A tractable genotype-phenotype map for the self-assembly of protein quaternary structure

The mapping between biological genotypes and phenotypes is central to the study of biological evolution. Here we introduce a rich, intuitive, and biologically realistic genotype-phenotype (GP) map, that serves as a model of self-assembling biological structures, such as protein complexes, and remains computationally and analytically tractable. Our GP map arises naturally from the self-assembly of polyomino structures on a 2D lattice and exhibits a number of properties: $\textit{redundancy}$ (genotypes vastly outnumber phenotypes), $\textit{phenotype bias}$ (genotypic redundancy varies greatly between phenotypes), $\textit{genotype component disconnectivity}$ (phenotypes consist of disconnected mutational networks) and $\textit{shape space covering}$ (most phenotypes can be reached in a small number of mutations). We also show that the mutational robustness of phenotypes scales very roughly logarithmically with phenotype redundancy and is positively correlated with phenotypic evolvability. Although our GP map describes the assembly of disconnected objects, it shares many properties with other popular GP maps for connected units, such as models for RNA secondary structure or the HP lattice model for protein tertiary structure. The remarkable fact that these important properties similarly emerge from such different models suggests the possibility that universal features underlie a much wider class of biologically realistic GP maps.Comment: 12 pages, 6 figure

Characterization of hormone and protein release from alpha-toxin- permeabilized chromaffin cells in primary culture

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+- dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha- toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells

Genetic Correlations Greatly Increase Mutational Robustness and Can Both Reduce and Enhance Evolvability.

Mutational neighbourhoods in genotype-phenotype (GP) maps are widely believed to be more likely to share characteristics than expected from random chance. Such genetic correlations should strongly influence evolutionary dynamics. We explore and quantify these intuitions by comparing three GP maps-a model for RNA secondary structure, the HP model for protein tertiary structure, and the Polyomino model for protein quaternary structure-to a simple random null model that maintains the number of genotypes mapping to each phenotype, but assigns genotypes randomly. The mutational neighbourhood of a genotype in these GP maps is much more likely to contain genotypes mapping to the same phenotype than in the random null model. Such neutral correlations can be quantified by the robustness to mutations, which can be many orders of magnitude larger than that of the null model, and crucially, above the critical threshold for the formation of large neutral networks of mutationally connected genotypes which enhance the capacity for the exploration of phenotypic novelty. Thus neutral correlations increase evolvability. We also study non-neutral correlations: Compared to the null model, i) If a particular (non-neutral) phenotype is found once in the 1-mutation neighbourhood of a genotype, then the chance of finding that phenotype multiple times in this neighbourhood is larger than expected; ii) If two genotypes are connected by a single neutral mutation, then their respective non-neutral 1-mutation neighbourhoods are more likely to be similar; iii) If a genotype maps to a folding or self-assembling phenotype, then its non-neutral neighbours are less likely to be a potentially deleterious non-folding or non-assembling phenotype. Non-neutral correlations of type i) and ii) reduce the rate at which new phenotypes can be found by neutral exploration, and so may diminish evolvability, while non-neutral correlations of type iii) may instead facilitate evolutionary exploration and so increase evolvability.This work was funded under EP/P504287/1 by the Engineering and Physical Sciences Research Council (https://www.epsrc.ac.uk). SEA is supported by The Royal Society (https://royalsociety.org/).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pcbi.100477

Social network analysis predicts health behaviours and self-reported health in African villages.

The provision of healthcare in rural African communities is a highly complex and largely unsolved problem. Two main difficulties are the identification of individuals that are most likely affected by disease and the prediction of responses to health interventions. Social networks have been shown to capture health outcomes in a variety of contexts. Yet, it is an open question as to what extent social network analysis can identify and distinguish among households that are most likely to report poor health and those most likely to respond to positive behavioural influences. We use data from seven highly remote, post-conflict villages in Liberia and compare two prominent network measures: in-degree and betweenness. We define in-degree as the frequency in which members from one household are named by another household as a friends. Betweenness is defined as the proportion of shortest friendship paths between any two households in a network that traverses a particular household. We find that in-degree explains the number of ill family members, whereas betweenness explains engagement in preventative health. In-degree and betweenness independently explained self-reported health and behaviour, respectively. Further, we find that betweenness predicts susceptibility to, instead of influence over, good health behaviours. The results suggest that targeting households based on network measures rather than health status may be effective for promoting the uptake of health interventions in rural poor villages.This work was supported by N.W.O. grant #W07.68.116 to Professor Erwin Bulte.This is the final version, originally published by PLOS at http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0103500

Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells

Additive Nonparametric Reconstruction of Dynamical Systems from Time Series

We present a nonparametric way to retrieve a system of differential equations in embedding space from a single time series. These equations can be treated with dynamical systems theory and allow for long term predictions. We demonstrate the potential of our approach for a modified chaotic Chua oscillator.Comment: accepted for Phys. Rev. E, Rapid Com

VGLUT1 binding to endophilin or intersectin1 and dynamin phosphorylation in a diurnal context

Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins.VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A–E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light–dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1