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.

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.

No abstract

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.

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.quantum rods | single molecule imaging S ingle molecule fluorescence imaging techniques are being increasingly used to observe the dynamic properties of single molecules like spatial orientation, which provides information on a protein's three-dimensional (3D) motility and its conformational changes (1, 2). These techniques can be grouped into two different classes: intensity distribution techniques and interference pattern techniques. In the first class, 3D orientation is determined by comparing fluorescence intensities among several excitation or detection polarizations. For example, in single-molecule fluorescence polarization microscopy (3), the dye is excited by multiple polarized beams incident from different directions. The resulting emission is split with respect to its polarization and detected with avalanche photodiodes, which allows for sensitivity to the 3D orientation of a single dye's transition dipole moments. The second class takes advantage of single molecule emission patterns created by defocusing. Defocused imaging reveals additional structures in single-molecule emission patterns that depend on the orientation of the emitting dipole (4-7). The 3D orientation is obtained by comparing the defocused images with corresponding calculated model images. This technique can be used to determine both the position and orientation of the dye. However, because the defocused image is spread over a greater number of pixels, the image inherently has poor position accuracy. Furthermore, in most orientation-determination techniques, time consuming fitting...

The regulation of glucose metabolism by pulse stimulations of insulin is compared with the effect of ramp stimulations. Specific network motifs mediate the differential response to these temporal patterns of stimulations that mimic in vivo patterns of insulin secretion.

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.

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.