Dynamic microtubules slow down during their shrinkage phase.

Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but rather a multistep process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multistate, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth, which led to multistep catastrophe. This suggests that the aging associated with growth is also manifested during shrinkage, with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip.

Surfing on Membrane Waves: Microvilli, Curved Membranes, and Immune Signaling.

Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.

Purification of Ciliary Tubulin from Chlamydomonas reinhardtii.

Cilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule-based structure known as the axoneme, which serves as a scaffold and is crucial for the function of cilia. Despite their key roles, the biochemical and biophysical properties of the ciliary proteins are poorly understood. To address this issue, we have developed a novel method to purify functional tubulins from different parts of the axoneme, namely the central pair and B-tubule. We use the biflagellate green alga Chlamydomonas reinhardtii, a model organism for studying cilia due to the conserved structure of this organelle, availability of genetic tools and a large collection of mutant strains. Our method yields highly purified functional axonemal tubulins in sufficient quantities to be used for in vitro biochemical and biophysical studies, such as microtubule dynamic assays. It takes 7 to 8 days to grow enough cells; the isolation of the flagella and the purification of the axonemal tubulins require an additional two full days.© 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and harvest of large volume of cell culture Support Protocol: Assembly of homemade concentration apparatus Basic Protocol 2: Isolation of flagella Basic Protocol 3: Tubulin extraction and purification.

The dynamic and structural properties of axonemal tubulins support the high length stability of cilia.

Cilia and flagella play essential roles in cell motility, sensing and development. These organelles have tightly controlled lengths, and the axoneme, which forms the core structure, has exceptionally high stability. This is despite being composed of microtubules that are often characterized as highly dynamic. To understand how ciliary tubulin contribute to stability, we develop a procedure to differentially extract tubulins from different components of axonemes purified from Chlamydomonas reinhardtii, and characterize their properties. We find that the microtubules support length stability by two distinct mechanisms: low dynamicity, and unusual stability of the protofilaments. The high stability of the protofilaments manifests itself in the formation of curved tip structures, up to a few microns long. These structures likely reflect intrinsic curvature of GTP or GDP·Pi tubulin and provide structural insights into the GTP-cap. Together, our study provides insights into growth, stability and the role of post-translational modifications of axonemal microtubules.

Programmed DNAzyme-Triggered Dissolution of DNA-Based Hydrogels: Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades.

Acrylamide/acrylamide-modified nucleic acid copolymer chains provide building units for the construction of acrylamide-DNA hydrogels. Three different hydrogels are prepared by the cross-linking of the acrylamide-DNA chains with metal ion-dependent DNAzyme sequences and their substrates. The metal ion-dependent DNAzyme sequences used in the study include the Cu(2+)-, Mg(2+)-, and Zn(2+)-dependent DNAzymes. In the presence of the respective metal ions, the substrates of the respective DNAzymes are cleaved, leading to the separation of the cross-linking units and to the dissolution of the hydrogel. The different hydrogels were loaded with a fluorophore-modified dextran or with a fluorophore-functionalized glucose oxidase. Treatment of the different hydrogels with the respective ions led to the release of the loaded dextran or the enzyme, and the rates of releasing of the loaded macromolecules followed the order of Cu(2+) > Mg(2+) > Zn(2+). Also, the different hydrogels were loaded with the enzymes ß-galactosidase (ß-Gal), glucose oxidase (GOx), or horseradish peroxidase (HRP). In the presence of the appropriate metal ions, the respective hydrogels were dissolved, resulting in the activation of the ß-Gal/GOx or GOx/HRP bienzyme cascades and of the ß-Gal/GOx/HRP trienzyme cascade.

Gossypol-cross-linked boronic acid-modified hydrogels: a functional matrix for the controlled release of an anticancer drug.

Anticancer drug gossypol cross-links phenylboronic acid-modified acrylamide copolymer chains to form a hydrogel matrix. The hydrogel is dissociated in an acidic environment (pH 4.5), and its dissociation is enhanced in the presence of lactic acid (an α-hydroxy carboxylic acid) as compared to formic acid. The enhanced dissociation of the hydrogel by lactic acid is attributed to the effective separation of the boronate ester bridging groups through the formation of a stabilized complex between the boronic acid substituent and the lactic acid. Because lactic acid exists in cancer cells in elevated amounts and the cancer cells' environment is acidic, the cross-linked hydrogel represents a stimuli-responsive matrix for the controlled release of gossypol. The functionality is demonstrated and characterized by rheology and other spectroscopic means.

Catalytic nucleic acids (DNAzymes) as functional units for logic gates and computing circuits: from basic principles to practical applications.

This feature article addresses the implementation of catalytic nucleic acids as functional units for the construction of logic gates and computing circuits, and discusses the future applications of these systems. The assembly of computational modules composed of DNAzymes has led to the operation of a universal set of logic gates, to field programmable logic gates and computing circuits, to the development of multiplexers/demultiplexers, and to full-adder systems. Also, DNAzyme cascades operating as logic gates and computing circuits were demonstrated. DNAzyme logic systems find important practical applications. These include the use of DNAzyme-based systems for sensing and multiplexed analyses, for the development of controlled release and drug delivery systems, for regulating intracellular biosynthetic pathways, and for the programmed synthesis and operation of cascades.

pH-stimulated DNA hydrogels exhibiting shape-memory properties.

Nucleic acid-functionalized polyacrylamide chains that are cooperatively cross-linked by i-motif and nucleic acid duplex units yield, at pH 5.0, DNA hydrogels exhibiting shape-memory properties. Separation of the i-motif units at pH 8.0 dissolves the hydrogel into a quasi-liquid phase. The residual duplex units provide, however, a memory code in the quasi-liquid allowing the regeneration of the hydrogel shape at pH 5.0.

Ternary DNA computing using 3 × 3 multiplication matrices.

Non-Boolean computations implementing operations on multi-valued variables beyond base 2 allow enhanced computational complexity. We introduce DNA as a functional material for ternary computing, and in particular demonstrate the use of three-valued oligonucleotide inputs to construct a 3 × 3 multiplication table. The system consists of two three-valued inputs of -1; 0; +1 and a fluorophore/quencher functional hairpin acting as computational and reporter module. The interaction of the computational hairpin module with the different values of the inputs yields a 3 × 3 multiplication matrix consisting of nine nanostructures that are read out by three distinct fluorescence intensities. By combining three different hairpin computational modules, each modified with a different fluorophore/quencher pair, and using different sets of inputs, the parallel operation of three multiplication tables is demonstrated.

Multiplexed analysis of genes and of metal ions using enzyme/DNAzyme amplification machineries.

The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg(2+)-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag(+), Hg(2+)). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic acid scaffolds that include recognition sites for the two genes and replication tracks that yield the nicking domains for Nt.BbvCI and two different Mg(2+)-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg(2+)-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F1 and/or F2 as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TP53 genes correspond to 0.1 nM. Similarly, two different nucleic acid scaffolds that include Ag(+)-ions or Hg(2+)-ions recognition sequences and the replication tracks that yield the Nt.BbvCI nicking domains and the respective Mg(2+)-dependent DNAzyme sequences are implemented as nicking/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds.

Switchable bifunctional stimuli-triggered poly-N-isopropylacrylamide/DNA hydrogels.

DNA-tethered poly-N-isopropylacrylamide copolymer chains, pNIPAM, that include nucleic acid tethers have been synthesized. They are capable of inducing pH-stimulated crosslinking of the chains by i-motif structures or to be bridged by Ag(+) ions to form duplexes. The solutions of pNIPAM chains undergo crosslinking at pH 5.2 or in the presence of Ag(+) ions to form hydrogels. The hydrogels reveal switchable hydrogel-to-solution transitions by the reversible crosslinking of the chains at pH 5.2 and the separation of the crosslinking units at pH 7.5, or by the Ag(+) ion-stimulated crosslinking of the chains and the reverse dissolution of the hydrogel by the cysteamine-induced elimination of the Ag(+) ions. The DNA-crosslinked hydrogels are thermosensitive and undergo reversible temperature-controlled hydrogel-to-solid transitions. The solid pNIPAM matrices are protected against the OH(-) or cysteamine-stimulated dissociation to the respective polymer solutions.

Ion-responsive hemin-G-quadruplexes for switchable DNAzyme and enzyme functions.

Programmed nucleic acid sequences undergo K(+) ion-induced self-assembly into G-quadruplexes and separation of the supramolecular structures by the elimination of K(+) ions by crown ether or cryptand ion-receptors. This process allows the switchable formation and dissociation of the respective G-quadruplexes. The different G-quadruplex structures bind hemin, and the resulting hemin-G-quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K(+) ion/receptor switchable systems are described: 1) The K(+) -induced self-assembly of the Mg(2+) -dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G-quadruplexes as bridging unit. 2) The K(+) -induced stabilization of the anti-thrombin G-quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K(+) -induced opening of DNA tweezers through the stabilization of G-quadruplexes on the "tweezers' arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin-G-quadruplex DNAzyme).

Reversible Ag(+)-crosslinked DNA hydrogels.

DNA hydrogels, consisting of Y-shaped nucleic acid subunits or of nucleic acid-functionalized acrylamide chains, undergo switchable gel-to-solution transitions. The Ag(+)-stimulated formation of cytosine-Ag(+)-cytosine complexes results in the crosslinking of the units to yield the hydrogels, while the cysteamine-induced elimination of the Ag(+) ions dissociates the hydrogels into a solution phase.

Self-assembly of luminescent Ag nanocluster-functionalized nanowires.

Two different methods to self-assemble red- or yellow-luminescent nucleic acids-stabilized Ag nanoclusters (NCs) nanowires are presented. By one method, the autonomous hybridization-polymerization process between two nucleic acids leads to polymer chains consisting of sequence-specific loops for the stabilization of the red- or yellow-emitting Ag NCs. By the other method, the nucleic acid-triggered hybridization chain reaction (HCR) involving the cross-opening of two functional hairpins leads to sequence-specific DNA loops and a nucleic acid scaffold that stabilize the respective red- or yellow-emitting Ag NCs. The micrometer-long luminescent Ag NC-functionalized nanowires are imaged by AFM and confocal microscopy.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

Fluorescent DNA hydrogels composed of nucleic acid-stabilized silver nanoclusters.

Y-shaped DNA units functionalized with Ag-nanoclusters are crosslinked by nucleic acids to yield fluorescent hydrogels with controlled luminescence properties.

Switchable catalytic acrylamide hydrogels cross-linked by hemin/G-quadruplexes.

Copolymer chains consisting of acrylamide units and guanine (G)-containing oligonucleotide-tethered acrylamide units undergo, in the presence of K(+) ions, cross-linking by G-quadruplexes to yield a hydrogel. The hydrogel is dissociated upon addition of 18-crown-6 ether that traps the K(+) ions. Reversible formation and dissociation of the hydrogel is demonstrated by the cyclic addition of K(+) ions and 18-crown-6 ether, respectively. Formation of the hydrogel in the presence of hemin results in a hemin/G-quadruplex-cross-linked catalytic hydrogel mimicking the function of horseradish peroxidase, reflected by the catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), ABTS(2-), by H2O2 to ABTS(·-) and by the catalyzed generation of chemiluminescence in the presence of luminol/H2O2. Cyclic "ON" and "OFF" activation of the catalytic functions of the hydrogel are demonstrated upon the formation of the hydrogel in the presence of K(+) ions and its dissociation by 18-crown-6 ether, respectively. The hydrogel is characterized by rheology measurements, circular dichroism, and probing its chemical and photophysical properties.

Logic reversibility and thermodynamic irreversibility demonstrated by DNAzyme-based Toffoli and Fredkin logic gates.

The Toffoli and Fredkin gates were suggested as a means to exhibit logic reversibility and thereby reduce energy dissipation associated with logic operations in dense computing circuits. We present a construction of the logically reversible Toffoli and Fredkin gates by implementing a library of predesigned Mg(2+)-dependent DNAzymes and their respective substrates. Although the logical reversibility, for which each set of inputs uniquely correlates to a set of outputs, is demonstrated, the systems manifest thermodynamic irreversibility originating from two quite distinct and nonrelated phenomena. (i) The physical readout of the gates is by fluorescence that depletes the population of the final state of the machine. This irreversible, heat-releasing process is needed for the generation of the output. (ii) The DNAzyme-powered logic gates are made to operate at a finite rate by invoking downhill energy-releasing processes. Even though the three bits of Toffoli's and Fredkin's logically reversible gates manifest thermodynamic irreversibility, we suggest that these gates could have important practical implication in future nanomedicine.

Detection of metal ions (Cu2+, Hg2+) and cocaine by using ligation DNAzyme machinery.

The Cu(2+)-dependent ligation DNAzyme is implemented as a biocatalyst for the colorimetric or chemiluminescence detection of Cu(2+) ions, Hg(2+) ions, or cocaine. These sensing platforms are based on the structural tailoring of the sequence of the Cu(2+)-dependent ligation DNAzyme for specific analytes. The tethering of a subunit of the hemin/G-quadruplex DNAzyme to the ligation DNAzyme sequence, and the incorporation of an imidazole-functionalized nucleic-acid sequence, which acts as a co-substrate for the ligation DNAzyme that is tethered to the complementary hemin/G-quadruplex subunit. In the presence of different analytes, Cu(2+) ions, Hg(2+) ions, or cocaine, the pretailored Cu(2+)-dependent ligation DNAzyme sequence stimulates the respective ligation process by combining the imidazole-functionalized co-substrate with the ligation DNAzyme sequence. These reactions lead to the self-assembly of stable hemin/G-quadruplex DNAzyme nanostructures that enable the colorimetric analysis of the substrate through the DNAzyme-catalyzed oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS(2-), by H(2)O(2) into the colored product ABTS(·-), or the chemiluminescence detection of the substrate through the DNAzyme-catalyzed oxidation of luminol by H(2)O(2). The detection limits for the sensing of Cu(2+) ions, Hg(2+) ions, and cocaine correspond to 1 nM, 10 nM and 2.5 µM, respectively. These different sensing platforms also reveal impressive selectivities.

Nucleic acid driven DNA machineries synthesizing Mg2+-dependent DNAzymes: an interplay between DNA sensing and logic-gate operations.

Polymerase/nicking enzymes and nucleic-acid scaffolds are implemented as DNA machines for the development of amplified DNA-detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase-induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg(2+)-dependent DNAzyme or the Mg(2+)-dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher-functionalized nucleic-acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled-AND gates, and the DNA-analyte targets represent two nucleic acid sequences of the smallpox viral genome.