Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Peering inside lipid rafts and caveolae

Clustering of proteins into membrane microdomains, such as lipid rafts and caveolae, could act as a mechanism for regulating cell signaling and other cellular functions. Certain lipid modifications are hypothesized to target proteins to these domains on the cytoplasmic leaflet of the plasma membrane. This concept has now been tested in living cells using an assay sensitive to the lateral distribution of proteins in membranes over sub-micron distances.     

A role of microtubules during the formation of cell processes in neuronal and non-neuronal cells

This review discusses the role of microtubules in the formation of processes from neuronal and non-neuronal cells. In elongating axons of the neuron, tubulin molecules are transported toward the end of pre-existing microtubules, which may be nucleated at the centrosome, via a mechanism called slow axonal flow. Two different hypotheses are presented to explain this mechanism; the transport of soluble monomers and/or oligomers versus the transport of polymerized microtubules. The majority of tubulin seems to be transported as small oligomers as shown by the data presented so far. Alternatively, an active transport of polymerized microtubules driven by microtubule-based motor proteins is postulated as being responsible for the non-uniform polarity of microtubule bundles in dendrites of the neuron. Microtubule-associated proteins (MAPs) play a crucial role in stabilizing the microtubular arrays, whereas the non-uniform polarity of microtubules may be established with the aid of microtubule-based motor proteins. The signals activating centrosomal proteins and MAPs, resulting in process formation, include phosphorylation and dephosphorylation of these proteins. Not only neuronal cells, but also renal glomerular podocytes develop prominent cell processes equipped with well-organized microtubular cytoskeletons, and intermediate and actin filaments. A novel cell culture system for podocytes, in which process formation can be induced, should provide further evidence that microtubules play a pivotal role in process formation of non-neuronal cells.     

In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein

BackgroundThe structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells.MethodsSingle-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET.ResultssmFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples.ConclusionsCytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF.General significanceStructural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.     

Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments

The study of protein--protein interactions is central to understanding the chemical machinery that makes up the living cell. Until recently, facile methods to study these processes in intact, living cells have not existed. Furthermore, the assignment of function to novel proteins relies on demonstrating interactions of these proteins with proteins of known function. This review describes an experimental strategy, devised to study protein--protein interactions in any intact living cells based on protein-fragment complementation assays. Applications to quantitative analysis of interactions, allosteric processes and cDNA library screening are discussed. Recently, the feasibility of employing this strategy in genome-wide biochemical pathway mapping efforts has been demonstrated.     

细胞核内蛋白质动态变化研究进展

漂白后荧光恢复和漂白荧光丢失技术是蛋白质动态变化研究中常用的两项技术.近年来,利用这两项技术对细胞核内蛋白质动态变化的研究表明：一些蛋白质在细胞核内是运动的,能和各自所在的区域快速结合和解离;并且这种运动主要以被动扩散的方式进行,不消耗代谢的能量;另外蛋白质的共价修饰可对某些蛋白质的运动产生影响.细胞核内蛋白质的动态变化对细胞核的结构组成和基因表达的调控都具有重要的意义,但详细的机制还有待于进一步的研究.     

Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins

Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of proteins in living cells. Although the technique is relatively old, its application to studying endogenous intracellular proteins in living cells is relatively recent and is a consequence of the newly developed fluorescent protein-based living cell protein tags. This is particularly true for nuclear proteins, in which endogenous protein mobility has only recently been studied. Here we examine the experimental design and analysis of FRAP experiments. Mathematical modeling of FRAP data enables the experimentalist to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion coefficient of the molecule under study. As experimentalists begin to dissect the relative influence of protein domains within individual proteins, this approach will allow a quantitative assessment of the relative influences of different molecular interactions on the steady-state distribution and protein function in vivo.     

Biogenic isoprene emission as expression of dissipativity,a fundamental cell property

Energy dynamics of isoprene biosynthesis and the mechanism of isoprene emission are discussed in view of their fundamental role in dissipativity of living cells. The significance of basic principles of colloidal chemistry for biological energy conversion is emphasized. The idea is put forward of the existence in living cells of the universal energy-dynamic structural unit, termed “biological micelle,” that accounts for the transport and distribution of protons over the cell volume. This unit is responsible for the creation and maintenance of physiological pH at any metabolically active site within the cell. Particular attention is paid to the involvement of F-type ATPase in the active proton transport from the thylakoid interior to the F1 domain of ATP-synthase and to recycling of protons from the outer cell surface to the thylakoid lumen due to H+-pumping activity of the thylakoid ATPase. The mechanism responsible for the outflow of entropy deS through the production of isoprene by protonation of dimethylallyl pyrophosphate (DMAPP) has been found. The stable steady-state condition of any thermodynamic system, including the living system, is correlated with the maximum entropy production. The rate of isoprene emission increases with temperature, which compensates for the decrease in outflow of thermal entropy deS. When the ambient temperature is increased, the sum of deS removed as heat and deS removed with isoprene emission remains constant. Thus, photobiosynthesis of isoprene is a special case of the entropy deS dissipation that provides a stable stationary state to the cell.     

A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.     

Finding one of a kind: advances in single-protein production

An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies.     

Degradation of target protein in living cells by small-molecule proteolysis inducer

Ubiquitin-dependent proteolysis of cellular proteins is one of the major pathways to regulate protein function posttranslationally. Here we demonstrate a potentially general method of degrading any targeted proteins by the ubiquitin-dependent proteolysis in living cells, using small-molecule proteolysis inducer (SMPI).     

Group normalization for genomic data

Data normalization is a crucial preliminary step in analyzing genomic datasets. The goal of normalization is to remove global variation to make readings across different experiments comparable. In addition, most genomic loci have non-uniform sensitivity to any given assay because of variation in local sequence properties. In microarray experiments, this non-uniform sensitivity is due to different DNA hybridization and cross-hybridization efficiencies, known as the probe effect. In this paper we introduce a new scheme, called Group Normalization (GN), to remove both global and local biases in one integrated step, whereby we determine the normalized probe signal by finding a set of reference probes with similar responses. Compared to conventional normalization methods such as Quantile normalization and physically motivated probe effect models, our proposed method is general in the sense that it does not require the assumption that the underlying signal distribution be identical for the treatment and control, and is flexible enough to correct for nonlinear and higher order probe effects. The Group Normalization algorithm is computationally efficient and easy to implement. We also describe a variant of the Group Normalization algorithm, called Cross Normalization, which efficiently amplifies biologically relevant differences between any two genomic datasets.     

Spatial pattern formation of prey-predator populations

Summary Formation of a non-uniform spatial distribution pattern of prey and predator populations in a heterogeneous environment is mathematically investigated. Both populations are distributed in linearly connected compartments. Furthermore it is assumed that only the predator species (animal) can randomly diffuse across the boundaries but the prey species (plant) are confined in each compartment. When the prey-predator relation is given by a simple Volterra type interaction it is known that the system cannot establish a non-uniform stationary distribution in a homogeneous environment. However, in a heterogeneous environment, it can be analytically shown by constructing a Lyapunov function that the system asymptotically and globally tends to a non-uniform stationary distribution. Thus, the populations are stabilized by the heterogeneity of environment.     

Determinants of histone H1 mobility and chromatin binding in living cells

The dynamic interaction of chromatin-binding proteins with their nucleosome binding sites is an important element in regulating the structure and function of chromatin in living cells. Here we review the major factors regulating the intranuclear mobility and chromatin binding of the linker histone H1, the most abundant family of nucleosome-binding proteins. The information available reveals that multiple and diverse factors modulate the interaction of H1 with chromatin at both a local and global level. This multifaceted mode of modulating the interaction of H1 with nucleosomes is part of the mechanism that regulates the dynamics of the chromatin fiber in living cells.     

Intra-troop spacing mechanism of the wild Japanese monkeys of the Koshima troop

The average frequencies of communicative behavior, social behavior, and social encounters (inter-individual proximity within three meters) per hour for a monkey were obtained in their natural habitat by tracing several adult males and females of a Japanese monkey troop living in the Koshima islet. The spatial distribution patterns and the density of troop members within the expanse of the troop at any moment were investigated by tracing several adult femals. Frequency distributions of the monkeys found within five and 10 meters were compared with a Poisson distribution. The frequencies of social encounters and of social interactions of Japanese monkeys were distinctly low, except between mothers and their offspring. The density of monkeys within the expanse of the troop at any moment was very low. Both aggressive behavior and inter-individual proximity (within three meters) were distinctly low when monkeys were foraging for natural food. An avoiding mechanism among troop members plays an important role in maintaining the social structure of these Japanese monkeys. This mechanism works in two ways: each individual does not approach others too closely; the density of monkeys within the expanse of the troop is low at all times.     

Effect of short-range forces on the length distribution of fibrous cytoskeletal proteins

The length distribution of cytoskeletal filaments is an important physical parameter, which can modulate physiological cell functions. In both eukaryotic and prokaryotic cells various biological cytoskeletal polymers form supramolecular structures due to short-range forces induced mainly by molecular crowding or cross linking proteins, but their in vivo length distribution remains difficult to measure. In general, based on experimental evidence and mathematical modeling of actin filaments in aqueous solutions, the steady state length distribution of fibrous proteins is believed to be exponential. We performed in vitro TIRF- and electron-microscopy to demonstrate that in the presence of short-range forces, which are an integral part of any living cell, the steady state length distributions of the eukaryotic cytoskeletal biopolymer actin, its prokaryotic homolog ParM and microtubule homolog FtsZ deviate from the classical exponential and are either double-exponential or Gaussian, as recent theoretical modeling predicts. Double exponential or Gaussian distributions opposed to exponential can change for example the visco-elastic properties of actin networks within the cell, influence cell motility by decreasing the amount of free ends at the leading edge of the cell or effect the assembly of FtsZ into the bacterial Z-ring thus modulating membrane constriction.     

Structural basis of activation and GTP hydrolysis in Rab proteins

BACKGROUND: Rab proteins comprise a large family of GTPases that regulate vesicle trafficking. Despite conservation of critical residues involved in nucleotide binding and hydrolysis, Rab proteins exhibit low sequence identity with other GTPases, and the structural basis for Rab function remains poorly characterized. RESULTS: The 2. 0 A crystal structure of GppNHp-bound Rab3A reveals the structural determinants that stabilize the active conformation and regulate GTPase activity. The active conformation is stabilized by extensive hydrophobic contacts between the switch I and switch II regions. Serine residues in the phosphate-binding loop (P loop) and switch I region mediate unexpected interactions with the gamma phosphate of GTP that have not been observed in previous GTPase structures. Residues implicated in the interaction with effectors and regulatory factors map to a common face of the protein. The electrostatic potential at the surface of Rab3A indicates a non-uniform distribution of charged and nonpolar residues. CONCLUSIONS: The major structural determinants of the active conformation involve residues that are conserved throughout the Rab family, indicating a common mode of activation. Novel interactions with the gamma phosphate impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the large variation of GTPase activity within the Rab family. An asymmetric distribution of charged and nonpolar residues suggests a plausible orientation with respect to vesicle membranes, positioning predominantly hydrophobic surfaces for interaction with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab GTPases.