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Mechanical forces drive and modulate a wide variety of processes in eukaryotic cells including those occurring in the nucleus. Relevantly, forces are fundamental during development since they guide lineage specifications of embryonic stem cells. A sophisticated macromolecular machinery transduces mechanical stimuli received at the cell surface into a biochemical output; a key component in this mechanical communication is the cytoskeleton, a complex network of biofilaments in constant remodeling that links the cell membrane to the nuclear envelope. Recent evidence highlights that forces transmitted through the cytoskeleton directly affect the organization of chromatin and the accessibility of transcription-related molecules to their targets in the DNA. Consequently, mechanical forces can directly modulate transcription and change gene expression programs. Here, we will revise the biophysical toolbox involved in the mechanical communication with the cell nucleus and discuss how mechanical forces impact on the organization of this organelle and more specifically, on transcription. We will also discuss how live-cell fluorescence imaging is producing exquisite information to understand the mechanical response of cells and to quantify the landscape of interactions of transcription factors with chromatin in embryonic stem cells. These studies are building new biophysical insights that could be fundamental to achieve the goal of manipulating forces to guide cell differentiation in culture systems.
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The interactions between mitochondria and the cytoskeleton have been found to alter mitochondrial function; however, the mechanisms underlying this phenomenon are largely unknown. Here, we explored how the integrity of the cytoskeleton affects the cellular organization, morphology and mobility of mitochondria in Xenopus laevis melanocytes. Cells were imaged in control condition and after different treatments that selectively affect specific cytoskeletal networks (microtubules, F-actin and vimentin filaments). We observed that mitochondria cellular distribution and local orientation rely mostly on microtubules, positioning these filaments as the main scaffolding of mitochondrial organization. We also found that cytoskeletal networks mold mitochondria shapes in distinct ways: while microtubules favor more elongated organelles, vimentin and actin filaments increase mitochondrial bending, suggesting the presence of mechanical interactions between these filaments and mitochondria. Finally, we identified that microtubule and F-actin networks play opposite roles in mitochondria shape fluctuations and mobility, with microtubules transmitting their jittering to the organelles and F-actin restricting the organelles motion. All our results support that cytoskeleton filaments interact mechanically with mitochondria and transmit forces to these organelles molding their movements and shapes.
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Actinas , Citoesqueleto , Citoesqueleto de Actina , Filamentos Intermediários , Microtúbulos , Vimentina , AnimaisRESUMO
The study of the active forces acting on semiflexible filaments networks such as the cytoskeleton requires noninvasive tools able to explore the deformation of single filaments in their natural environment. We propose here a practical method based on the solution of the hydrodynamic beam equation in the presence of transverse forces. We found that the derivative of the local curvature presents discontinuities that match the location of the applied forces, in contrast to the smooth curvature function obtained for the case of compressing longitudinal forces. These patterns can be easily appreciated in a kymograph of the curvature, which also reflects the temporal behavior of the forces. We assessed the method performance with numerical simulations describing the deformation of single microtubules provoked by the action of intracellular active forces.
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DYRK1A triplication in Down's Syndrome (DS) and its overexpression in Alzheimer's Disease (AD) suggest a role for increased DYR1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of AD, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live-imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speeds transitions suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in DS and AD.Significance StatementAxonal transport defects are early events in the progression of neurodegenerative diseases such as Alzheimer's Disease (AD). However, the molecular mechanisms underlying transport defects remain elusive. DYRK1A kinase is triplicated in Down's Syndrome and overexpressed in AD, suggesting that DYRK1A dysfunction affects molecular pathways leading to early-onset neurodegeneration. Here, we show by live imaging of human-derived neurons that DYRK1A activity differentially regulates the intracellular trafficking of the amyloid precursor protein (APP). Further, single particle analysis revealed DYRK1A as a modulator of axonal transport and the configuration of active motors within the APP vesicle. Our work highlights DYRK1A as a regulator of APP axonal transport and metabolism; supporting DYRK1A inhibition as a therapeutic strategy to restore intracellular dynamics in AD.
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Uncovering the link between mitochondrial morphology, dynamics, positioning and function is challenging. Mitochondria are very flexible organelles that are subject to tension and compression within cells. Recent findings highlighted the importance of these mechanical aspects in the regulation of mitochondria dynamics, arising the question on which are the processes and mechanisms involved in their shape remodeling. In this work we explored in detail the morphological changes and spatio-temporal fluctuations of these organelles in livingXenopus laevismelanophores, a well-characterized cellular model. We developed an automatic method for the classification of mitochondria shapes based on the analysis of the curvature of the contour shape from confocal microscopy images. A persistence length of 2.1µm was measured, quantifying, for the first time, the bending plasticity of mitochondria in their cellular environment. The shape evolution at the single organelle level was followed during a few minutes revealing that mitochondria can bend and unbend in the seconds timescale. Furthermore, the inspection of confocal movies simultaneously registering fluorescent mitochondria and microtubules suggests that the cytoskeleton network architecture and dynamics play a significant role in mitochondria shape remodeling and fluctuations. For instance changes from sinuous to elongated organelles related to transitions from confined behavior to fast directed motion along microtubule tracks were observed.
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Citoesqueleto , Microtúbulos , Citoesqueleto/metabolismo , Microscopia Confocal , Microtúbulos/metabolismo , Mitocôndrias/fisiologia , OrganelasRESUMO
Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate tumor cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodial growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodial length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.
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Comunicação Celular , Forminas/química , Miosinas/química , Pseudópodes/fisiologia , Actinas , Algoritmos , Movimento Celular , Simulação por Computador , Citoplasma/metabolismo , Difusão , Humanos , Microscopia Confocal , Células PC-3 , Probabilidade , Transdução de Sinais , Processos EstocásticosRESUMO
The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.
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Citoesqueleto/metabolismo , Filamentos Intermediários/metabolismo , Polímeros/metabolismo , Vimentina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Forma Celular , Cricetinae , Citoesqueleto/ultraestrutura , Filamentos Intermediários/ultraestrutura , Microscopia Confocal , Microtúbulos/metabolismo , Estresse MecânicoRESUMO
Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100â¯ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.
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Cinesinas/metabolismo , Microtúbulos/fisiologia , Organelas/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Drosophila , Fluorescência , Cinética , Microtúbulos/metabolismo , Espectrometria de FluorescênciaRESUMO
Molecular motors play relevant roles on the regulation of mitochondria size and shape, essential properties for the cell homeostasis. In this work, we tracked single rod-shaped mitochondria with nanometer precision to explore the performance of microtubule motor teams during processive anterograde and retrograde transport. We analyzed simultaneously the organelle size and verified that mitochondria retracted during retrograde transport with their leading tip moving slower in comparison with the rear tip. In contrast, mitochondria preserved their size during anterograde runs indicating a different performance of plus-end directed teams. These results were interpreted considering the different performance of dynein and kinesin teams and provide valuable information on the collective action of motors during mitochondria transport.
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Homeostase/genética , Microtúbulos/genética , Mitocôndrias/genética , Forma das Organelas/genética , Animais , Dineínas/genética , Cinesinas/genética , Microtúbulos/metabolismo , Análise de Célula Única , Xenopus laevis/genéticaRESUMO
The indiscriminate use of herbal medicines to prevent or to heal diseases or even the use for questionable purposes such as weight loss has received both interest and scrutiny from the scientific community and general public alike. An increasing number of women put their own and the unborn child's health at risk due to a lack of knowledge about the phytochemical properties and adequate use of herbal medicine (phytomedicines or herbal supplements) and lack of communication with their healthcare provider. The purpose of this narrative review was to summarize the use of herbal medicines during pregnancy and their potential toxic effects to highlight the importance of caution when prescribing herbal medicines or supplements for women, because, in addition to suffering interactions and a great amount of information obtained in preclinical predictive studies, assessment of nephrotoxicity, neurotoxicity, hepatotoxicity, genotoxicity, and teratogenicity of traditional medicinal herbs still remains scarce in the clinical setting.
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Medicina Herbária , Fitoterapia , Gravidez , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Feminino , Medicina Herbária/métodos , Humanos , Fitoterapia/efeitos adversos , Fitoterapia/métodos , Plantas Medicinais/efeitos adversos , Plantas Medicinais/fisiologia , Complicações na Gravidez/induzido quimicamente , Complicações na Gravidez/epidemiologia , Fatores de Risco , Saúde da MulherRESUMO
Neurons rely on complex axonal transport mechanisms that mediate the intracellular dynamics of proteins, vesicles, and mitochondria along their high polarized structure. The fast improvement of live imaging techniques of fluorescent cargos allowed the identification of the diverse motion properties of different transported molecules. These properties arise as the result of molecular interactions between many players involved in axonal transport. Motor proteins, microtubule tracks, cargo association, and even axonal viscosity contribute to the proper axonal dynamics of different cargos. The unique properties in each cargo determine their distribution and location that is relevant to ensure neuronal cell activity and survival. This chapter provides a computational-based method for the generation of cargo trajectories and the identification of different motion regimes while cargo moves along axons. Then, the procedure to extract relevant parameters from active, diffusive, and confined motion is provided. These properties will allow a better comprehension of the nature and characteristics of cargo motion in living cells, therefore contributing to understanding the consequences of transport defects that arise during diseases of the nervous system.
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Axônios/metabolismo , Biologia Computacional/métodos , Neurônios/citologia , Animais , Transporte Axonal , Humanos , Camundongos , Imagem Molecular , Neurônios/metabolismo , Ratos , SoftwareRESUMO
Mesoporous oxide films are attractive frameworks in technological areas such as catalysis, sensing, environmental protection, and photovoltaics. Herein, we used fluorescence correlation spectroscopy to explore how the pore dimensions of hydrated TiO2 mesoporous calcined films modulate the molecular diffusion. Rhodamine B molecules in mesoporous films follow a Fickian process 2-3 orders slower compared to the probe in water. The mobility increases with the pore and neck radii reaching an approximately constant value for a neck radius >2.8 nm. However, the pore size does not control the dye diffusion at low ionic strength emphasizing the relevance of the probe interactions with the pore walls on dye mobility. In conclusion, our results show that the thermal conditioning of TiO2 mesoporous films provides an exceptional tool for controlling the pore and neck radii on the nanometer scale and has a major impact on molecular diffusion within the mesoporous network.
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BACKGROUND: Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. METHODS: We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-ß-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. RESULTS AND CONCLUSIONS: The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. GENERAL SIGNIFICANCE: Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport.
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Microtúbulos/fisiologia , Peroxissomos/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Drosophila , Fluidez de Membrana , beta-Ciclodextrinas/farmacologiaRESUMO
The distribution of the transcription machinery among different sub-nuclear domains raises the question on how the architecture of the nucleus modulates the transcriptional response. Here, we used fluorescence fluctuation analyses to quantitatively explore the organization of the glucocorticoid receptor (GR) in the interphase nucleus of living cells. We found that this ligand-activated transcription factor diffuses within the nucleus and dynamically interacts with bodies enriched in the coregulator NCoA-2, DNA-dependent foci and chromatin targets. The distribution of the receptor among the nuclear compartments depends on NCoA-2 and the conformation of the receptor as assessed with synthetic ligands and GR mutants with impaired transcriptional abilities. Our results suggest that the partition of the receptor in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets, and thus has an impact on transcription regulation.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Coativador 2 de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Sítios de Ligação , Núcleo Celular/genética , Células Cultivadas , Cromatina/genética , Humanos , Coativador 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Glucocorticoides/genética , Ativação TranscricionalRESUMO
Microtubules are filamentous biopolymers involved in essential biological processes. They form key structures in eukaryotic cells, and thus it is very important to determine the mechanisms involved in the formation and maintenance of the microtubule network. Microtubule bucklings are transient and localized events commonly observed in living cells and characterized by a fast bending and its posterior relaxation. Active forces provided by molecular motors have been indicated as responsible for most of these rapid deformations. However, the factors that control the shape amplitude and the time scales of the rising and release stages remain unexplored. In this work, we study microtubule buckling in living cells using Xenopus laevis melanophores as a model system. We tracked single fluorescent microtubules from high temporal resolution (0.3-2 s) confocal movies. We recovered the center coordinates of the filaments with 10-nm precision and analyzed the amplitude of the deformation as a function of time. Using numerical simulations, we explored different force mechanisms resulting in microtubule bending. The simulated events reproduce many features observed for microtubules, suggesting that a mechanistic model captures the essential processes underlying microtubule buckling. Also, we studied the interplay between actively transported vesicles and the microtubule network using a two-color technique. Our results suggest that microtubules may affect transport indirectly besides serving as tracks of motor-driven organelles. For example, they could obstruct organelles at microtubule intersections or push them during filament mechanical relaxation.
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Fenômenos Mecânicos , Microtúbulos/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Sobrevivência Celular , Modelos Biológicos , Movimento , Xenopus laevisRESUMO
Tau, as a microtubule (MT)-associated protein, participates in key neuronal functions such as the regulation of MT dynamics, axonal transport, and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) MT binding repeats. Although tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated with the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here, we addressed this issue using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content, neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectory analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Conversely, the imbalance toward the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoform imbalances with APP abnormal metabolism in neurodegenerative processes. SIGNIFICANCE STATEMENT: The tau protein has a relevant role in the transport of cargos throughout neurons. Dysfunction in tau metabolism underlies several neurological disorders leading to dementia. In the adult human brain, two tau isoforms are found in equal amounts, whereas changes in such equilibrium have been associated with neurodegenerative diseases. We investigated the role of tau in human neurons in culture and found that perturbations in the endogenous balance of tau isoforms were sufficient to impair the transport of the Alzheimer's disease-related amyloid precursor protein (APP), although neuronal morphology was normal. Our results provide evidence of a direct relationship between tau isoform imbalance and defects in axonal transport, which induce an abnormal APP metabolism with important implications in neurodegeneration.
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Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/fisiologia , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Neurônios/ultraestrutura , Isoformas de Proteínas , Tauopatias/metabolismoRESUMO
Fluorescence fluctuation-based methods are non-invasive microscopy tools especially suited for the study of dynamical aspects of biological processes. These methods examine spontaneous intensity fluctuations produced by fluorescent molecules moving through the small, femtoliter-sized observation volume defined in confocal and multiphoton microscopes. The quantitative analysis of the intensity trace provides information on the processes producing the fluctuations that include diffusion, binding interactions, chemical reactions and photophysical phenomena. In this review, we present the basic principles of the most widespread fluctuation-based methods, discuss their implementation in standard confocal microscopes and briefly revise some examples of their applications to address relevant questions in living cells. The ultimate goal of these methods in the Cell Biology field is to observe biomolecules as they move, interact with targets and perform their biological action in the natural context. © 2016 IUBMB Life, 69(1):8-15, 2017.
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Células/ultraestrutura , Citoplasma/ultraestrutura , Microscopia de Fluorescência , Imagem Molecular/métodos , Transporte Biológico , FluorescênciaRESUMO
Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.
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Comunicação Celular/genética , Redes Reguladoras de Genes , Heme Oxigenase-1/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Pseudópodes/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Cristalografia por Raios X , Meios de Cultivo Condicionados/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Ligação Proteica/efeitos dos fármacos , Proteômica , Pseudópodes/efeitos dos fármacos , Análise de Sequência de RNA , Espectrometria de Massas em Tandem , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
In the present paper, the avoidance behavior of pedestrians was characterized by controlled experiments. Several conflict situations were studied considering different flow rates and group sizes in crossing and head-on configurations. Pedestrians were recorded from above, and individual two-dimensional trajectories of their displacement were recovered after image processing. Lateral swaying amplitude and step lengths were measured for free pedestrians, obtaining similar values to the ones reported in the literature. Minimum avoidance distances were computed in two-pedestrian experiments. In the case of one pedestrian dodging an arrested one, the avoidance distance did not depend on the relative orientation of the still pedestrian with respect to the direction of motion of the first. When both pedestrians were moving, the avoidance distance in a perpendicular encounter was longer than the one obtained during a head-on approach. It was found that the mean curvature of the trajectories was linearly anticorrelated with the mean speed. Furthermore, two common avoidance maneuvers, stopping and steering, were defined from the analysis of the acceleration and curvature in single trajectories. Interestingly, it was more probable to observe steering events than stopping ones, also the probability of simultaneous steering and stopping occurrences was negligible. The results obtained in this paper can be used to validate and calibrate pedestrian dynamics models.