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1.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34812842

RESUMO

Epithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.


Assuntos
Junções Aderentes/metabolismo , Citoesqueleto/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Células CACO-2 , Proteínas de Ligação a DNA , Cães , Forminas/metabolismo , Humanos , Células Madin Darby de Rim Canino , Modelos Biológicos , Quinase de Cadeia Leve de Miosina/metabolismo , Quinases Associadas a rho/metabolismo
2.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119139, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34624436

RESUMO

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca2+ ([Ca2+]c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca2+]c, whereas pharmacological inhibition of these channels reduced Ca2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.


Assuntos
Sinalização do Cálcio , Movimento Celular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trofoblastos/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Hormônios Peptídicos/farmacologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/fisiologia
3.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119161, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655689

RESUMO

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Nuclear/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Embrião de Galinha , Proteínas Intrinsicamente Desordenadas/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membrana Nuclear/ultraestrutura , Proteoma/genética , Proteoma/metabolismo , Peixe-Zebra
4.
Cells ; 10(12)2021 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-34944085

RESUMO

Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei-mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei-mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Lactobacillus casei/fisiologia , Pressão Osmótica , Junções Íntimas/patologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Células CACO-2 , Receptores ErbB/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pressão Osmótica/efeitos dos fármacos , Proteína Quinase C/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
5.
Cells ; 10(12)2021 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-34944092

RESUMO

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.


Assuntos
Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/metabolismo , Análise de Célula Única , Transcriptoma/genética , Citoesqueleto de Actina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Humanos , Modelos Biológicos , RNA-Seq
6.
PLoS One ; 16(12): e0252845, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34962917

RESUMO

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.


Assuntos
Proteínas Contráteis/metabolismo , Citocinese , Embrião não Mamífero/metabolismo , Miosina Tipo II/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/metabolismo , Septinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Anticorpos/metabolismo , Proteínas Contráteis/química , Proteínas Contráteis/imunologia , Imageamento Tridimensional , Domínios Proteicos , Septinas/imunologia , Proteínas rho de Ligação ao GTP/metabolismo
7.
Exp Cell Res ; 408(1): 112853, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34597679

RESUMO

Docetaxel could inhibit the proliferation of tumor cells by targeting microtubules. The extension of cellular microtubules plays an important role in the invasion and metastasis of tumor cells. This paper aims to study the distribution and mechanical properties of cytoskeletal proteins with low concentration of docetaxel. MTT assay was used to detect the minimum drug activity concentration of docetaxel on SKOV-3 cells, fluorescence staining was used to analyze the distribution of cytoskeleton proteins, scanning electron microscopy(SEM) was used to observe the morphology of single cells, and atomic force microscopy(AFM) was used to determine the microstructure and mechanical properties of cells. The results showed that the IC10 of docetaxel was 1 ng/ml. Docetaxel can effectively inhibit the formation of cell pseudopodia, hinder the indirectness between cells, reduce the cell extension area, and make the cells malformed. In addition, when AFM analyzes the effects of drugs on cell microstructure and mechanical properties, the average cell surface roughness and cell height are positively correlated with the concentration of docetaxel. Especially when the concentration was 100 ng/ml, the adhesion decreased by 37.04% and Young's modulus increased by 1.57 times compared with the control group. This may be because docetaxel leads to microtubule remodeling and membrane protein aggregation, which affects cell microstructure and increases cell strength, leading to significant changes in the mechanical properties of ovarian cells. This is of great significance to the study of the formation mechanism of tumor cell invasion and migration activities mediated by actin.


Assuntos
Citoesqueleto/efeitos dos fármacos , Docetaxel/farmacologia , Microtúbulos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Humanos , Microtúbulos/metabolismo , Neoplasias Ovarianas/metabolismo
8.
FASEB J ; 35(11): e21983, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34662453

RESUMO

FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.


Assuntos
Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/farmacologia , Citoesqueleto de Actina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Podócitos/citologia , Ratos , Ratos Wistar
9.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638705

RESUMO

A cell should deal with the changing external environment or the neighboring cells. Inevitably, the cell surface receives and transduces a number of signals to produce apt responses. Typically, cell surface receptors are activated, and during this process, the subplasmalemmal actin cytoskeleton is often rearranged. An intriguing point is that some signaling enzymes and ion channels are physically associated with the actin cytoskeleton, raising the possibility that the subtle changes of the local actin cytoskeleton can, in turn, modulate the activities of these proteins. In this study, we reviewed the early and new experimental evidence supporting the notion of actin-regulated enzyme and ion channel activities in various cell types including the cells of immune response, neurons, oocytes, hepatocytes, and epithelial cells, with a special emphasis on the Ca2+ signaling pathway that depends on the synthesis of inositol 1,4,5-trisphosphate. Some of the features that are commonly found in diverse cells from a wide spectrum of the animal species suggest that fine-tuning of the activities of the enzymes and ion channels by the actin cytoskeleton may be an important strategy to inhibit or enhance the function of these signaling proteins.


Assuntos
Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Canais Iônicos/metabolismo , Animais , Humanos
10.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502558

RESUMO

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Germinação/efeitos dos fármacos , Hordeum/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Cloreto de Sódio/farmacologia , Vacúolos/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Endosperma/citologia , Endosperma/metabolismo , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Proteômica/métodos , Sementes/citologia , Sementes/metabolismo , Vacúolos/metabolismo
11.
Nat Commun ; 12(1): 5329, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504078

RESUMO

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Melanócitos/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/química , Actinas/genética , Animais , Sítios de Ligação , Bovinos , Citoesqueleto/ultraestrutura , Gelsolina/química , Gelsolina/genética , Gelsolina/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Melanócitos/citologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Modelos Moleculares , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citologia , Timo/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
12.
Biophys J ; 120(20): 4399-4417, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34509503

RESUMO

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.


Assuntos
Citoesqueleto de Actina , Actinas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Cinética , Polimerização
13.
FASEB J ; 35(10): e21925, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34569663

RESUMO

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Espermatogênese , Testículo/citologia , Testículo/metabolismo , Animais , Núcleo Celular/metabolismo , Hidrazinas/metabolismo , Indazóis/metabolismo , Masculino , Modelos Animais , Ratos , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Testículo/química
14.
Elife ; 102021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569933

RESUMO

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.


Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Imagem Individual de Molécula/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Músculo Estriado/metabolismo , Ligação Proteica , Processos Estocásticos , Troponina/metabolismo
15.
Int J Mol Sci ; 22(18)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34575851

RESUMO

Type 1 tunneling nanotubes (TNTs-1) are long, cytoplasmic protrusions containing actin, microtubules and intermediate filaments that provide a bi-directional road for the transport of various components between distant cells. TNT-1 formation is accompanied by dramatic cytoskeletal reorganization offering mechanical support for intercellular communication. Although the centrosome is the major microtubule nucleating center and also a signaling hub, the relationship between the centrosome and TNTs-1 is still unexplored. We provide here the first evidence of centrosome localization and orientation towards the TNTs-1 protrusion site, which is implicated in TNT-1 formation. We also envision a model whereby synchronized reorientation of the Golgi apparatus along with the centrosome towards TNTs-1 ensures effective polarized trafficking through TNTs-1. Furthermore, using immunohistochemistry and live imaging, we observed for the first time the movement of an extra centrosome within TNTs-1. In this regard, we hypothesize a novel role for TNTs-1 as a critical pathway serving to displace extra centrosomes and potentially to either protect malignant cells against aberrant centrosome amplification or contribute to altering cells in the tumor environment. Indeed, we have observed the increase in binucleation and proliferation markers in receiving cells. The fact that the centrosome can be both as the base and the user of TNTs-1 offers new perspectives and new opportunities to follow in order to improve our knowledge of the pathophysiological mechanisms under TNT control.


Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte Biológico , Biomarcadores , Linhagem Celular , Núcleo Celular/genética , Transformação Celular Neoplásica , Imunofluorescência , Humanos , Imuno-Histoquímica , Modelos Biológicos
16.
Dev Cell ; 56(18): 2579-2591.e4, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34525342

RESUMO

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. Here, we report that PtdIns(3,4,5)P3 is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP3, and disruption of PIP3 reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP3 enrichments are developmentally regulated, with mesodermal cells having high apical PIP3 while germband cells have higher interfacial PIP3. Finally, we show that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Our results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues.


Assuntos
Actomiosina/metabolismo , Forma Celular/fisiologia , Células Epiteliais/metabolismo , Morfogênese/fisiologia , Fosfatidilinositóis/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo
17.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502098

RESUMO

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.


Assuntos
Adesão Celular , Lamina Tipo A/metabolismo , Miofibroblastos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Lamina Tipo A/genética , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Nanotubos/química , Pinças Ópticas , Ratos , Ratos Sprague-Dawley
18.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502296

RESUMO

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.


Assuntos
Acetilcolina/metabolismo , Microtúbulos/fisiologia , Mioblastos/fisiologia , Junção Neuromuscular/fisiologia , Neuropeptídeos/farmacologia , Receptores Colinérgicos/metabolismo , Sinapses/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Camundongos , Microtúbulos/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Receptores Colinérgicos/genética , Transmissão Sináptica
19.
Dev Cell ; 56(17): 2419-2426.e4, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34473942

RESUMO

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes.


Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Dev Cell ; 56(17): 2486-2500.e6, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34480876

RESUMO

During cytokinesis, animal cells rapidly remodel the equatorial cortex to build an aligned array of actin filaments called the contractile ring. Local reorientation of filaments by active equatorial compression is thought to underlie the emergence of filament alignment during ring assembly. Here, combining single molecule analysis and modeling in one-cell C. elegans embryos, we show that filaments turnover is far too fast for reorientation of individual filaments by equatorial compression to explain the observed alignment, even if favorably oriented filaments are selectively stabilized. By tracking single formin/CYK-1::GFP particles to monitor local filament assembly, we identify a mechanism that we call filament-guided filament assembly (FGFA), in which existing filaments serve as templates to orient the growth of new filaments. FGFA sharply increases the effective lifetime of filament orientation, providing structural memory that allows cells to build highly aligned filament arrays in response to equatorial compression, despite rapid turnover of individual filaments.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
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