Signaling in the crowded cell

High-resolution technologies have clarified some of the principles underlying cellular actions. However, understanding how cells receive, communicate, and respond to signals is still challenging. Questions include how efficient regulation of assemblies, which execute cell actions at the nanoscales, transmits productively at micrometer scales, especially considering the crowded environment, and how the cell organization makes it happen. Here, we describe how cells can navigate long-range diffusion-controlled signaling via association/dissociation of spatially proximal entities. Dynamic clusters can span the cell, engaging in most signaling steps. Effective local concentration, allostery, scaffolding, affinities, and the chemical and mechanical properties of the macromolecules and the environment play key roles. Signaling strength and duration matter, for example, deciding if a mutation promotes cancer or developmental syndromes.     

Volume exclusion effect as a driving force for reverse proteolysis. Implications for polypeptide assemblage in a macromolecular crowded milieu

Macromolecular crowding, in principle, should affect any reaction that is accompanied by significant reduction in excluded volume. Here we have examined the influence of crowding on reverse proteolysis. We show that proteosynthesis of a polypeptide product with an interacting folding motif such as coiled coil is facilitated in crowded media as a consequence of the volume exclusion effect. Further, we demonstrate that crowding could also effect the conversion of a noncovalent protein complex (fragment complementing protein) obtained by limited proteolysis to the native covalent form, but only if the formation of the native protein results in large compaction leading to a substantial volume exclusion effect. Subtilisin-catalyzed reformation of native triosephosphate isomerase (TIM) from multiple fragments is facilitated by crowding. However, a single nick in ribonuclease S (RNase S) could not be ligated under similar conditions. The failure of generation of RNase A from RNase S is consistent with the fact that the crystal structure of the two forms are almost superimposable, and hence no significant difference of volume exclusion exists between reactant (RNase S) and product (RNase A). In contrast, considerable compaction, and consequently large reduction in excluded volume, is attained through the assembly of a TIM barrel structure. Taken together, these results have implications for both in vitro as well as in vivo polypeptide assemblage by reverse proteolysis.     

Entropic tension in crowded membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than [Formula: see text] in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.     

Concentration sensing in crowded environments

Signal transduction within crowded cellular compartments is essential for the physiological function of cells. Although the accuracy with which receptors can probe the concentration of ligands has been thoroughly investigated in dilute systems, the effect of macromolecular crowding on the inference of concentration remains unclear. In this work, we develop an algorithm to simulate reversible reactions between reacting Brownian particles. Our algorithm facilitates the calculation of reaction rates and correlation times for ligand-receptor systems in the presence of macromolecular crowding. Using this method, we show that it is possible for crowding to increase the accuracy of estimated ligand concentration based on receptor occupancy. In particular, we find that crowding can enhance the effective association rates between small ligands and receptors to a degree sufficient to overcome the increased chance of rebinding due to caging by crowding molecules. For larger ligands, crowding decreases the accuracy of the receptor’s estimate primarily by decreasing the microscopic association and dissociation rates.     

Enzyme activity during the metabolism of glycogen

Summary Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii.After incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO₄ (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO₄, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Protein aggregation in crowded environments

The generic tendency of proteins to aggregate into non-functional, and sometimes cytotoxic, structures poses a universal problem for all types of cell. This tendency is greatly exacerbated by the high total concentration of macromolecules found within most intracellular compartments, a phenomenon referred to as macromolecular crowding. This review discusses the quantitative effects of crowding on protein aggregation and the role of molecular chaperones in combating this problem.     

Understanding enzyme behavior in a crowded scenario through modulation in activity,conformation and dynamics

Macromolecular crowding, inside the physiological interior, modulates the energy landscape of biological macromolecules in multiple ways. Amongst these, enzymes occupy a special place and hence understanding the function of the same in the crowded interior is of utmost importance. In this study, we have investigated the manner in which the multidomain enzyme, AK3L1 (PDB ID: 1ZD8), an isoform of adenylate kinase, has its features affected in presence of commonly used crowders (PEG 8, Dextran 40, Dextran 70, and Ficoll 70). Michaelis Menten plots reveal that the crowders in general enhance the activity of the enzyme, with the K_m and V_max values showing significant variations. Ficoll 70, induced the maximum activity for AK3L1 at 100 g/L, beyond which the activity reduced. Ensemble FRET studies were performed to provide insights into the relative domain (LID and CORE) displacements in presence of the crowders. Solvation studies reveal that the protein matrix surrounding the probe CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin) gets restricted in presence of the crowders, with Ficoll 70 providing the maximum rigidity, the same being linked to the decrease in the activity of the enzyme. Through our multipronged approach, we have observed a distinct correlation between domain displacement, enzyme activity and associated dynamics. Thus, keeping in mind the complex nature of enzyme activity and the surrounding bath of dense soup that the biological entity remains immersed in, indeed more such approaches need to be undertaken to have a better grasp of the “enzymes in the crowd”.     

Electrochemical structure of the crowded cytoplasm

The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm.     

Enzyme activity in dialkyl phosphate ionic liquids

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.     

Enzyme indicator of the immunobiologic activity of antigens

The test-system has been elaborated for the determination of the ability of different antigens in the cultures of the mouse peritoneal macrophages. Measuring an intracellular acid phosphatase activity, reflecting the extent of the cell activation, was taken as a principle of this test-system. With the help of this test-system ability of a few antigens (lipopolysaccharide, polysaccharide, glycoprotein, protein and low molecular weight substances--peptide and glycopeptide) have been studied. It is testified that above mentioned antigens possessed different effects on the acid phosphatase activity.     

Enzyme activity of the formate hydrogenlyase complex in Citrobacter freundii

Citrobacter freundii 62 can grow in the absence of oxygen in media containing glucose, peptone, fumarate or malate. When the medium contained fumarate or malate, the culture could grow under anaerobic conditions only in the presence of molecular hydrogen, formate or nitrate. The highest activity of formatehydrogenlyase and hydrogenase was found when C. freundii grew in a medium with glucose and formate. The activity was lower in media with other organic substrates, particularly, in the absence of formate or H2. The activity of hydrogenase was very low in cells grown under aerobic conditions or in the presence of nitrates while the activity of formatehydrogenlyase was not found at all for all practical purposes. The activity of formate dehydrogenase assessed in the presence of methylene blue was rather high irrespective of the conditions under which the culture was grown. However, when the activity of formate dehydrogenase was determined in the presence of benzyl viologen, it was high only in cells grown in the medium with glucose and formate.     

Enzyme synthesis in the regulation of hepatic `malic'' enzyme activity

A homogeneous preparation of ;malic' enzyme (EC 1.1.1.40) from livers of thyroxine-treated rats was used to prepare in rabbits an antiserum to the enzyme that reacts monospecifically with the ;malic' enzyme in livers of rats in several physiological states. Changes in enzyme activity resulting from modification of the state of the animal are hence due to an altered amount of enzyme protein. The antiserum has been used to precipitate out ;malic' enzyme from heat-treated supernatant preparations of livers from both adult and neonatal rats, in a number of physiological conditions, that had been injected 30min earlier with l-[4,5-(3)H]leucine. The low incorporations of radioactivity into the immunoprecipitable enzyme have permitted the qualitative conclusion that changed enzyme activity in adult rats arises mainly from alterations in the rate of enzyme synthesis. The marked increase in ;malic' enzyme activity that occurs naturally or as a result of thyroxine treatment of the weanling rat is likewise due to a marked increase in the rate of enzyme synthesis possibly associated with a concurrent diminished rate of enzyme degradation.     

Macromolecular diffusion in crowded solutions.

The effects of crowding on the self or tracer diffusion of macromolecules in concentrated solutions is an important but difficult problem, for which, so far, there has been no rigorous treatment. Muramatsu and Minton suggested a simple model to calculate the diffusion coefficient of a hard sphere among other hard spheres. In this treatment, scaled particle theory is used to evaluate the probability that the target volume for a step in a random walk is free of any macromolecules. We have improved this approach by using a more appropriate target volume which also allows the calculation to be extended to the diffusion of a hard sphere among hard spherocylinders. We conclude that, to the extent that proteins can be approximated as hard particles, the hindrance of globular proteins by other proteins is reduced when the background proteins aggregate (the more so the greater the decrease in particle surface area), the hindrance due to rod-shaped background particles is reduced slightly if the rod-like particles are aligned, and the anisotropy of the diffusion of soluble proteins among cytoskeletal proteins will normally be small.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Modeling the growth of individuals in crowded plant populations

Aims We present an improved model for the growth of individuals in plant populations experiencing competition.Methods Individuals grow sigmoidally according to the Birch model, which is similar to the more commonly used Richards model, but has the advantage that initial plant growth is always exponential. The individual plant growth models are coupled so that there is a maximum total biomass for the population. The effects of size-asymmetric competition are modeled with a parameter that reflects the size advantage that larger individual have over smaller individuals. We fit the model to data on individual growth in crowded populations of Chenopodium album .Important findings When individual plant growth curves were not coupled, there was a negative or no correlation between initial growth rate and final size, suggesting that competitive interactions were more important in determining final plant size than were plants' initial growth rates. The coupled growth equations fit the data better than individual, uncoupled growth models, even though the number of estimated parameters in the coupled competitive growth model was far fewer, indicating the importance of modeling competition and the degree of size-asymmetric growth explicitly. A quantitative understanding of stand development in terms of the growth of individuals, as altered by competition, is within reach.