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bioRxiv2016

Assembly of photoactive Orange Carotenoid Protein from its domains unravels a carotenoid shuttle mechanism

The Orange Carotenoid Protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCPO to the red OCPR form. Cyanobacterial genomes frequently harbor, besides genes for Orange Carotenoid Proteins (OCPs), several genes encoding homologs of OCPs N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. The CTD itself is a novel, dimeric carotenoid-binding protein, which effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein, assigning physiological roles to CTD homologs and explaining the evolutionary process of OCP formation.\n\nOne Sentence SummaryThe C-domain of cyanobacterial OCP dimerizes, binds a carotenoid, and delivers it to the N-domain forming photoactive OCP.

biophysics↗

bioRxiv2016

Label-free high-resolution 3-D imaging of gold nanoparticles inside live cells using optical diffraction tomography

Delivery of gold nanoparticles (GNPs) into live cells has high potentials, ranging from molecular-specific imaging, photodiagnostics, to photothermal therapy. However, studying the long-term dynamics of cells with GNPs using conventional fluorescence techniques suffers from phototoxicity and photobleaching. Here, we present a method for 3-D imaging of GNPs inside live cells exploiting refractive index (RI) as imaging contrast. Employing optical diffraction tomography, 3-D RI tomograms of live cells with GNPs are precisely measured for an extended period with sub-micrometer resolution. The locations and contents of GNPs in live cells are precisely addressed and quantified due to their distinctly high RI values, which was validated by confocal fluorescence imaging of fluorescent dye conjugated GNPs. In addition, we perform quantitative imaging analysis including the segmentations of GNPs in the cytosol, the volume distributions of aggregated GNPs, and the temporal evolution of GNPs contents in HeLa and 4T1 cells.\n\nAbbreviations

biophysics↗

bioRxiv2017

Monitoring Microtubule Mechanical Vibrations via Optomechanical Coupling

The possible disruption of a microtubule during mitosis can control the duplication of a cancer cell. Cancer detection and treatment may be possible based on the detection and control of microtubule mechanical oscillations in cells throughexternal fields (e.g. electromagnetic or ultrasound). However, little is known about the dynamic (high-frequency) mechanical properties of microtubules. Here we propose to control the vibrations of a doubly clamped microtubule by tip electrodes and to detect its motion via the optomechanical coupling between the vibrational modes of the microtubule and an optical cavity. In the presence of a red-detuned strong pump laser, this coupling leads to optomechanical induced transparency of an optical probe field, which can be detected with state-of-the art technology. The center frequency and linewidth of the transparency peak give the resonance frequency and damping rate of the microtubule respectively, while the height of the peak reveals information about the microtubule-cavity field coupling. Our method should yield new knowledge about the physical properties of microtubules, which will enhance our capability to design physical cancer treatment protocols as alternatives to chemotherapeutic drugs.

biophysics↗

bioRxiv2017

Bacterial Conversations and Pattern Formation

It has long been recognized that certain bacterial groups exhibit cooperative behavioral patterns. Bacteria accomplish such communication via exchange of extracellular signaling molecules called pheromones(autoinducer or quorum sensing molecules). As the bacterial culture grows, signal molecules are released into extracellular milieu accumulate, changing water fluidity. Under such threshold conditions swimming bacterial suspensions impose a coordinated water movement on a length scale of the order 10 to 100 micrometers compared with a bacterial size of the order of 3 micrometers.Here, we investigate the non-local hydrodynamics of the quorum state and pattern formation using forced Burgers equation with Kwak transformation. Such approach resulted in the conversion of the Burgers equation paradigm into a reaction-diffusion system. The examination of the dynamics of the quorum sensing system, both analytically as well as numerically result in similar long-time dynamical behaviour.

biophysics↗

bioRxiv2017

How does Mg2+ modulate the RNA folding mechanism - a case study of G:C W:W Trans base pair.

Reverse Watson-Crick G:C base pairs (G:C W:W Trans) occur frequently in different functional RNAs. It is one of the few base pairs whose gas phase optimized isolated geometry is inconsistent with the corresponding experimental geometry. Several earlier studies indicate that accumulation of positive charge near N7 of guanine, through posttranscriptional modification, direct protonation or coordination with Mg2+, can stabilize the experimental geometry. Interestingly, recent studies reveal significant variation in the position of putatively bound Mg2+. This, in conjunction with recently raised doubts regarding some of the Mg2+ assignments near the imino nitrogen of guanine, is suggestive of the existence of multiple Mg2+ binding modes for this base pair. Our detailed investigation of Mg2+ bound G:C W:W Trans pairs, occurring in high resolution RNA crystal structures, show that they occur in 14 different contexts, 8 out of which display Mg2+ binding at the Hoogsteen edge of guanine. Further examination of occurrences in these 8 contexts led to the characterization of three different Mg2+ binding modes, (i) direct binding via N7 coordination, (ii) direct binding via O6 coordination and (iii) binding via hydrogen bonding interaction with the first shell water molecules. In the crystal structures, the latter two modes are associated with a buckled and propeller twisted geometry of the base pair. Interestingly, respective optimized geometries of these different Mg2+ binding modes (optimized at B3LYP) are consistent with their corresponding experimental geometries. Subsequent interaction energy calculations at MP2 level, and decomposition of its components, suggest that for G:C W:W Trans, Mg2+ binding can fine tune the base pair geometries without compromising with their stability. Our results, therefore, underline the importance of the mode of binding of Mg2+ ions in shaping RNA structure, folding and function.

biophysics↗

bioRxiv2017

Active diffusion and advection in the Drosophila ooplasm result from the interplay of the actin and microtubule cytoskeletons

Transport in cells occurs via a delicate interplay of passive and active processes, including diffusion, directed transport and advection. Despite progresses in super-resolution microscopy, discriminating and quantifying these processes is a challenge, requiring tracking of rapidly moving, sub-diffraction objects in a crowded, noisy environment. Here we use Differential Dynamic Microscopy with different contrast mechanisms to provide a thorough characterization of the dynamics in the Drosophila oocyte. We study the movement of vesicles and the elusive motion of a cytoplasmic F-actin mesh, a known regulator of cytoplasmic flows. We find that cytoplasmic motility constitutes a combination of directed motion and random diffusion. While advection is mainly attributed to microtubules, we find that active diffusion is driven by the actin cytoskeleton, although it is also enhanced by the flow. We also find that an important dynamic link exists between vesicles and cytoplasmic F-actin motion, as recently suggested in mouse oocytes.

biophysics↗

bioRxiv2017

Nucleotide-dependent stiffness suggests role of interprotofilament bonds in microtubule assembly

Many eukaryotic cell functions depend on dynamic instability, meaning the nucleotide-driven assembly and disassembly of microtubules. Assembly requires the constituent tubulin dimers to bind the nucleotide GTP, and its subsequent hydrolysis to GDP induces disassembly. The underlying structural mechanisms, however, are not well understood. Here, we determine the strength of contacts in the microtubule lattice by combining high precision measurements of the bending stiffness of analogues of GTP and GDP microtubules with a recent theoretical model. While previous structural studies have focussed on how the curvature of the tubulin dimer is affected by nucleotide binding, we present evidence of a dramatic regulation of the lateral interactions between the parallel protofilaments that dimers form in the microtubule. We conclude that the shear coupling between neighboring protofilaments is at least two orders of magnitude stronger in the GTP state than in the GDP state, and discuss the implications for the microtubule assembly.

biophysics↗

bioRxiv2017

Reverse Watson-Crick purine-purine base pairs : the Sharp-turn motif and other structural consequences in functional RNAs

Identification of static and/or dynamic roles of different noncanonical base pairs is essential for a comprehensive understanding of the sequence-structure-function space of RNA. In this context, reverse Watson-Crick purine-purine base pairs (A:A, G:G&A:GW:W Trans) constitute an interesting class of noncanonical base pairs in RNA due to their characteristic C1'-C1' distance (highest among all base pairing geometries) and parallel local strand orientation. Structural alignment of the RNA stretches containing these W:W Trans base pairs with their corresponding homologous sites in a non-redundant set of RNA crystal structures show that, as expected, these base pairs are associated with specific structural folds or functional roles. Detailed analysis of these contexts further revealed a bimodal distribution in the local backbone geometry parameters associated with these base pairs. One mode, populated by both A:A and G:G W:W Trans pairs, manifests itself as a characteristic backbone fold. We call this fold a Sharp-turn motif. The other mode is exclusively associated with A:A W:W Trans pairs involved in mediating higher order interactions. The same trend is also observed in available solution NMR structures. We have also characterized the importance of recurrent hydrogen bonding interactions between adenine and guanine in W:W Trans geometry. Quantum chemical calculations performed at M05-2X/6-31++(2d,2p) level explain how the characteristic electronic properties of these W:W Trans base pairs facilitate their occurrence in such exclusive structural folds that are important for RNA functionalities.

biophysics↗

bioRxiv2017

Structural landscape of base pairs containing post-transcriptional modifications in RNA

Base pairs involving post-transcriptionally modified nucleobases are believed to play important roles in a wide variety of functional RNAs. Here we present our attempts towards understanding the structural and functional role of naturally occurring modified base pairs by analyzing their distribution in different RNA classes, with the help of crystal structure and sequence database analyses. In addition, we quantify the variation in geometrical features of modified base pairs within RNA structures, and characterize their optimum geometries and binding energies using advanced quantum chemical methods. Further comparison of modified base pairs with their unmodified counterparts illustrates the effect of steric and electronic structure alterations due to base modifications. Analysis of specific structural contexts of modified base pairs in RNA crystal structures revealed several interesting scenarios, including those at the tRNA:rRNA interface, antibiotic-binding site and the three-way junctions within tRNA, which when analyzed in context of available experimental data, allowed us to correlate the occurrence and strength of modified base pairs with the specific functional roles they play in context of RNA macromolecules.

biophysics↗

bioRxiv2017

Accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

biophysics↗

bioRxiv2017

Direct visualization of transcriptional activation by physical enhancer-promoter proximity

A long-standing question in metazoan gene regulation is how remote enhancers communicate with their target promoters over long distances. Combining genome editing and quantitative live imaging we simultaneously visualize physical enhancer-promoter communication and transcription in Drosophila embryos. Enhancers regulating pair rule stripes of even-skipped expression activate transcription of a reporter gene over a distance of 150 kb. We show in individual cells that activation only occurs after the enhancer comes into close proximity with its regulatory target and that upon dissociation transcription ceases almost immediately. We further observe distinct topological conformations of the eve locus, depending on the spatial identity of the activating stripe enhancer. In addition, long-range activation results in transcriptional competition at the endogenous eve locus, causing corresponding developmental defects. Overall, we demonstrate that sustained physical proximity and enhancer-promoter engagement are required for enhancer action, and we provide a path to probe the implications of long-range regulation on cellular fates.

biophysics↗

bioRxiv2017

Sinusoidal Voltage Protocols For Rapid Characterization Of Ion Channel Kinetics

Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics -- the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fit a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells over-expressing hERG1a. The model is then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprised of a collection of physiologically-relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.\n\nTable of Contents CategoryTechniques for Physiology1\n\nKey PointsO_LIIon current kinetics are commonly represented by current-voltage relationships, time-constant voltage relationships, and subsequently mathematical models fitted to these. These experiments take substantial time which means they are rarely performed in the same cell.\nC_LIO_LIRather than traditional square-wave voltage clamps, we fit a model to the current evoked by a novel sum-of-sinusoids voltage clamp that is only 8 seconds long.\nC_LIO_LIShort protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions.\nC_LIO_LIThe new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically-relevant series of action potential clamps is predicted extremely well.\nC_LIO_LIThe short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time.\nC_LI

biophysics↗

bioRxiv2017

RNA Gelation in Repeat Expansion Disorders

Expansions of short nucleotide repeats in the protein coding and non-coding regions of >30 genes produce a variety of neurological and neuromuscular disorders including Huntingtons disease (CAG repeats), muscular dystrophy (CTG repeats) and amyotrophic lateral sclerosis (GGGGCC repeats) [1-3]. Expression of expanded repeats alone is sufficient to recapitulate disease pathology in animal models [4-6]. Repeat-containing transcripts accumulate in the nucleus as aberrant \"RNA foci\" [7-10] and sequester numerous RNA binding proteins [11,12], leading to a disruption of cellular homeostasis [13,14]. Interestingly, RNA foci, as well as the disease symptoms, only manifest at a critical threshold of nucleotide repeats: >30 for CAG/CTG expansions [1] and >7 for the GGGGCC expansion [15]. However, the reason for this characteristic threshold, as well as the molecular mechanism of foci formation, remain unresolved [16]. Here, we show that nucleotide repeat expansions in RNA create templates for multivalent Watson-Crick (CAG/CUG expansions) or Hoogsteen (GGGGCC expansion) base-pairing. These multivalent interactions cause purified RNAs containing repeat expansions to undergo a sol-gel transition and form micron-sized clusters. Reflecting an increase in the valency for intermolecular hybridization, the gelation of purified RNA only occurs above a critical number of trinucleotide or hexanucleotide repeats. These thresholds for in vitro RNA gelation are similar to those associated with manifestation of disease. By visualizing RNA in live cells, we show that nuclear foci form as a result of phase separation of the repeat-containing RNA and that these foci can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

biophysics↗

bioRxiv2017

Structural-mechanical remodelling of GDP-microtubules by kinesin

Kinesin-1 is a nanoscale molecular motor that walks towards the fast growing (plus) ends of microtubules (MTs), hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organisation of eukaryotic cells and shows great promise as a tool for nano-engineering1,2. Recent work hints that kinesin may also play a role in modulating the stability of its MT track, both in vitro3-5 and in vivo6, but results are conflicting7-9 and mechanisms are unclear. Here we report a new dimension to the kinesin-MT interaction, whereby strong-state (ATP-bound and apo) kinesin-1 motor domains inhibit the shrinkage of GDP-MTs by up to 2 orders of magnitude and expand their lattice spacing by ~1.6%. Our data reveal an unexpected new mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, allowing motile kinesins to influence the structure, stability and mechanics of their MT track.

biophysics↗

bioRxiv2017

Tracking multiple genomic elements using correlative CRISPR imaging and sequential DNA FISH

Live imaging of genome has offered important insights into the dynamics of the genome organization and gene expression. The demand to image simultaneously multiple genomic loci has prompted a flurry of exciting advances in multi-color CRISPR imaging, although color-based multiplexing is limited by the need for spectrally distinct fluorophores. Here we introduce an approach to achieve highly multiplexed live recording via correlative CRISPR imaging and sequential DNA fluorescence in situ hybridization (FISH). This approach first performs one-color live imaging of multiple genomic loci and then uses sequential rounds of DNA FISH to determine the loci identity. We have optimized the FISH protocol so that each round is complete in 1 min, demonstrating the identification of 7 genomic elements and the capability to sustain reversible staining and washing for up to 20 rounds. We have also developed a correlation-based algorithm to faithfully register live and FISH images. Our approach keeps the rest of the color palette open to image other cellular phenomena of interest, as demonstrated by our simultaneous live imaging of genomic loci together with a cell cycle reporter. Furthermore, the algorithm to register faithfully between live and fixed imaging is directly transferrable to other systems such as multiplex RNA imaging with RNA-FISH and multiplex protein imaging with antibody-staining.

biophysics↗

bioRxiv2017

A new and accurate continuum description of moving fronts

Processes that involve moving fronts of populations are prevalent in ecology and cell biology. A common approach to describe these processes is a lattice-based random walk model, which can include mechanisms such as crowding, birth, death, movement and agent-agent adhesion. However, these models are generally analytically intractable and it is computationally expensive to perform sufficiently many realisations of the model to obtain an estimate of average behaviour that is not dominated by random fluctuations. To avoid these issues, both mean-field and corrected mean-field continuum descriptions of random walk models have been proposed. However, both continuum descriptions are inaccurate outside of limited parameter regimes, and corrected mean-field descriptions cannot be employed to describe moving fronts. Here we present an alternative description in terms of the dynamics of groups of contiguous occupied lattice sites and contiguous vacant lattice sites. Our description provides an accurate prediction of the average random walk behaviour in all parameter regimes. Critically, our description accurately predicts the persistence or extinction of the population in situations where previous continuum descriptions predict the opposite outcome. Furthermore, unlike traditional mean-field models, our approach provides information about the spatial clustering within the population and, subsequently, the moving front.

biophysics↗

bioRxiv2017

Convection - Diffusion model of talking bacteria

Quorum sensing is cell to cell communication process through chemical signals formally known as autoinducers. When the concentration of quorum sensing molecules reached threshold concentration bacteria are in active state or quorum state. In this article, we propose a mathematical model of quorum sensing systems and study this biological system numerically. Moreover, we compare the different numerical scheme with the batch culture of P.aeruginosa. We observed a negative diffusion coefficient which plays an important role in the quorum sensing mechanism.

biophysics↗

bioRxiv2017

Computing Osmotic Permeabilities of Aquaporins AQP4, AQP5, and GlpF from Near-Equilibrium Simulations

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC=\"FIGDIR/small/102343_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@c57ff0org.highwire.dtl.DTLVardef@527a79org.highwire.dtl.DTLVardef@e6bde6org.highwire.dtl.DTLVardef@bd53cf_HPS_FORMAT_FIGEXP M_FIG C_FIG Measuring or computing the single-channel permeability of aquaporins/aquaglyceroporins (AQPs) has long been a challenge. The measured values scatter over an order of magnitude but the corresponding Arrhenius activation energies converge in the current literature. Osmotic flux through an AQP was simulated as water current forced through the channel by kilobar hydraulic pressure or theoretically approximated as single-file diffusion. In this paper, we report large scale simulations of osmotic current under sub M gradient through three water channels (the water-specific AQP4 and AQP5 along with aquaglyceroporin GlpF) using the mature particle mesh Ewald technique (PME) for which the established force fields have been optimized with known accuracy. These simulations were implemented with hybrid periodic boundary conditions devised to avoid the artifactitious mixing across the membrane in a regular PME simulation. The computed single-channel permeabilities at 5{degrees}C and 25{degrees}C are in agreement with recently refined experiments on GlpF. The Arrhenius activation energies extracted from our simulations for all the three AQPs agree with the in vitro measurements. The single-file diffusion approximations from our large-scale simulations are consistent with the current literature on smaller systems. From these unambiguous agreements among the in vitro and in silico studies, we observe the quantitative accuracy of the all-atom force fields of the current literature for water-channel biology. We also observe that AQP4, that is particularly rich in the central nervous system, is more efficient in water conduction and more temperature-sensitive than other water-specific channel proteins.

biophysics↗