processing (weaving, knitting, braiding, nonwovens) using microfilaments. However, these filaments lack nano-topographical features to improve bone cell adhesion and proliferation. Moreover, the diameter of such filaments should be higher than that used for classical textiles (10−30 µm) to enable adhesion and the efficient spreading of the osteoblast cell (>30 µm diameter). We report, for the first time, the fabrication of biodegradable nanostructured cylindrical PLLA (poly-L-Lactic acid) microfilaments of diameters 100 µm and 230 µm, using a single step melt-spinning process for straightforward integration of nano-scale ridge-like structures oriented in the fiber length direction. Appropriate drawing speed and temperature used during the filament spinning allowed for the creation of instabilities giving rise to nanofibrillar ridges, as observed by AFM (Atomic Force Microscopy). These micro-filaments were hydrophobic, and had reduced crystallinity and mechanical strength, but could still be processed into 2D/3D textile scaffolds of various shapes. Biological tests carried out on the woven scaffolds made from these nano-structured micro filaments showed excellent human bone cell MG 63 adhesion and proliferation, better than on smooth 30 µm- diameter fibers. Elongated filopodia of the osteoblast, intimately anchored to the nano-structured filaments, was observed. The filaments also induced in vitro osteogenic expression, as shown by the expression of osteocalcin and bone sialoprotein after 21 days of culture. This work deals with the fabrication of a new generation of nano-structured micro-filament for use as scaffolds of different shapes suited for bone cell engineering.
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Engenharia Tecidual , Alicerces Teciduais , Adesão Celular , Proliferação de Células , Humanos , Poliésteres/farmacologia , Têxteis , Alicerces Teciduais/químicaRESUMO
Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1-10 µM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate.
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Adipócitos , Células-Tronco , Adipogenia/fisiologia , Tecido Adiposo , Citocalasina B/farmacologia , HumanosRESUMO
The delivery of sperm cells via tip-growing pollen tubes is an innovation of seed plants and shows the importance of pollen tubes for reproduction and their specific growth kinetics. Fast-growing pollen tubes demand an extensive and dynamic vesicular trafficking network to build new cell membrane and wall, to deliver proteins among endomembrane compartments, and also to respond to external stimuli for growth adjustment. In this review, we summarize current findings on endomembrane compartments and vesicular trafficking routes of pollen tubes, comparing and contrasting their features with those of most somatic cells. We discuss the importance of membrane homeostasis, either at the plasma membrane (PM) or between PM-targeted trafficking and vacuolar trafficking, for pollen tube growth. We also provide perspectives to facilitate future studies of vesicular trafficking in pollen tubes.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tubo Polínico , Sementes/metabolismo , Vacúolos/metabolismoRESUMO
Cell and tissue nanomechanics, being inspired by progress in high-resolution physical mapping, has recently burst into biomedical research, discovering not only new characteristics of normal and diseased tissues, but also unveiling previously unknown mechanisms of pathological processes. Some parallels can be drawn between early development and carcinogenesis. Early embryogenesis, up to the blastocyst stage, requires a soft microenvironment and internal mechanical signals induced by the contractility of the cortical actomyosin cytoskeleton, stimulating quick cell divisions. During further development from the blastocyst implantation to placenta formation, decidua stiffness is increased ten-fold when compared to non-pregnant endometrium. Organogenesis is mediated by mechanosignaling inspired by intercellular junction formation with the involvement of mechanotransduction from the extracellular matrix (ECM). Carcinogenesis dramatically changes the mechanical properties of cells and their microenvironment, generally reproducing the structural properties and molecular organization of embryonic tissues, but with a higher stiffness of the ECM and higher cellular softness and fluidity. These changes are associated with the complete rearrangement of the entire tissue skeleton involving the ECM, cytoskeleton, and the nuclear scaffold, all integrated with each other in a joint network. The important changes occur in the cancer stem-cell niche responsible for tumor promotion and metastatic growth. We expect that the promising concept based on the natural selection of cancer cells fixing the most invasive phenotypes and genotypes by reciprocal regulation through ECM-mediated nanomechanical feedback loop can be exploited to create new therapeutic strategies for cancer treatment.
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Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.
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Citoesqueleto de Actina , Actinas , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Lisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Premature meiotic arrest during in vitro maturation (IVM) of porcine oocytes after germinal vesicle breakdown is associated with microfilament degradation. We aimed to clarify (1) if such arrest occurs at the metaphase-I (MI) stage or the oocyte progresses to a so-called diploid metaphase-II (MII) stage and (2) if microfilament degradation is the cause or result of the meiotic arrest. The number and morphology of chromosomes in oocytes showing premature meiotic arrest at 44 h IVM (38 monovalents) was similar to those cultured in the presence of the actin polymerization-inhibitor cytochalasin-B, but different from those of MI-stage (19 bivalents), and MII-stage oocytes (19 monovalents) at 33 and 44 h of IVM, respectively. Immunostaining revealed similar frequencies of microfilament degradation in prematurely arrested and cytochalasin-B-treated oocytes (58.7% and 57.2%, respectively), which were higher (P < 0.05) than those in MI- and MII-stage oocytes (10.6% and 6.8%, respectively). Induction of MI-arrest by nocodazole did not affect microfilament morphology. ATP and mRNA levels of microfilament-related genes in oocytes were similar among all groups. These results suggest that altered microfilament dynamics contribute to the formation of diploid metaphase spindles in oocytes, which fail to reach the MII stage. However, the cause of microfilament degeneration remains unclear.