RESUMO
Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.
Assuntos
Microscopia de Fluorescência/métodos , Miosina Tipo V/metabolismo , Pontos Quânticos , Actinas/metabolismo , Polarização de FluorescênciaRESUMO
Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC(ex)). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC(ex) was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.
Assuntos
Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Insulina/farmacologia , Glicogênio Hepático/biossíntese , Animais , Linhagem Celular Tumoral , Simulação por Computador , Retroalimentação Fisiológica , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/metabolismo , Insulina/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Myosin-V processively walks on actin filaments in a hand-over-hand fashion. The identical structures of the heads predict a symmetric hand-over-hand mechanism where regular, unidirectional rotation occurs during a 36-nm step. We investigated this by observing how fixed myosin-V rotates actin filaments. Actin filaments randomly rotated 90 degrees both clockwise and counter-clockwise during each step. Furthermore, ATP-dependent rotations were regularly followed by ATP-independent ones. Kinetic analysis indicated that the two 90 degrees rotations relate to the coordinated unbinding and rebinding of the heads with actin. We propose a 'brownian rotation hand-over-hand' model, in which myosin-V randomly rotates by thermally twisting its elastic neck domains during the 36-nm step. The brownian rotation may be advantageous for cargo transport through a crowded actin meshwork and for carrying cargoes reliably via multiple myosin-V molecules in the cell.
Assuntos
Actinas , Miosina Tipo V , Actinas/metabolismo , Actinas/ultraestrutura , Trifosfato de Adenosina/metabolismo , Humanos , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Miosina Tipo V/metabolismo , Miosina Tipo V/ultraestrutura , Ligação Proteica , RotaçãoRESUMO
Therapeutic monoclonal antibodies (mAbs) have been a promising therapeutic approach for several diseases and a wide variety of mAbs are being evaluated in clinical trials. To accelerate clinical development and improve the probability of success, pharmacokinetics and pharmacodynamics (PKPD) in humans must be predicted before clinical trials can begin. Traditionally, empirical-approach-based PKPD prediction has been applied for a long time. Recently, modeling and simulation (M&S) methods have also become valuable for quantitatively predicting PKPD in humans. Although several models (e.g., the compartment model, Michaelis-Menten model, target-mediated drug disposition model, and physiologically based pharmacokinetic model) have been established and used to predict the PKPD of mAbs in humans, more complex mechanistic models, such as the quantitative systemics pharmacology model, have been recently developed. This review summarizes the recent advances and future direction of M&S-based approaches to the quantitative prediction of human PKPD for mAbs.
RESUMO
Homeostatic control of blood glucose is regulated by a complex feedback loop between glucose and insulin, of which failure leads to diabetes mellitus. However, physiological and pathological nature of the feedback loop is not fully understood. We made a mathematical model of the feedback loop between glucose and insulin using time course of blood glucose and insulin during consecutive hyperglycemic and hyperinsulinemic-euglycemic clamps in 113 subjects with variety of glucose tolerance including normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). We analyzed the correlation of the parameters in the model with the progression of glucose intolerance and the conserved relationship between parameters. The model parameters of insulin sensitivity and insulin secretion significantly declined from NGT to IGT, and from IGT to T2DM, respectively, consistent with previous clinical observations. Importantly, insulin clearance, an insulin degradation rate, significantly declined from NGT, IGT to T2DM along the progression of glucose intolerance in the mathematical model. Insulin clearance was positively correlated with a product of insulin sensitivity and secretion assessed by the clamp analysis or determined with the mathematical model. Insulin clearance was correlated negatively with postprandial glucose at 2h after oral glucose tolerance test. We also inferred a square-law between the rate constant of insulin clearance and a product of rate constants of insulin sensitivity and secretion in the model, which is also conserved among NGT, IGT and T2DM subjects. Insulin clearance shows a conserved relationship with the capacity of glucose disposal among the NGT, IGT and T2DM subjects. The decrease of insulin clearance predicts the progression of glucose intolerance.
Assuntos
Intolerância à Glucose/fisiopatologia , Glucose/metabolismo , Insulina/metabolismo , Adulto , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Progressão da Doença , Jejum/metabolismo , Jejum/fisiologia , Feminino , Técnica Clamp de Glucose/métodos , Intolerância à Glucose/metabolismo , Teste de Tolerância a Glucose/métodos , Humanos , Resistência à Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial/fisiologiaRESUMO
Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.
Assuntos
Insulina/fisiologia , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Regulação Alostérica , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Redes e Vias Metabólicas , Metaboloma , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Transdução de SinaisRESUMO
Cells use common signaling molecules for the selective control of downstream gene expression and cell-fate decisions. The relationship between signaling molecules and downstream gene expression and cellular phenotypes is a multiple-input and multiple-output (MIMO) system and is difficult to understand due to its complexity. For example, it has been reported that, in PC12 cells, different types of growth factors activate MAP kinases (MAPKs) including ERK, JNK, and p38, and CREB, for selective protein expression of immediate early genes (IEGs) such as c-FOS, c-JUN, EGR1, JUNB, and FOSB, leading to cell differentiation, proliferation and cell death; however, how multiple-inputs such as MAPKs and CREB regulate multiple-outputs such as expression of the IEGs and cellular phenotypes remains unclear. To address this issue, we employed a statistical method called partial least squares (PLS) regression, which involves a reduction of the dimensionality of the inputs and outputs into latent variables and a linear regression between these latent variables. We measured 1,200 data points for MAPKs and CREB as the inputs and 1,900 data points for IEGs and cellular phenotypes as the outputs, and we constructed the PLS model from these data. The PLS model highlighted the complexity of the MIMO system and growth factor-specific input-output relationships of cell-fate decisions in PC12 cells. Furthermore, to reduce the complexity, we applied a backward elimination method to the PLS regression, in which 60 input variables were reduced to 5 variables, including the phosphorylation of ERK at 10 min, CREB at 5 min and 60 min, AKT at 5 min and JNK at 30 min. The simple PLS model with only 5 input variables demonstrated a predictive ability comparable to that of the full PLS model. The 5 input variables effectively extracted the growth factor-specific simple relationships within the MIMO system in cell-fate decisions in PC12 cells.
Assuntos
Fenômenos Fisiológicos Celulares , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Biológicos , Transdução de Sinais , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Análise dos Mínimos Quadrados , Células PC12 , Ratos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
Robust transmission of information despite the presence of variation is a fundamental problem in cellular functions. However, the capability and characteristics of information transmission in signaling pathways remain poorly understood. We describe robustness and compensation of information transmission of signaling pathways at the cell population level. We calculated the mutual information transmitted through signaling pathways for the growth factor-mediated gene expression. Growth factors appeared to carry only information sufficient for a binary decision. Information transmission was generally more robust than average signal intensity despite pharmacological perturbations, and compensation of information transmission occurred. Information transmission to the biological output of neurite extension appeared robust. Cells may use information entropy as information so that messages can be robustly transmitted despite variation in molecular activities among individual cells.
Assuntos
Teoria da Informação , Transdução de Sinais , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células PC12 , Proteínas Proto-Oncogênicas c-fos/metabolismo , RatosRESUMO
We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , XenopusRESUMO
Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.