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1.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
2.
Nat Commun ; 11(1): 4828, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973141

RESUMO

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismo
3.
Nat Commun ; 11(1): 4640, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934232

RESUMO

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.


Assuntos
Microtúbulos/química , Paclitaxel/química , Linhagem Celular Tumoral , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paclitaxel/farmacologia
4.
Medicine (Baltimore) ; 99(39): e22172, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32991410

RESUMO

Osteoporosis is a severe chronic skeletal disorder that increases the risks of disability and mortality; however, the mechanism of this disease and the protein markers for prognosis of osteoporosis have not been well characterized. This study aims to characterize the imbalanced serum proteostasis, the disturbed pathways, and potential serum markers in osteoporosis by using a set of bioinformatic analyses. In the present study, the large-scale proteomics datasets (PXD006464) were adopted from the Proteome Xchange database and processed with MaxQuant. The differentially expressed serum proteins were identified. The biological process and molecular function were analyzed. The protein-protein interactions and subnetwork modules were constructed. The signaling pathways were enriched. We identified 209 upregulated and 230 downregulated serum proteins. The bioinformatic analyses revealed a highly overlapped functional protein classification and the gene ontology terms between the upregulated and downregulated protein groups. Protein-protein interactions and pathway analyses showed a high enrichment in protein synthesis, inflammation, and immune response in the upregulated proteins, and cell adhesion and cytoskeleton regulation in the downregulated proteins. Our findings greatly expand the current view of the roles of serum proteins in osteoporosis and shed light on the understanding of its underlying mechanisms and the discovery of serum proteins as potential markers for the prognosis of osteoporosis.


Assuntos
Mineração de Dados/métodos , Osteoporose/sangue , Proteoma/fisiologia , Biomarcadores , Adesão Celular/fisiologia , Biologia Computacional , Citoesqueleto/metabolismo , Regulação para Baixo , Humanos , Mediadores da Inflamação/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteômica , Regulação para Cima
5.
Phys Rev Lett ; 125(7): 078101, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32857554

RESUMO

The friction between cytoskeletal filaments is of central importance for the formation of cellular structures such as the mitotic spindle and the cytokinetic ring. This friction is caused by passive cross-linkers, yet the underlying mechanism and the dependence on cross-linker density are poorly understood. Here, we use theory and computer simulations to study the friction between two filaments that are cross-linked by passive proteins, which can hop between discrete binding sites while physically excluding each other. The simulations reveal that filaments move via rare discrete jumps, which are associated with free-energy barrier crossings. We identify the reaction coordinate that governs the relative microtubule movement and derive an exact analytical expression for the free-energy barrier and the friction coefficient. Our analysis not only elucidates the molecular mechanism underlying cross-linker-induced filament friction, but also predicts that the friction coefficient scales superexponentially with the density of cross-linkers.


Assuntos
Citoesqueleto/química , Citoesqueleto/fisiologia , Modelos Biológicos , Modelos Químicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Sítios de Ligação , Citoesqueleto/metabolismo , Fricção , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Termodinâmica
6.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
7.
Nat Commun ; 11(1): 4112, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807784

RESUMO

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.


Assuntos
Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Fagócitos/metabolismo , Pinocitose/genética , Pinocitose/fisiologia , Receptores Imunológicos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo
8.
PLoS One ; 15(8): e0237116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857787

RESUMO

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.


Assuntos
Calcificação Fisiológica , Adesão Celular , Neoplasias da Próstata/metabolismo , Animais , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Colágeno/metabolismo , Citoesqueleto/metabolismo , Dentina/química , Elefantes , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Tecidos Suporte/química , Vitamina D/metabolismo
9.
Nat Commun ; 11(1): 3457, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651364

RESUMO

Glioblastoma is a deadly cancer, with no effective therapies. Better understanding and identification of selective targets are urgently needed. We found that advillin (AVIL) is overexpressed in all the glioblastomas we tested including glioblastoma stem/initiating cells, but hardly detectable in non-neoplastic astrocytes, neural stem cells or normal brain. Glioma patients with increased AVIL expression have a worse prognosis. Silencing AVIL nearly eradicated glioblastoma cells in culture, and dramatically inhibited in vivo xenografts in mice, but had no effect on normal control cells. Conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes, supporting AVIL being a bona fide oncogene. We provide evidence that the tumorigenic effect of AVIL is partly mediated by FOXM1, which regulates LIN28B, whose expression also correlates with clinical prognosis. AVIL regulates the cytoskeleton through modulating F-actin, while mutants disrupting F-actin binding are defective in its tumorigenic capabilities.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas dos Microfilamentos/metabolismo , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citoesqueleto/metabolismo , Imunofluorescência , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
10.
Nat Commun ; 11(1): 3495, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661310

RESUMO

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.


Assuntos
Actinas/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Biologia Celular , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Organelas , Filogenia , Proteínas rab27 de Ligação ao GTP/genética
11.
Am J Physiol Heart Circ Physiol ; 319(2): H410-H421, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32648820

RESUMO

Cardiac t tubules undergo significant remodeling in various pathological and experimental conditions, which can be associated with mechanical or osmotic stress. In particular, it has been shown that removal of hyposmotic stress can lead to sealing of t tubules. However, the mechanisms underlying the sealing process remain essentially unknown. In this study we used dextran trapping assay to demonstrate that in adult mouse cardiomyocytes, t-tubular sealing can also be induced by hyperosmotic challenge and that both hypo- and hyperosmotic sealing display a clear threshold behavior requiring ≈100 mosmol/L minimal stress. Importantly, during both hypo- and hyperosmotic challenges, the sealing of t tubules occurs only during the shrinking phase. Analysis of the time course of t-tubular remodeling following removal of hyposmotic stress shows that t tubules become sealed essentially instantly, well before any significant reduction in cell size can be observed. Overall, the data support the hypothesis that the critical event in the process of t-tubular sealing during osmotic challenges is detachment (peeling) of the membrane from the underlying cytoskeleton due to suprathreshold stress.NEW & NOTEWORTHY This study provides new insights into how t-tubular membranes respond to osmotic forces. In particular, the data show that osmotically induced sealing of cardiac t tubules is a threshold phenomenon initiated by detachment of t-tubular membrane from the underlying cytoskeleton. The findings are consistent with the hypothesis that final sealing of t tubules is driven by negative hydrostatic intracellular pressure coincident with cell shrinking.


Assuntos
Membrana Celular/patologia , Tamanho Celular , Citoesqueleto/patologia , Miócitos Cardíacos/patologia , Pressão Osmótica , Vacúolos/patologia , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fatores de Tempo , Vacúolos/metabolismo
12.
Braz J Med Biol Res ; 53(7): e9207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520207

RESUMO

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.


Assuntos
Citoesqueleto/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Inativação Gênica , Vetores Genéticos , Humanos , Camundongos , Células RAW 264.7 , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transfecção
13.
Nat Commun ; 11(1): 3068, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555155

RESUMO

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.


Assuntos
Cálcio/química , Epitélio/metabolismo , Aderências Teciduais/metabolismo , Animais , Sinalização do Cálcio , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , Citoesqueleto/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias , Análise de Componente Principal , RNA Interferente Pequeno/metabolismo , Análise de Célula Única
14.
J Vis Exp ; (160)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32597842

RESUMO

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.


Assuntos
Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/citologia , Automação , Contagem de Células , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos
15.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510485

RESUMO

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.


Assuntos
Arabidopsis/citologia , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microtúbulos/metabolismo , Imagem Molecular , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Sobrevivência Celular , Parede Celular/metabolismo , Celulose/metabolismo , Meristema/citologia , Meristema/crescimento & desenvolvimento , Morfogênese
16.
Am J Physiol Cell Physiol ; 319(2): C300-C312, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520607

RESUMO

Skeletal muscle is a target of contraction-induced loading (CiL), leading to protein unfolding or cellular perturbations, respectively. While cytoskeletal desmin is responsible for ongoing structural stabilization, in the immediate response to CiL, alpha-crystallin B (CRYAB) is phosphorylated at serine 59 (pCRYABS59) by P38, acutely protecting the cytoskeleton. To reveal adaptation and deadaptation of these myofibrillar subsystems to CiL, we examined CRYAB, P38, and desmin regulation following resistance exercise at diverse time points of a chronic training period. Mechanosensitive JNK phosphorylation (pJNKT183/Y185) was determined to indicate the presence of mechanical components in CiL. Within 6 wk, subjects performed 13 resistance exercise bouts at the 8-12 repetition maximum, followed by 10 days detraining and a final 14th bout. Biopsies were taken at baseline and after the 1st, 3rd, 7th, 10th, 13th, and 14th bout. To assess whether potential desensitization to CiL can be mitigated, one group trained with progressive and a second with constant loading. As no group differences were found, all subjects were combined for statistics. Total and phosphorylated P38 was not regulated over the time course. pCRYABS59 and pJNKT183/Y185 strongly increased following the unaccustomed first bout. This exercise-induced pCRYABS59/pJNKT183/Y185 increase disappeared with the 10th until 13th bout. As response to the detraining period, the 14th bout led to a renewed increase in pCRYABS59. Desmin content followed pCRYABS59 inversely, i.e., was up- when pCRYABS59 was downregulated and vice versa. In conclusion, the pCRYABS59 response indicates increase and decrease in resistance to CiL, in which a reinforced desmin network could play an essential role by structurally stabilizing the cells.


Assuntos
Adaptação Fisiológica/genética , Desmina/genética , Músculo Esquelético/metabolismo , Cadeia B de alfa-Cristalina/genética , Adulto , Citoesqueleto/genética , Citoesqueleto/metabolismo , Desmina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fosforilação/genética , Treinamento de Resistência/efeitos adversos , Adulto Jovem , Cadeia B de alfa-Cristalina/metabolismo
17.
Chem Biol Interact ; 325: 109109, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376239

RESUMO

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 µM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 µM vs. >100 µM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs.


Assuntos
Apoptose/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Flavonas/farmacologia , Gardenia/química , Melanoma/patologia , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Flavonas/química , Flavonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(21): 11257-11264, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404423

RESUMO

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Citoesqueleto/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos
19.
Proc Natl Acad Sci U S A ; 117(23): 12817-12825, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444491

RESUMO

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.


Assuntos
Citoesqueleto/metabolismo , Módulo de Elasticidade , Mecanotransdução Celular , Tecidos Suporte/química , Actinina/metabolismo , Polaridade Celular , Reagentes para Ligações Cruzadas/química , Citoesqueleto/ultraestrutura , Fibroblastos/metabolismo , Humanos , Modelos Teóricos , Miosinas/metabolismo
20.
PLoS Pathog ; 16(5): e1008106, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463830

RESUMO

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.


Assuntos
Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Parasitos/metabolismo , Transporte Proteico , Proteínas de Protozoários , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
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