May 09, 2017

Volume 112Issue 9p1737-2020
Open Archive
Cover picture: Calcium mediates the structural state of the Caulobacter crescentus surface layer protein, RsaA. With calcium, RsaA assembles into a 2D crystal, whereas without calcium, RsaA forms an amorphous aggregate. Both states can exist in vivo, and the aggregated state is implicated in protecting the cell against calcium deficiency stress. Artist: Gregory Stewart....
Cover picture: Calcium mediates the structural state of the Caulobacter crescentus surface layer protein, RsaA. With calcium, RsaA assembles into a 2D crystal, whereas without calcium, RsaA forms an amorphous aggregate. Both states can exist in vivo, and the aggregated state is implicated in protecting the cell against calcium deficiency stress. Artist: Gregory Stewart.

New and Notable

  • New and Notable

    How to Make a Worm Twitch

    • Philipp J. Keller
    Understanding how the nervous system generates behavior is a long-standing goal of neuroscience. The early nervous system develops alongside other tissues and organs in the course of embryogenesis and is crucial for establishing the organism’s early behavioral repertoire, including its ability to perform coordinated movements. The roundworm Caenorhabditis elegans has long been an important model system for studying developmental processes and offers several powerful features that aid such investigations, including its rapid embryonic development and its known and invariant cell lineage (1).
  • New and Notable

    Tug of War at the Cell-Matrix Interface

    • Paolo P. Provenzano
    Mechanical interactions between a cell and its environment, or between cells, influence key developmental and physiologic processes as well as many aspect of disease (1,2). Indeed the ability of a cell to sense, produce, and respond to mechanical cues has emerged as a fundamental regulator of cell behaviors such as differentiation, proliferation, survival, and migration. In mechanical terms the interactions governing these behaviors are regulated by intracellular and extracellular physical events that are orchestrated by complex biochemical and mechanical signals.

Comprehensive Review

  • Comprehensive Review

    Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles

    • Velia M. Fowler,
    • Roberto Dominguez
    Cytoskeletal structures characterized by actin filaments with uniform lengths, including the thin filaments of striated muscles and the spectrin-based membrane skeleton, use barbed and pointed-end capping proteins to control subunit addition/dissociation at filament ends. While several proteins cap the barbed end, tropomodulins (Tmods), a family of four closely related isoforms in vertebrates, are the only proteins known to specifically cap the pointed end. Tmods are ∼350 amino acids in length, and comprise alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, and ABS2).

Computational Tools

  • Computational Tool

    AESOP: A Python Library for Investigating Electrostatics in Protein Interactions

    • Reed E.S. Harrison,
    • Rohith R. Mohan,
    • Ronald D. Gorham Jr.,
    • Chris A. Kieslich,
    • Dimitrios Morikis
    Electric fields often play a role in guiding the association of protein complexes. Such interactions can be further engineered to accelerate complex association, resulting in protein systems with increased productivity. This is especially true for enzymes where reaction rates are typically diffusion limited. To facilitate quantitative comparisons of electrostatics in protein families and to describe electrostatic contributions of individual amino acids, we previously developed a computational framework called AESOP.
  • Computational Tool

    Microvessel Chaste: An Open Library for Spatial Modeling of Vascularized Tissues

    • James A. Grogan,
    • Anthony J. Connor,
    • Bostjan Markelc,
    • Ruth J. Muschel,
    • Philip K. Maini,
    • Helen M. Byrne,
    • Joe M. Pitt-Francis
    Spatial models of vascularized tissues are widely used in computational physiology. We introduce a software library for composing multiscale, multiphysics models for applications including tumor growth, angiogenesis, osteogenesis, coronary perfusion, and oxygen delivery. Composition of such models is time consuming, with many researchers writing custom software. Recent advances in imaging have produced detailed three-dimensional (3D) datasets of vascularized tissues at the scale of individual cells.

Biophysical Letter

  • Biophysical Letter

    Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH

    • Yodai Takei,
    • Sheel Shah,
    • Sho Harvey,
    • Lei S. Qi,
    • Long Cai
    Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call “track first and identify later” for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization.

Nucleic Acids and Genome Biophysics

  • Article

    Molecular Counting with Localization Microscopy: A Bayesian Estimate Based on Fluorophore Statistics

    • Daniel Nino,
    • Nafiseh Rafiei,
    • Yong Wang,
    • Anton Zilman,
    • Joshua N. Milstein
    Superresolved localization microscopy has the potential to serve as an accurate, single-cell technique for counting the abundance of intracellular molecules. However, the stochastic blinking of single fluorophores can introduce large uncertainties into the final count. Here we provide a theoretical foundation for applying superresolved localization microscopy to the problem of molecular counting based on the distribution of blinking events from a single fluorophore. We also show that by redundantly tagging single molecules with multiple, blinking fluorophores, the accuracy of the technique can be enhanced by harnessing the central limit theorem.


  • Article

    Simultaneous Determination of Two Subdomain Folding Rates Using the “Transfer-Quench” Method

    • Gil Rahamim,
    • Dan Amir,
    • Elisha Haas
    The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements.
  • Article

    The N-Terminal Domain of Ribosomal Protein L9 Folds via a Diffuse and Delocalized Transition State

    • Satoshi Sato,
    • Jae-Hyun Cho,
    • Ivan Peran,
    • Rengin G. Soydaner-Azeloglu,
    • Daniel P. Raleigh
    The N-terminal domain of L9 (NTL9) is a 56-residue mixed α-β protein that lacks disulfides, does not bind cofactors, and folds reversibly. NTL9 has been widely used as a model system for experimental and computational studies of protein folding and for investigations of the unfolded state. The role of side-chain interactions in the folding of NTL9 is probed by mutational analysis. ϕ-values, which represent the ratio of the change in the log of the folding rate upon mutation to the change in the log of the equilibrium constant for folding, are reported for 25 point mutations and 15 double mutants.
  • Article

    Fast Protein Translation Can Promote Co- and Posttranslational Folding of Misfolding-Prone Proteins

    • Fabio Trovato,
    • Edward P. O’Brien
    Chemical kinetic modeling has previously been used to predict that fast-translating codons can enhance cotranslational protein folding by helping to avoid misfolded intermediates. Consistent with this prediction, protein aggregation in yeast and worms was observed to increase when translation was globally slowed down, possibly due to increased cotranslational misfolding. Observation of similar behavior in molecular simulations would confirm predictions from the simpler chemical kinetic model and provide a molecular perspective on cotranslational folding, misfolding, and the impact of translation speed on these processes.
  • Article

    Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A

    • Takahiro Kawamura,
    • Takuro Wakamoto,
    • Soichiro Kitazawa,
    • Shun Sakuraba,
    • Tomoshi Kameda,
    • Ryo Kitahara
    Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain.
  • Article

    Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

    • Zackary N. Scholl,
    • Weitao Yang,
    • Piotr E. Marszalek
    Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation.
  • Article

    Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer

    • Jonathan Herrmann,
    • Fatemeh Jabbarpour,
    • Paul G. Bargar,
    • John F. Nomellini,
    • Po-Nan Li,
    • Thomas J. Lane,
    • Thomas M. Weiss,
    • John Smit,
    • Lucy Shapiro,
    • Soichi Wakatsuki
    Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures.
  • Article

    Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase

    • Michael J. Reddish,
    • Robert Callender,
    • R. Brian Dyer
    Enzymes are known to exhibit conformational flexibility. An important consequence of this flexibility is that the same enzyme reaction can occur via multiple reaction pathways on a reaction landscape. A model enzyme for the study of reaction landscapes is lactate dehydrogenase. We have previously used temperature-jump (T-jump) methods to demonstrate that the reaction landscape of lactate dehydrogenase branches at multiple points creating pathways with varied reactivity. A limitation of this previous work is that the T-jump method makes only small perturbations to equilibrium and may not report conclusively on all steps in a reaction.


  • Article

    Antioxidant and Membrane Binding Properties of Serotonin Protect Lipids from Oxidation

    • Slim Azouzi,
    • Hubert Santuz,
    • Sandrine Morandat,
    • Catia Pereira,
    • Francine Côté,
    • Olivier Hermine,
    • Karim El Kirat,
    • Yves Colin,
    • Caroline Le Van Kim,
    • Catherine Etchebest,
    • Pascal Amireault
    Serotonin (5-hydroxytryptamine, 5-HT) is a well-known neurotransmitter that is involved in a growing number of functions in peripheral tissues. Recent studies have shown nonpharmacological functions of 5-HT linked to its chemical properties. Indeed, it was reported that 5-HT may, on the one hand, bind lipid membranes and, on the other hand, protect red blood cells through a mechanism independent of its specific receptors. To better understand these underevaluated properties of 5-HT, we combined biochemical, biophysical, and molecular dynamics simulations approaches to characterize, at the molecular level, the antioxidant capacity of 5-HT and its interaction with lipid membranes.

Molecular Machines, Motors, and Nanoscale Biophysics

  • Article

    Integrated Analysis of Intracellular Dynamics of MenaINV Cancer Cells in a 3D Matrix

    • Michael Mak,
    • Sarah Anderson,
    • Meghan C. McDonough,
    • Fabian Spill,
    • Jessica E. Kim,
    • Alexandra Boussommier-Calleja,
    • Muhammad H. Zaman,
    • Roger D. Kamm
    The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data.
  • Article

    Mechanosensitive Conformation of Vinculin Regulates Its Binding to MAPK1

    • Kiavash Garakani,
    • Hengameh Shams,
    • Mohammad R.K. Mofrad
    Extracellular matrix stiffness sensing by living cells is known to play a major role in a variety of cell mechanobiological processes, such as migration and differentiation. Various membrane and cytoplasmic proteins are involved in transmitting and transducing environmental signals to biochemical cascades. Protein kinases play a key role in regulating the activity of focal adhesion proteins. Recently, an interaction between mitogen-activated protein kinase (MAPK1) and vinculin was experimentally shown to mediate this process.

Cell Biophysics

  • Article

    Laser-Activated Polymeric Microcapsules for Ultrasound Imaging and Therapy: In Vitro Feasibility

    • Guillaume Lajoinie,
    • Tom van Rooij,
    • Ilya Skachkov,
    • Emilie Blazejewski,
    • Gert Veldhuis,
    • Nico de Jong,
    • Klazina Kooiman,
    • Michel Versluis
    Polymeric microcapsules with a light-absorbing dye incorporated in their shell can generate vapor microbubbles that can be spatiotemporally controlled by pulsed laser irradiation. These contrast agents of 6–8 μm in diameter can circulate through the vasculature, offering possibilities for ultrasound (molecular) imaging and targeted therapies. Here, we study the impact of such vapor bubbles on human endothelial cells in terms of cell poration and cell viability to establish the imaging and therapeutic windows.
  • Article

    Rolling Adhesion of Schizont Stage Malaria-Infected Red Blood Cells in Shear Flow

    • Anil K. Dasanna,
    • Christine Lansche,
    • Michael Lanzer,
    • Ulrich S. Schwarz
    To avoid clearance by the spleen, red blood cells infected with the human malaria parasite Plasmodium falciparum (iRBCs) adhere to the vascular endothelium through adhesive protrusions called “knobs” that the parasite induces on the surface of the host cell. However, the detailed relation between the developing knob structure and the resulting movement in shear flow is not known. Using flow chamber experiments on endothelial monolayers and tracking of the parasite inside the infected host cell, we find that trophozoites (intermediate-stage iRBCs) tend to flip due to their biconcave shape, whereas schizonts (late-stage iRBCs) tend to roll due to their almost spherical shape.
  • Article

    Nuclear Positioning and Its Translational Dynamics Are Regulated by Cell Geometry

    • A.V. Radhakrishnan,
    • Doorgesh S. Jokhun,
    • Saradha Venkatachalapathy,
    • G.V. Shivashankar
    The collective activity of several molecular motors and other active processes generate large forces for directional motion within the cell, which is vital for a multitude of cellular functions such as migration, division, contraction, transport, and positioning of various organelles. These processes also generate a background of fluctuating forces, which influence intracellular dynamics and thereby create unique biophysical signatures, which are altered in many diseases. In this study, we have used the nucleus as a probe particle to understand the microrheological properties of altered intracellular environments by using micropatterning to confine cells in two structurally and functionally extreme geometries.
  • Article

    Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

    • Boqun Liu,
    • Christoffer Åberg,
    • Floris J. van Eerden,
    • Siewert J. Marrink,
    • Bert Poolman,
    • Arnold J. Boersma
    Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Förster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size.
  • Article

    Near-Membrane Refractometry Using Supercritical Angle Fluorescence

    • Maia Brunstein,
    • Lopamudra Roy,
    • Martin Oheim
    Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the “footprint” region of the cell during image acquisition.

Systems Biophysics

  • Article

    Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

    • Christopher Y. Ko,
    • Michael B. Liu,
    • Zhen Song,
    • Zhilin Qu,
    • James N. Weiss
    Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach.
  • Article

    Mechanotransduction Dynamics at the Cell-Matrix Interface

    • Seth H. Weinberg,
    • Devin B. Mair,
    • Christopher A. Lemmon
    The ability of cells to sense and respond to mechanical cues from the surrounding environment has been implicated as a key regulator of cell differentiation, migration, and proliferation. The extracellular matrix (ECM) is an oft-overlooked component of the interface between cells and their surroundings. Cells assemble soluble ECM proteins into insoluble fibrils with unique mechanical properties that can alter the mechanical cues a cell receives. In this study, we construct a model that predicts the dynamics of cellular traction force generation and subsequent assembly of fibrils of the ECM protein fibronectin (FN).
  • Article

    Visualizing Calcium Flux in Freely Moving Nematode Embryos

    • Evan L. Ardiel,
    • Abhishek Kumar,
    • Joseph Marbach,
    • Ryan Christensen,
    • Rishi Gupta,
    • William Duncan,
    • Jonathan S. Daniels,
    • Nico Stuurman,
    • Daniel Colón-Ramos,
    • Hari Shroff
    The lack of physiological recordings from Caenorhabditis elegans embryos stands in stark contrast to the comprehensive anatomical and gene expression datasets already available. Using light-sheet fluorescence microscopy to address the challenges associated with functional imaging at this developmental stage, we recorded calcium dynamics in muscles and neurons and developed analysis strategies to relate activity and movement. In muscles, we found that the initiation of twitching was associated with a spreading calcium wave in a dorsal muscle bundle.
  • Article

    Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System

    • Dominique Zander,
    • Daniel Samaga,
    • Ronny Straube,
    • Katja Bettenbrock
    The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system.
  • Article

    Rate-Dependent Role of IKur in Human Atrial Repolarization and Atrial Fibrillation Maintenance

    • Martin Aguilar,
    • Jianlin Feng,
    • Edward Vigmond,
    • Philippe Comtois,
    • Stanley Nattel
    The atrial-specific ultrarapid delayed rectifier K+ current (IKur) inactivates slowly but completely at depolarized voltages. The consequences for IKur rate-dependence have not been analyzed in detail and currently available mathematical action-potential (AP) models do not take into account experimentally observed IKur inactivation dynamics. Here, we developed an updated formulation of IKur inactivation that accurately reproduces time-, voltage-, and frequency-dependent inactivation. We then modified the human atrial cardiomyocyte Courtemanche AP model to incorporate realistic IKur inactivation properties.
  • Article

    Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae

    • Mark Jelcic,
    • Balázs Enyedi,
    • João B. Xavier,
    • Philipp Niethammer
    Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100–300 μm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells.