The Dynamic Behavior Possibilities of Raft-Like Domains in Biological Membranes

A possible scenario of the behavior of a raft-like domain system oscillating near the phase transition point of the Verchulst transition type, when the form of the stationary distribution for the concentration of domains changes stepwise, has been considered. A stationary state of the system is also possible at the indicated phase transition point, as well as fluctuations in the state of the system between the modes of extinction and survival, if the analogy with the Verhulst model is applied. The system behavior is explored in the framework of the stochastic storage model. This model is compared with the Verhulst model of a biological population. Similarities and differences between the models are highlighted. There are no bifurcations and transition to chaos in the domain system. Other features and characteristics of the dynamic behavior and stationary states of the raft-like domain system are considered.     

Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling

Mammalian cell entry (mce) operons, implicated in the entry of mycobacteria into host cells, are present in pathogenic and saprophytic species. It is likely that the genes in these operons have functions other than those required for entry into host cells. Using in silico analysis we have identified domains within the mce operons that might justify their occurrence in saprophytic species like Mycobacterium smegmatis. Our analysis identified in addition to the mce domain, the presence of the Ttg2B and Ttg2C domains, typical of proteins involved in transport. We have also analysed and compared the expression profile between mce operons of Mycobacterium tuberculosis, Mycobacterium bovis and M. smegmatis under different growth conditions. In case of M. smegmatis, each operon presented domain truncation for at least one gene. We observe differential expression among the operons in M. smegmatis growing under different culture conditions. Bacilli growing in nutritionally rich medium with aeration, only the mce4 operon was expressed while during stationary phase of a standing culture, all four mce operons were expressed. In M. bovis, in addition to the absence of the mce3 operon, several protein domains encoded by the other operons were truncated. We detected expression of the mce2 operon in the exponential and stationary growth phase, while the mce1 operon was only expressed in the stationary growth phase. Differential expression of mce operons and their redundancy in the genome of the majority members of mycobacteria are discussed in view of our results.     

Macroscopic domain formation during cooling in the platelet plasma membrane: An issue of low cholesterol content

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 °C, but not at 37 °C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (～ 15 mol%), appears to be crucial for the formation of large domains during cooling.     

Genome-Wide Analysis of Gene Expression in Stationary Phase and Genetic Characterization of Stationary-Phase-Dependent Halocin Gene Expression in the Haloarchaeon Haloferax mediterranei

The stationary phase of microbial growth is a very complex state regulated by various environmental and physiological factors.An intensive study of stationary phase could promote a comprehensive understanding of the complete life cycle of microorganisms,and may provide important insights into their adaptation to harsh and nutrient-depleted conditions.Although the underlying mechanisms have been well-studied in bacteria and yeasts (Herman,2002;Navarro Llorens et al.,2010),less is known about this growth phase in archaea yet.The haloarchaeon Haloferax mediterranei has served as a good model for studying haloarchaeal physiology and metabolism for several decades because of its accelerated growth,remarkable metabolic ability and genomic stability (Han et al.,2012).During stationary phase,H.mediterranei can produce halocin H4 (Cheung et al.,1997),synthesize gas vesicles (J(a)ger et al.,2002),secrete extracellular polysaccharide (Antón et al.,1988) and accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)(Cai et al.,2012).Due to these specific features,we selected H.mediterranei as a model system to investigate the archaeal gene expression and regulation during the stationary phase.     

Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures

Understanding the phase behavior of biological membranes is helped by the study of more simple systems. Model membranes that have as few as 3 components exhibit complex phase behavior that can be well described, providing insight for biological membranes. A number of different studies are in agreement on general findings for some compositional phase diagrams, in particular, those that model the outer leaflet of animal cell plasma membranes. These model mixtures include cholesterol, together with one high-melting lipid and one low-melting lipid. An interesting finding is of two categories of such 3-component mixtures, leading to what we term Type I and Type II compositional phase diagrams. The latter have phase regions of macroscopic coexisting domains of {Lα + Lβ + Lo} and of {Lα + Lo}, with domains resolved under the light microscope. Type I mixtures have the same phase coexistence regions, but the domains seem to be nanoscopic. Type I mixtures are likely to be better models for biological membranes.     

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.     

Bifurcation analysis of a neural network model

This paper describes the analysis of the well known neural network model by Wilson and Cowan. The neural network is modeled by a system of two ordinary differential equations that describe the evolution of average activities of excitatory and inhibitory populations of neurons. We analyze the dependence of the model's behavior on two parameters. The parameter plane is partitioned into regions of equivalent behavior bounded by bifurcation curves, and the representative phase diagram is constructed for each region. This allows us to describe qualitatively the behavior of the model in each region and to predict changes in the model dynamics as parameters are varied. In particular, we show that for some parameter values the system can exhibit long-period oscillations. A new type of dynamical behavior is also found when the system settles down either to a stationary state or to a limit cycle depending on the initial point.     

Expression of a pepT homologue from Bacillus subtilis

Abstract We isolated pepT from Bacillus subtilis , a gene with homology to various tripeptidases from different bacterial sources, pepT is preceded by genes encoding a two component regulatory system. Its expression is activated during stationary phase. In minimal medium this activation is boosted in the presence of phosphate. The response regulator is preceded by putative promoter consensus sequences recognized by the stationary phase specific sigma factors σ ^H, σ ^F, and σ ^G. This is in accordance with the initiation of expression at the beginning of stationary phase. Inactivation of pepT causes no obvious phenotype.     

Identification of YhdA as a regulator of the Escherichia coli carbon storage regulation system

The Escherichia coli BarA-UvrY two-component system, which controls adaptation via the CsrB and CsrC sRNAs, is induced at the entry of the stationary phase by an unknown stimulus. Using a csrB-lacZ fusion, we demonstrated that the factors RelA, SpoT and LuxS, previously suggested to act on orthologues of this system, have no role in BarA-UvrY induction. However, using a transposon screen, we identified the hypothetical protein YhdA as a new regulator of CsrB and CsrC expression. The YhdA protein is predicted to be membrane-bound and to harbor GGDEF and EAL domains, which, however, are highly divergent from the consensus motifs.     

Sorting of streptavidin protein coats on phase-separating model membranes

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.     

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.     

Seeing spots: complex phase behavior in simple membranes

Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes.     

Time-Related Transcriptome Analysis of B. subtilis 168 During Growth with Glucose

Gene expression in Bacillus subtilis from late exponential to stationary phase was monitored by DNA microarrays with samples taken from the culture in LB broth with glucose supplement to prevent sporulation. Three major patterns of gene expression as revealed in this study were consistent to the expression profiling of PerR/Spx regulons and three major sigma factors—SigA, SigB, and SigW. Expression of most SigA-dependent house-keeping genes was significantly decreased and remained at low levels in the stationary phase. The sigB gene and additional genes of the SigB regulon for stress response exhibited a distinct pattern of transient induction with a peak in transition phase. The majority of induced genes after cessation of SigB-dependent surge were subjected to regulation by SigW, PerR, and Spx in response to oxidative stress. No induction of spo0A and skfA regulons supports the suppression of sporulation and cannibalism processes in the stationary phase by glucose supplement. In summary, these results depicted complicated strategies by cells to adapt changes from the fast growing exponential phase toward the stationary phase. The absence of programed cell death and sporulation greatly facilitated data analysis and the identification of distinct expression patterns in the stationary phase of growth in B. subtilis.     

N-terminal Domain of TDP43 Enhances Liquid-Liquid Phase Separation of Globular Proteins

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43′s NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.