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bioRxiv2017

Redundancy principle for optimal random search in biology

Chemical activation rate is traditionally determined by the diffusion flux into an absorbing ball, as computed by Smoluchowski in 1916. Thus the rate is set by the mean first passage time (MFPT) of a Brownian particle to a small target. This paradigm is shifted in this manuscript to set the time scale of activation in cellular biology to the mean time of the first among many arrivals of particles at the activation site. This rate is very different from the MFPT and depends on different geometrical parameters. The shift calls for the reconsideration of physical modeling such as deterministic and stochastic chemical reactions based on the traditional forward rate, especially for fast activation processes occurring in living cells. Consequently, the biological activation time is not necessarily exponential. The new paradigm clarifies the role of population redundancy in accelerating search processes and in defining cellular-activation time scales. This is the case, for example, in cellular transduction or in the nonlinear dependence of fertilization rate on the number of spermatozoa. We conclude that statistics of the extreme set the new laws of biology, which can be very different from the physical laws derived for individuals.

biophysics↗

bioRxiv2017

Study on Linear Combination of Long Memory Processes Corrupted by Additive Noises for fMRI Time Series Analysis

Estimating the long memory parameter of the fMRI time series enables us to understand the fractal behavior of neural activity of the brain through fMRI time series. However, the existence of white noise and physiological noise compounds which also have fractal properties prevent us from making the estimation precise. As basic strategies to overcome noises, we address how to estimate the long memory parameter in the presence of additive noises, and how to estimate the long memory parameters of linearly combined long memory processes.

biophysics↗

bioRxiv2017

Cross talk between photo-pigments and graphene electron cloud - Designing a biodiode

We report emergence of a new electrical material by growing photosynthetic biofilm on a Dirac material, graphene. The material showed new conducting as well as semiconducting properties. Frequency dependent capacitive spectra further indicated presence of electrical isosbestic points(at 0.8 and 9MHz), implying two state dieletric transitions at critical frequencies. A notable reult was a Schottky diode like behavior in the IV curve. Voltage dependent conductance with conductance peaks near the Schottky diode threshold was observed. We obtained facilitated growth of photosynthetic biofilm in presence of graphene. Lastly higher bacterial metabolism was seen in graphene incorporated biofilm. For this zero band gap Dirac material this can only be interpreted as coupling of the electron transport chain of the bacterial biofilm and the graphene electron cloud.

biophysics↗

bioRxiv2017

Auxiliary ATP binding sites power rapid unwinding by RecBCD

RecBCD, responsible for the initiation of double stranded break repair in bacteria, is a processive DNA helicase with an unwinding rate approaching [~]1,600 bp{middle dot}s-1. The mechanism enabling RecBCD to achieve such fast unwinding rate is not known. We employed a combination of equilibrium and time-resolved binding experiments, and ensemble and single molecule activity assays to uncover the molecular mechanism underlying RecBCDs rapid catalysis. We report the existence of auxiliary binding sites, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. The catalytic rate of RecBCD is reduced both by preventing and by strengthening ATP binding to these sites, suggesting that the dynamics of ATP at these sites modulates the enzymes rate. We propose a model by which RecBCD achieves its fast unwinding rate by utilizing the weaker binding sites to increase the flux of ATP to its catalytic sites.

biophysics↗

bioRxiv2017

Forced-rupture of Cell-Adhesion Complexes Reveals abrupt switch between two Brittle States

Cell adhesion complexes (CACs), which are activated by ligand binding, play key roles in many cellular functions ranging from cell cycle regulation to mediation of cell extracellular matrix adhesion. Inspired by single molecule pulling experiments using atomic force spectroscopy on leukocyte function-associated antigen-1 (LFA-1), expressed in T-cells, bound to intercellular adhesion molecules (ICAM), we performed constant loading rate (rf) and constant force (F) simulations using the Self-Organized Polymer (SOP) model to describe the mechanism of ligand rupture from CACs. The simulations reproduce the major experimental finding on the kinetics of the rupture process, namely, the dependence of the most probable rupture forces (f*s) on ln rf (rf is the loading rate) exhibits two distinct linear regimes. The first, at low rf, has a shallow slope whereas the the slope at high rf is much larger, especially for LFA-1/ICAM-1 complex with the transition between the two occurring over a narrow rf range. Locations of the two transition states (TSs), extracted from the simulations show an abrupt change from a high value at low rf or F to a low value at high rf or F. This unusual behavior in which the CACs switch from one brittle (TS position is a constant over a range of forces) state to another brittle state is not found in forced-rupture in other protein complexes. We explain this novel behavior by constructing the free energy profiles, F ({Lambda})s, as a function of a collective reaction coordinate ({Lambda}), involving many key charged residues and a critical metal ion (M g2+). The TS positions in F({Lambda}), which quantitatively agree with the parameters extracted using the Bell-Evans model, change abruptly at a critical force, demonstrating that it, rather than the molecular extension is a good reaction coordinate. Our combined analyses using simulations performed in both the pulling modes (constant rf and force) reveal a new mechanism for the two loading regimes observed in the rupture kinetics in CACs.

biophysics↗

bioRxiv2017

Antiferromagnetic switch in serum

1.Ferritin contains naturally occurring iron oxide nanoparticle surrounded by a structured spherical array of peptide residues that provides tremendous stability to this iron storage protein. We use synthetic citrate coated Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) and static magnetic field in exploring the Ferritin induced magnetic environment of human serum samples with varying ferritin level collected from thalassemic patients. We report anti-ferromagnetic properties of serum in patients with iron overloading. Magnetic pulling by an external magnetic field showed a cusp-like behavior with increasing concentration of serum Ferritin measured by standard ELISA based kit. A reduction in the extent of pulling after a threshold concentration of Ferritin (1500 ng/ml) suggests a Ferritin dependent magnetic switching.Negative magnetization (anti-ferromagnetization) was confirmed by Vibrating Sample Magnetometric (VSM) analysis of SPION-serum mixture containing very high level of Ferritin. Such magnetic switching may have a possible role in iron homeostasis during overloading of Ferritin.\n\n3. AbbreviationsSPIONs: Super Paramagnetic Iron Oxide Nanoparticles, VSM: Vibrating Sample Magnetometry, SQUID: Super conducting Quantum Interference Device, PCS: Photon Correlation Spectroscopy

biophysics↗

bioRxiv2017

The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity

The influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6-13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures.\n\nIn this paper, it was shown that PB1(6-13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6-13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6-13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC=\"FIGDIR/small/211284_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (77K):\norg.highwire.dtl.DTLVardef@185db47org.highwire.dtl.DTLVardef@ff2fd2org.highwire.dtl.DTLVardef@19c8587org.highwire.dtl.DTLVardef@10282b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins additionally exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether sorting platform complexes exist in the cytosol, we measured the diffusive properties of the two central sorting platform proteins, SctQ and SctL, using live cell high-throughput 3D single-molecule tracking microscopy. Single-molecule trajectories, measured in wild-type and mutant Yersinia enterocolitica cells, reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. Our findings provide the first diffusive state-resolved insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the type 3 secretion system.

biophysics↗

bioRxiv2017

Blue light is a universal tactic signal for Escherichia coli

Blue light has been shown to elicit a tumbling response in E. coli, a non-phototrophic bacterium. The exact mechanism of this phototactic response is still unknown, and its biological significance remains unclear. Here, we quantify phototaxis in E. coli by analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure. Bacterial strains expressing only one type of chemoreceptor reveal that all five E. coli receptors - Aer, Tar, Tsr, Tap and Trg - are capable of mediating a response to light. In particular, light exposure elicits a running response in Tap-only strain, the opposite of the tumbling response observed for all other strains. Light therefore emerges as a universal stimulus for all E. coli chemoreceptors. We also show that blue light exposure causes a reversible decrease in swimming velocity, a proxy for proton motive force. We hypothesize that rather than sensing light directly, chemoreceptors sense light-induced perturbations in proton motive force.\n\nImportanceOur findings provide new insights on the mechanism of E. coli phototaxis, showing that all five chemoreceptor types respond to light and that their interactions play an important role in cell behavior. Our results also open up new avenues for examining and manipulating E. coli taxis. Since light is a universal stimulus, it may provide a way to quantify interactions between different types of receptors. Since light is easier to control spatially and temporally than chemicals, it may be used to study swimming behavior in complex environments. Since phototaxis can cause migration of E. coli bacteria in light gradients, light may be used to control bacterial density for studying density-dependent processes in bacteria.

biophysics↗

bioRxiv2017

Molecular mechanism of the dual regulation of bacterial iron sulfur cluster biogenesis by CyaY and IscX

IscX (or YfhJ) is a protein of unknown function which takes part in the iron-sulfur cluster assembly machinery, a highly specialised and essential metabolic pathway. IscX binds to iron with low affinity and interacts with IscS, the desulfurase central to cluster assembly. Previous studies have suggested a competition between IscX and CyaY, the bacterial ortholog of frataxin, for the same binding surface of IscS. This competition could suggest a link between the two proteins with a functional significance. Using a hybrid approach, we show here that IscX is a modulator of the inhibitory properties of CyaY: by competing for the same site on IscS, the presence of IscX rescues the rates of enzymatic cluster formation which are inhibited by CyaY. The effect is stronger at low iron concentrations, whereas it becomes negligible at high iron concentrations. These results strongly suggest that iron-sulfur cluster assembly is an exquisite example of an enzymatic process which requires a double regulation under the control of iron as the effector.

biophysics↗

bioRxiv2017

The fluid membrane determines mechanics of red blood cell extracellular vesicles and is softened in hereditary spherocytosis

Extracellular vesicles (EVs) are widely studied regarding their role in cell-to-cell communication and disease, as well as for applications as biomarker or drug delivery vehicle. EVs contain both membrane and intraluminal proteins, affecting their structural properties and thereby likely their functioning. Here, we use atomic force microscopy for the mechanical characterization of red blood cell (RBC) EVs from healthy individuals as well as from a patient with hereditary spherocytosis (HS) due to ankyrin deficiency. We show that the EVs are packed with proteins, yet their response to indentation is similar to that of a fluid lipid vesicle lacking proteins. The bending modulus of RBC EVs of healthy donors is ~15 kbT, agreeing well with the bending modulus of the RBC membrane. Surprisingly, whereas RBCs become more rigid in HS, the excreted vesicles of a patient with this blood disorder have a significantly (~50%) lower bending modulus than donor EVs. These results shed new light on the mechanism and effects of EV budding and may underlie the reported increase in vesiculation and stiffening of RBCs in hereditary spherocytosis patients.

biophysics↗

bioRxiv2017

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

biophysics↗

bioRxiv2017

Fractional Dynamics in Bioscience and Biomedicine and the Physics of Cancer

Almost all phenomena and structures in nature exhibit some degrees of fractionality or fractality. Fractional calculus and fractal theory are two interrelated concepts. In this article we study the memory effects in nature and particularly in biological structures. Based on this fact that natural way to incorporate memory effects in the modeling of various phenomena and dealing with complexities is using of fractional calculus, in this article we present different examples in various branch of science from cosmology to biology and we investigate this idea that are we able to describe all of such these phenomena using the well-know and powerful tool of fractional calculus. In particular we focus on fractional calculus approach as an effective tool for better understanding of physics of living systems and organism and especially physics of cancer.

biophysics↗

bioRxiv2017

Theory of site-specific DNA-protein interactions in the presence of nucleosome roadblocks

We show that nucleosomes can efficiently control the relative search times spent by transcription factors (TFs) on one- (1D) and three-dimensional (3D) diffusion routes towards locating their cognate sites on DNA. Our theoretical results suggest that the roadblock effects of nucleosomes are dependent on the relative position on DNA with respect to TFs and their cognate sites. Especially, nucleosomes exert maximum amount of hindrance to the 1D diffusion dynamics of TFs when they are positioned in between TFs and their cognate sites. The effective 1D diffusion coefficient ({chi}TF) associated with the dynamics of TFs in the presence of nucleosome decreases with the free energy barrier ({micro}) associated the sliding dynamics of nucleosomes as [Formula]. Subsequently the mean first passage time ({eta}L) that is required by TFs to scan L number of binding sites on DNA via 1D diffusion increases with as [Formula]. When TFs move close to nucleosomes then they exhibit a typical sub-diffusive dynamics. Nucleosomes can enhance the search dynamics of TFs when TFs present in between nucleosomes and transcription factor binding sites (TFBS). The level of enhancement effects of nucleosomes seems to be much lesser than the level of retardation effects when nucleosomes present in between TFs and their cognate sites. These results suggest that nucleosome depleted regions around the cognate sites of TFs is mandatory for an efficient site-specific interactions of TFs with DNA. Remarkably the genome wide positioning pattern of TFs shows maximum at their specific binding sites and the positioning pattern of nucleosome shows minimum at the specific binding sites of TFs under in vivo conditions. This seems to be a consequence of increasing level of breathing dynamics of nucleosome cores and decreasing levels of fluctuations in the DNA binding domains of TFs as they move across TFBS. Since the extent of breathing dynamics of nucleosomes and fluctuations in the DBDs of TFs are directly linked with their respective 1D diffusion coefficients, the dynamics of TFs becomes slow as they approach their cognate sites so that TFs form tight site-specific complex. Whereas the dynamics of nucleosomes becomes rapid so that they pass through the cognate sites of TFs. Several in vivo datasets on genome wide positioning pattern of nucleosomes as well as TFs seem to agree well with our arguments. We further show that the condensed conformational state of DNA can significantly decrease the retarding effects of nucleosome roadblocks. The retarding effects of nucleosomes on the 1D diffusion dynamics of TFs can be nullified when the degree of condensation of the genomic DNA is such that it can permit a jump size associated with the dynamics of TFs beyond k > 150 bps.

biophysics↗

bioRxiv2017

New Cell Fate Potentials and Switching Kinetics Uncovered in a Classic Bistable Genetic Switch

Bistable switches are common gene regulatory motifs directing two mutually exclusive gene expression states, and consequently distinct cell fates. Theoretical studies suggest that the simple circuitry of bistable switches is sufficient to encode more than two cell fates due to the non-equilibrium, heterogeneous cellular environment, allowing a high degree of adaptation and differentiation. However, new cell fates arising from a classic bistable switch without rewiring the circuitry have not been experimentally observed. By developing a new, dual single-molecule gene-expression reporting system in live E. coli cells, we investigated the expression dynamics of two mutually repressing transcription factors, CI and Cro, in the classic genetic switch of bacteriophage {lambda}. We found that in addition to the two expected high-Cro and high-CI production states, there existed two new ones, in which neither CI nor Cro was produced, or both CI and Cro were produced. We constructed the corresponding potential landscape and mapped the transition kinetics between the four production states, providing insight into possible state-switching rates and paths. These findings uncover new cell fate potentials beyond the classical picture of {lambda} switch, and open a new window to explore the genetic and environmental origins of the cell fate decision-making process in gene regulatory networks.

biophysics↗

bioRxiv2017

Spin coated chitin films for biosensors and its analysis are depended on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.

biophysics↗

bioRxiv2017

PKD2 is an essential ion channel subunit in the primary cilium of the renal collecting duct epithelium

Mutations in either Pkd1 or Pkd2 result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although PKD2 is proposed to be an ion channel subunit, recordings of PKD2 ion channels conflict in their properties. Using a new ADPKD mouse model, we observe primary cilia are abnormally long in cells associated with cysts. Using primary cultures of collecting duct epithelial cells, we show that PKD2, but not PKD1, is a required subunit for primary cilia ion channel. The ciliary PKD2 channel conducts potassium and sodium ions, but little calcium. We also demonstrate that PKD2 is not constitutively active in the plasma membrane, but PKD2 channels are functional in primary cilia and are sensitized by high cilioplasmic [Ca2+]. We introduce a novel method for measuring PKD2 channels heterologously expressed in primary cilia of HEK-293 cells, which will have utility characterizing Pkd2 variants that cause ADPKD in their native ciliary membrane.

biophysics↗

bioRxiv2017

Tumorcode - A framework to simulate vascularized tumors

During the past years our group published several articles using computer simulations to address the complex interaction of tumors and the vasculature as underlying transport network. Advances in imaging and lab techniques pushed in vitro research of tumor spheroids forward and animal models as well as clinical studies provided more insights to single processes taking part in tumor growth, however, an overall picture is still missing. Computer simulations are a none-invasive option to cumulate current knowledge and form a quasi in vivo system. In our software, several known models were assembled into a multi-scale approach which allows to study length scales relevant for clinical applications.\n\nWe release our code to the public domain, together with a detailed description of the implementation and several examples, with the hope of usage and futher development by the community. Justification for the included algorithms and the biological models was obtained in previous publications, here we summarize technical aspects following the workflow of a typical simulation procedure.

biophysics↗