Hook Region Research Articles

CESTA, a positive regulator of brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroid hormones that are essential for the development of plants. A tight control of BR homeostasis is vital for modulating their impact on growth responses. Although it is recognized that the rapid adaptation of de novo synthesis has a key role in adjusting required BR levels, our knowledge of the mechanisms governing feedback control is limited. In this study, we identify the transcription factor CESTA as a regulator of BR biosynthesis. ces-D was isolated in a screen of Arabidopsis mutants by BR over-accumulation phenotypes. Loss-of-function analysis and the use of a dominant repressor version revealed functional overlap among CESTA and its homologues and confirmed the role of CESTA in the positive control of BR-biosynthetic gene expression. We provide evidence that CESTA interacts with its homologue BEE1 and can directly bind to a G-box motif in the promoter of the BR biosynthesis gene CPD. Moreover, we show that CESTA subnuclear localization is BR regulated and discuss a model, in which CESTA interplays with BEE1 to control BR biosynthesis and other BR responses.

Hook formation and mechanical properties in AZ31 friction stir spot welds

Hook formation and the failure load properties of AZ31 friction stir spot welds are investigated in friction stir spot welds made between wrought and heat-treated AZ31 sheets having widely different average grain sizes. Tool shoulder penetration into the surface of the upper sheet and pin penetration into the lower sheet during the dwell period provides the driving force for the displacement of lower sheet material upwards, the formation of hook regions and increasing stir zone and bonded widths. Once formed, hook regions are displaced outwards from the axis of the rotating tool in AZ31 spot welds made using dwell times from 1 s to 4 s, and are located at the boundary between the thermal-mechanical affected zone (TMAZ) and the heat affected zone (HAZ). Particularly poor failure load properties are found in AZ31 spot welds made without the application of a dwell period, since the bonded width is extremely small (about 280 μm). The failure load properties are highest in AZ31 spot welds made using a dwell time of 1 s and are decreased when the dwell period is further extended, since the beneficial influence of increasing bonded width is outweighed by the detrimental effects resulting from the increased distance from the sheet intersection to the tip of the hook region, angle of curvature of the hook region and shoulder penetration depth values.

Microstructures and mechanical properties in dissimilar AZ91/AZ31 spot welds

The stir zone microstructures and mechanical properties of dissimilar AZ91/AZ31 friction stir spot welds made using different tool designs and tool rotational speed settings are investigated. Intermingled AZ91 and AZ31 lamellae are formed in the stir zones of dissimilar spot welds made using threaded, three-flat/0.7 mm/threaded and three-flat/no-thread tools and tool rotational speeds ranging from 1500 to 3000 rpm. The intermingled lamellae have chemical compositions, which are similar to those of the upper and lower sheets in the dissimilar sandwich. The flats on the rotating tool facilitate the downward transfer of upper and lower sheet materials in the location close to the pin periphery and therefore intermingled AZ91 and AZ31 lamellae are formed in the stir zones of dissimilar spot welds produced using a three-flat tool without a thread. The distance (Y) from the tip of the hook region to the keyhole periphery has a dominant influence on the mechanical properties of dissimilar AZ91/AZ31 spot welds, since the hook regions are curved inwards towards the axis of the rotating tool. The highest failure load properties and largest Y-values are found in dissimilar spot welds made using threaded and three-flat/0.7 mm/threaded tools and tool rotational speeds from 1500 to 3000 rpm. Dissimilar spot welds made using a rotational speed of 1000 rpm have the smallest Y-values and the lowest failure load properties.

Microstructural features and mechanical properties of AM60 and AZ31 friction stir spot welds

The microstructural features and mechanical properties of AM60 and AZ31 friction stir spot welds are investigated in joints made using different tool designs (threaded and three-flat/threaded tools) and dwell time settings. Since the hook regions are curved inwards towards the keyhole periphery in AM60 friction stir spot welds made using threaded and three-flat/threaded tools and different dwell time settings, the distance from the tip of the hook region to the keyhole periphery mainly determines their failure load properties. In contrast, the hook regions are curved outwards from the axis of the rotating tool in AZ31 friction stir spot welds and their failure strength properties are determined by the bonded width, the distance from the tip of the hook region to the sheet intersection, the depth of tool shoulder penetration into the surface of the upper sheet and the distance from the tip of the hook region to the top of the welded joint. The highest failure load properties are found in AM60 friction stir spot welds made using a three-flat/threaded tool and dwell times of 3 and 4 s, since the distance from the tip of the hook region to the keyhole periphery is highest in these joints. The highest failure load properties are found in AZ31 friction stir spot welds made using the three-flat/threaded tool and a dwell time of 1 s, since these joints have the largest bonded widths, distances from the tips of the hook regions to the sheet intersection, and limited penetration of the tool shoulder into the surface of the upper sheet (about 0.5 mm).

Structural studies of phosphoglucose isomerase fromMycobacterium tuberculosisH37Rv

Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.

Chronic Reduction of Endocochlear Potential Reduces Auditory Nerve Activity: Further Confirmation of an Animal Model of Metabolic Presbyacusis

Gerbils aged in quiet show a decline of the endocochlear potential (EP) and elevated auditory nerve compound action potential (CAP) thresholds. However, establishing a direct relationship between an age-related reduction in the EP and changes in the activities of primary auditory neurons is difficult owing to the complexity of age-related histological changes in the cochlea. To address this issue, we developed a young gerbil model of "metabolic" presbyacusis that uses an osmotic pump to deliver furosemide into the round window niche for 7 days, resulting in a chronically reduced EP. In this model, the only major histopathologic changes were restricted to the hook region of the cochlea and consisted of loss of strial intermediate cells and massive edema in the lateral wall. The morphological and physiological evidence suggests that the cochlea can adapt to furosemide application over time. The morphology of spiral ganglion cells and hair cells appeared normal throughout the cochlea. CAP responses and EP values in this model are similar to those of quiet-aged ears. The spontaneous activity of single auditory fibers (n = 188) was assessed in 15 young gerbils treated with furosemide for 7 days. The percentage of recorded low-spontaneous rate (SR) fibers at characteristic frequencies (CFs) > or = 6 kHz was significantly lower in furosemide-treated than in control ears. Recovery function tests of CAP responses after prior stimulation also showed a decline in activity of the low-SR population with CFs > or = 6 kHz in the treated cochleas. A similar loss in the activity of low-SR fiber has been previously shown in quiet-aged gerbils. These results suggest that dysfunction of the cochlear lateral wall and subsequent chronic reduction in the EP can directly affect the activity patterns of primary auditory neurons in a manner similar to that seen in aged gerbils.

Influence of tool design on mechanical properties of AZ31 friction stir spot welds

The influence of tool design on the energy output, microstructural features and overlap shear strength properties of friction stir spot welds made of AZ31 base material is examined. The mechanical properties of AZ31 friction stir spot welds made using three-flat/threaded tools are superior to those in joints made using a tool with a threaded pin at all tool rotational speed settings. It is proposed that the failure load properties are optimised when the friction stir spot welding operation is carried out in such a manner that it produces a large bonded width, a small v/t ratio (the height of the hook region above the sheet intersection divided by the thickness of the upper sheet) and a hook region, which is curved outwards from the tool axis.

Extension of chains composed of freely joined elastic segments

A relatively simple formula for the mean distance between the ends of a model chain composed of elastic freely joined segments as a function of an extending force applied to its ends was derived. It was assumed that the stiffness coefficient, which characterizes the ability of the segment to extend and contract, is rather high. In the limit of infinite stiffness the relationship obtained transforms into the Langevin function. The formula predicts the existence of two Hook regions in the force-relative extension dependence. The first of them is characterized by small extensions, whereas the second one, by extensions exceeding the equilibrium contour length. The existence of the latter is confirmed by recent experiments. The magnitude of fluctuations of the relative extension was estimated. The obtained relationships for the relative extension and its scatter are in close agreement with the results of computer simulations performed using collisional molecular dynamics for chains composed of 25, 50, 100, and 200 segments.

Coprinus cinereus rad50Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and Meiotic Recombination

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.

Structural requirements for calmodulin binding to membrane‐associated guanylate kinase homologs

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.

Determinants of the voltage dependence of G protein modulation within calcium channel beta subunits.

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone.

Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis

The archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella-associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function (flaC, flaD, flaE, flaF, flaG and flaH), an ATPase (flaI) and a membrane protein (flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full-length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella-associated genes. Markerless in-frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the DeltaFlaB3 and FlaD M(191)I strains, all mutants were non-motile by light microscopy and non-flagellated by electron microscopy. A detailed examination of the DeltaFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function.

Functional Modularity of the β-Subunit of Voltage-Gated Ca 2+ Channels

The β-subunit of voltage-gated Ca 2+ channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and α interaction domain (AID), the high-affinity binding site in the pore-forming α 1 subunit. Here we show that the AID-GK domain interaction is necessary for β-subunit-stimulated Ca 2+ channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four β-subunit subfamilies in modulating P/Q-type Ca 2+ channel gating and demonstrate that the β-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the β-subunit to the α 1 subunit, enabling α 1- β pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four β-subunit subfamilies its distinctive modulatory properties.

Anatomy of the Round Window and Hook Region of the Cochlea With Implications for Cochlear Implantation and Other Endocochlear Surgical Procedures

The goal of this study was to create a three-dimensional model of the anatomy of the hook region to identify the optimal site for cochleostomy in cochlear implant surgery. The anatomy of the hook region is complex, and spatial relationships can be difficult to evaluate using two-dimensional histological slides or cadaveric temporal bones. The right temporal bone of a 14-year-old adolescent boy was used to create a three-dimensional model. Sections containing the round window membrane (RWM) and surrounding cochlear structures were stained, digitized, and imported into a general purpose three-dimensional rendering and analysis software program (Amira, version 4.1). Three-dimensional models of the RWM, basilar membrane, osseous spiral lamina, spiral ligament, cochlear aqueduct, inferior cochlea vein, scala media, ductus reuniens, scala vestibuli, scala tympani, and surrounding bone were generated. The relationship between these structures and the RWM and adjacent otic capsule was evaluated. Histological sections from a different temporal bone were also analyzed. This temporal bone was sectioned in a plane perpendicular to the axis corresponding to the surgical view of the RWM, seen through the facial recess. The anteroinferior margin of the RWM or adjacent otic capsule was identified as the site for a cochleostomy that will avoid damage to critical cochlear structures and allow implantation directly into the scala tympani. The model can be downloaded from: https://research.meei.harvard.edu/otopathology/3dmodels. This three-dimensional model has implications for surgical procedures to the inner ear that aim to minimize insertional trauma.

Anatomy of the Round Window and Hook Region of the Cochlea With Implications for Cochlear Implantation and Other Endocochlear Surgical Procedures

Anatomy of the Round Window and Hook Region of the Cochlea With Implications for Cochlear Implantation and Other Endocochlear Surgical Procedures

Dimerization of the Rad50 protein is independent of the conserved hook domain

The Mre11 complex (Mre11-Rad50-Nbs1) is involved in a diverse array of DNA metabolic processes including the response to DNA double-strand breaks (DSBs). The structure of Rad50 plays a key role in the DNA-binding and end-bridging activity of the complex. An interesting feature within the central portion of the Rad50 protein is the Rad50 hook region that is defined by the highly conserved CXXC motif. The structure of the Pyrococcus furiosus Rad50 hook region revealed an intermolecular dimerization of Rad50 through the coordination of a zinc ion by the four cysteines. Biochemical and genetic analysis in Saccharomyces cerevisiae have shown that mutations in the conserved cysteines impact all functions of the Mre11 complex including interaction with Mre11, increased sensitivity to DSB inducing agents, telomere maintenance and intrachromosomal association. Mutations in the yeast hook domain can lead to increased chromosome fragmentation, suggesting that the hook domain of Rad50 is essential for the tethering of chromosome ends. In this study, we have examined the effects of mutating the key cysteine residues in the hook domain of human Rad50 (hRad50), focusing on the interactions Rad50 has with itself, Mre11 and DNA. Our results reveal that mutation of the conserved cysteine residues abrogates dimerization at the hook domain in hRad50; however, disrupting dimerization at this domain does not appear to impair the interaction of full-length hRad50 with itself and hMre11 or affect DNA-binding activity of the hMre11-Rad50 complex.

Structural Basis for Rab11-mediated Recruitment of FIP3 to Recycling Endosomes

The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.

Physiological genomics in PG and beyond: October to December 2005

physiological genomics ( PG ) publishes, on a quarterly basis, brief editorial perspectives entitled “Physiological genomics in PG and beyond.” These editorial perspectives provide brief, accessible highlights of the studies published in PG in the most recent quarter, and relate those studies to

Differential Expression of KCNQ4 in Inner Hair Cells and Sensory Neurons Is the Basis of Progressive High-Frequency Hearing Loss

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Time course of auditory impairment in mice lacking the electroneutral sodium bicarbonate cotransporter NBC3 ( slc4a7)

Mice with a targeted disruption of the gene encoding the stilbene-insensitive electroneutral sodium bicarbonate cotransporter (NBC3; slc4a7) exhibit cochlear and retinal degeneration. To establish the progressive nature of sensory cells loss in slc4a7 −/− deficient mice, we studied the morphology of cochleas of slc4a7 −/− and slc4a7 +/+ mice from postnatal day two (P2) to ninety (P90). Cell death was evaluated in slc4a7 −/− cochleas using the TUNEL technique and caspase-3 immunoreactivity. The time course of NBC3 expression in the cochlea was assessed by immunohistochemistry using an antibody against NBC3. Between P2 and P8, slc4a7 −/− mice cochlea exhibit normal morphology. There was a normal complement of inner and outer hair cells from the hook to the apical region. At P15, slc4a7 −/− mice cochlea inner and outer hair cells were still present at the hook region, and vacuoles were seen underneath Hensen's cells. At P21, inner and outer hair cells were degenerated in this region. Between P30 and P90, there was a pronounced loss of hair cells and spiral ganglia neurons. Morphological analysis of the spiral ligament showed a progressive loss of type II and IV fibrocytes beginning at day 21. Transmission electron microscopy observations at P30 and P90 revealed that type II and IV fibrocytes showed shrinkage and vacuolization. In addition, hair cells were deteriorated with evidence of shrinkage and picnotic nuclei. TUNEL staining showed apoptotic cells at P8 in the organ of Corti at the basal region of the cochlea. At P15, caspase-3 immunoreactivity was present in supporting cells of the organ of Corti. NBC3 mild immunoreactivity was detected in the organ of Corti at P11. There was an increase in the expression of NBC3 in the spiral ligament between P17 and P19. From P21 to P90, NBC3 expression was confined to the spiral ligament and inner and outer sulcus cells. The vestibular sensory epithelia from slc4a7 −/− mice were normal from P2 to P90. Damage of the sensory epithelia at the high frequency zone of the cochlea suggests that NBC3 may play an important physiological role in this region.