Outer Junction Research Articles

Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets.

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis (Tv) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv-DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, such as TvFAP40 and TvFAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of TvFAP40 and has characteristics of a drug molecule. This first atomic model of the Tv-DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform rational design of therapeutics.

Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets.

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis ( Tv ) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv- DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, like Tv FAP40 and Tv FAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of Tv FAP40 and has characteristics of a drug molecule. This first atomic model of the Tv -DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform the rational design of therapeutics.

Doublet microtubule inner junction protein FAP20 recruits tubulin to the microtubule lattice

Doublet microtubules of eukaryotic cilia and flagella are made up of a complete A- and an incomplete B-tubule that are fused together. Of the two fusion points, the outer junction is made of tripartite tubulin connections, while the inner junction contains non-tubulin elements. The latter includes flagellar-associated protein 20 (FAP20) and Parkin co-regulated gene protein (PACRG) that together link the A- and B-tubule at the inner junction. While structures of doublet microtubules reveal molecular details, their assembly is poorly understood. In this study, we purified recombinant FAP20 and characterized its effects on microtubule dynamics. We use invitro reconstitution and cryo-electron microscopy to show that FAP20 recruits free tubulin to the existing microtubule lattice. Our cryo-electron microscopy reconstruction of microtubule:FAP20:tubulin complex reveals the mode of tubulin recruitment by FAP20 onto microtubules, providing insights into assembly steps of B-tubule closure during doublet microtubule formation.

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications.

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography

Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments.

Stability diagrams and turnstile operations of single-common-gate triple-dot single-electron devices with outer junction capacitances different from inner ones

Single-common-gate triple-dot single-electron devices with outer and inner junction capacitances, Ca and Cb, respectively, have been investigated. By changing the Ca/Cb ratio, the stability regions and overlap regions between neighboring stability regions change their shapes and then the sequences of single-electron transfers around the overlap regions also change. The most interesting phenomenon is that the arrangement of the stability regions along the gate voltage axis, which normally follows the total excess electron number in the three islands, is partially inverted. That is, the electron configuration in the three islands with the smaller total excess electron number is stable with a higher gate voltage range. Even when in such a situation, the turnstile operation is still possible though the sequence of single-electron transfer changes. The inversion of the arrangement can be explained in terms of the charging energies.

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

Electrical control of capacitance dispersion for single-electron turnstile operation in common-gated junction arrays

We have studied single-electron turnstile operation in common-gated one-dimensional arrays of four tunnel junctions (three dots) having inhomogeneous junction capacitances. Analytical calculations show that the source-drain voltage range with a current plateau due to single-electron turnstile operation is increased when the outer two tunnel capacitances are adjusted to be smaller than the inner ones. In fact, we have demonstrated in phosphorous-doped silicon-on-insulator field-effect transistors (FETs) that back-gate voltage works to assist the turnstile operation, which is primarily ascribed to electrical control of junction capacitance dispersion, i.e., reduction in outer junction capacitances. As a result, postfabrication control of capacitance dispersion in multijunction FETs can be achieved, resulting in successful turnstile operation.

Phase synchronization in an array of driven Josephson junctions

We consider an array of N Josephson junctions connected in parallel and explore the condition for chaotic synchronization. It is found that the outer junctions can be synchronized while they remain uncorrelated to the inner ones when an external biasing is applied. The stability of the solution is found out for the outer junctions in the synchronization manifold. Symmetry considerations lead to a situation wherein the inner junctions can synchronize for certain values of the parameter. In the presence of a phase difference between the applied fields, all the junctions exhibit phase synchronization. It is also found that chaotic motion changes to periodic in the presence of phase differences.

Floral Nectar Secretion and Ploidy in Brassica rapaand B. napus(Brassicaceae). II. Quantified Variability of Nectary Structure and Function in Rapid-cycling Lines

Haploid, diploid and tetraploid lines of Brassica rapaL. (syn. campestris), and allotetraploid B. napusL., were examined to determine the influence of ploidy on floral features, particularly nectary morphology and anatomy, and to relate nectary structure to nectar production capacity. Except for haploids, all lines were rapid-cycling. Average flower dry weight, and petal length and width, were in the descending order B. napus> B. rapa (4n) >2 n> n. Pollen grains of 4 nplants were larger than those of 2 nplants; haploids lacked pollen. All lines developed nectaries. Typically, each flower produced two pairs of nectaries, of different types and nectar production capacity. Normally, each lateral gland was located above the base of a short stamen, and together this pair yielded most of a flower 's nectar carbohydrate. Each median nectary arose at the outer junction of the bases of two adjacent long stamens. All lateral nectaries received a vascular supply of phloem alone, but median glands received reduced amounts of phloem, or lacked vasculature altogether. Most nectaries were solitary, but 14% of all flowers, and especially those of 2 n B. rapa,had at least one median and lateral gland connected. Obvious variation existed in nectary morphology between ploidy levels, between flowers of the same plant, and even within flowers. Ten forms of each nectary type were recognized. Plants producing the most nectar carbohydrate had high frequencies of lateral nectaries which were symmetrical, unfurrowed swellings. Tetraploids of B. rapahad both the highest frequencies of furrowed lateral glands, and of isolated segments of nectarial tissue at that position. Even these separated nectarial outgrowths received phloem and produced a nectar droplet. At the median location, nectaries were commonly of two forms: peg- or fan-shaped. Lobes on median nectaries, up to four per nectary, were detected in almost half of glands of 4 nflowers examined; lobes were absent in haploids.

The anatomy of flagellar microtubules: polarity, seam, junctions, and lattice.

Although the overall structures of flagellar and cytoplasmic microtubules are understood, many details have remained a matter of debate. In particular, studies of the arrangement of tubulin subunits have been hampered by the low contrast of the tubulin subunits. This problem can now be addressed by the kinesin decoration technique. We have shown previously that the recombinant kinesin head domain binds to beta-tubulin, thus enhancing the contrast between alpha- and beta-tubulin in the electron microscope; this allows one to study the arrangement of tubulin dimers. Here we describe the lattices of the four different types of microtubules in eukaryotic flagellar axonemes (outer doublet A and B, central pair C1 and C2). They could all be labeled with kinesin head with an 8-nm axial periodicity (the tubulin dimer repeat), and all of them showed the B-surface lattice. This lattice is characterized by a 0.92-nm stagger between adjacent protofilaments. The B-lattice was observed on the axonemal microtubules as well as on extensions made by polymerizing porcine brain tubulin onto axonemal microtubules in the proximal and distal directions. This emphasizes that axonemal microtubules serve as high fidelity templates for seeding microtubules. The presence of a B-lattice implies that there must be a helical discontinuity ("seam") in the wall. This discontinuity is now placed near protofilaments A1 and A2 of the A-tubule, close to the inner junction between A- and B-microtubules. The two junctions differ in structure: the protofilaments of the inner junction (A1-B10) are staggered roughly by half a dimer, those of the outer junction (A10-B1) are roughly in register. Of the two junctions the inner one appears to have the stronger bonds, whereas the outer one is more labile and opens up easily, generating "composite sheets" with chevron patterns from which the polarity can be deduced (arrow in the plus direction). Decorated microtubules have a clear polarity. We find that all flagellar microtubules have the same polarities. The orientation of the dimers is such that the plus end terminates with a crown of alpha subunits, the minus end terminates with beta subunits which thus could be in contact with gamma-tubulin at the nucleation centers.

Transient analysis of p-n-p-n optoelectronic devices

p-n-p-n optoelectronic devices were analyzed using a coupled junction model, an equivalent circuit model, and a physical model. The accuracy of these models was confirmed by comparison with experimental data. The result of the authors' analysis was a new understanding of the dynamic properties of p-n-p-n devices. The authors found that the onset of switching depends only on the voltage across the forward-biased outer junctions, not on the total applied bias. Turn-on delay is dependent on the rate of voltage change across the outer junctions and the efficiency of the laser emitter. Fast turn-off of two-terminal devices is theoretically possible by applying a reverse bias during the turn-off transient. dV/dt induced switching can be avoided by proper design. Based on the authors' models, the maximum large-signal operating frequency of two-terminal p-n-p-n devices was estimated to be approximately 240 MHz. >

Quantum interference Josephson logic devices

Experiments with latching and nonlatching quantum interference logic devices are described. The device is a three-junction interferometer, switching with control currents of <0.5 mA. Simple logic functions have been performed with dc-powered nonlatching devices having a power dissipation of <40 nW in continuous operation.