Double-faced micelles from water-soluble polymers.
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Magnetic Resonance Spectroscopy↗
Biomedical subjects
Stephen M King
Publications and source records attributed to Stephen M King.
Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.
Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+ -binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.
Animals↗
Upper critical solution temperature phase behavior, composition fluctuations, and complex formation in poly (vinyl methyl ether)/D2O solutions: small-angle neutron-scattering experiments and wertheim lattice thermodynamic perturbation theory predictions.
Small-angle neutron-scattering measurements are presented for homogeneous mixtures of poly (methyl vinyl ether) (PVME) and deuterium oxide (D(2)O) at high polymer concentrations and for temperatures lower than the equilibrium melting point of the solvent. The experimental data are analyzed to give values for the second-order compositional derivative of the Gibbs energy and the Ornstein-Zernike correlation length. The experimental data together with earlier SANS data determined at higher temperatures cannot be represented with an extended Flory-Huggins (F-H) interaction function depending on composition and temperatures. The experimental data confirm the existence of a narrow upper critical solution temperature (UCST) miscibility gap at high concentrations in agreement with theoretical predictions of the Wertheim lattice thermodynamic perturbation theory (LTPT). The Wertheim LTPT incorporates the influence of hydrogen bonding and predicts not only the existence of bimodal lower critical solution temperature (LCST) phase behavior but also the occurrence of highly unconventional two narrow adjacent UCST miscibility gaps. Finally, the experimental data do not support the existence of a stable molecular complex at the investigated temperatures and compositions. Even at the lowest investigated temperature, the energy required to induce typical Ornstein-Zernike-like concentration fluctuations is smaller than the thermal energy. Also, in this case, the Wertheim LTPT provides a theoretical basis to understand the formation of polymer solvent associations in PVME/water.
Journal Article↗
Characterising the size and shape of polyamidoamines in solution as a function of pH using neutron scattering and pulsed-gradient spin-echo NMR.
Bioresponsive polymers are being developed as synthetic viral mimetics to enhance the intracellular delivery of macromolecular therapeutic agents such as genes, proteins and peptides. In this context we have designed pH-responsive, amphoteric polyamidoamines (PAAs) which change conformation on passing from a neutral pH (extracellular) to an acidic pH (endosomal and lysosomal) environments. PAAs have already demonstrated cytosolic delivery of genes and non-permeant toxins (e.g. gelonin and ricin A chain). The aim of this study was to use small-angle neutron scattering (SANS) to investigate the most likely shape of the hydrochloride salt form of one particular PAA (ISA23) in solution, under pH conditions that mimic those the polymer would be expected to encounter during endocytic internalisation (pH 7.4-3). It was shown that models based on a Gaussian coil representation of the polymer conformation described the SANS data better over this pH range than models based on a rod-like conformation. The conformation of ISA23 at 37 degrees C was expanded (radius of gyration approximately 80A) at pH approximately 3 but collapsed with an increase in pH (radius of gyration approximately 20A at pH 7.4), a conclusion also reached in a model-free analysis of the neutron data. Outside this pH range - at the extremes of high and low pH - the polymer coil collapsed and interpretation of the scattering was slightly complicated by the presence of a very weak structure factor indicating that the polymer coils are highly charged. The PAA concentration did not significantly affect the polymer size over the concentration range 10-50mg/ml. Characterisation of the dynamics of these polymer solutions - diffusion coefficients and viscosity -ostensibly suggest a very different conclusion with the polymer expanding as the pH is increased, but this arises due to weak aggregation of the amphoteric polymer coils.
Hydrogen-Ion Concentration↗
Radial spoke proteins of Chlamydomonas flagella.
The radial spoke is a ubiquitous component of '9+2' cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes.
Amino Acid Sequence↗
Axonemal protofilament ribbons, DM10 domains, and the link to juvenile myoclonic epilepsy.
Juvenile myoclonic epilepsy (JME) is a common neurological disorder that results in short uncontrolled muscle contractions and sometimes more severe seizures. Genetic studies have suggested that JME may be caused by mutations in EFHC1. The Efhc1 protein consists of three DM10 domains and a C-terminal region containing a potential Ca2+ -binding motif. In Chlamydomonas, a protein (Rib72) of almost identical domain structure is a component of the protofilament ribbons within the doublet microtubules of the flagellar axoneme. Here I discuss recent work that supports assignment of human Efhc1 as a ciliary component and the resulting implications for the mechanism of disease causation.
Adolescent↗
Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2.
Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.
Animals↗
Prospective evaluation of a critical care pathway for clearance of the cervical spine using the bolster and active range-of-motion flexion/extension techniques.
BACKGROUND: Clearance of the cervical spine after blunt trauma remains controversial in patients with normal radiologic evaluation. METHODS: Blunt trauma patients with midline boney cervical tenderness and plain films that disclose no abnormalities and computed tomography (CT) scans were entered into a care pathway for spinal clearance using the Bolster or active range-of-motion (AROM) flexion/extension techniques. The quality of films between the two techniques was then compared. RESULTS: In all, 159 patients entered the pathway with 14 patients (9%) unable to complete the examination secondary to pain. The Bolster was used in 129 patients (89%) and AROM was used in 16 (11%). The total range of motion was significantly better with AROM at 51.4 +/- 19.4 degrees of motion compared with 32.0 +/- 13.0 degrees with the Bolster (p < or = 0.05). The most distal level visualized was not different between groups with 6.6 +/- 0.8 cervical vertebrae visualized on average in the Bolster group and 6.8 +/- 0.7 in the AROM group. Positive findings occurred in five patients (3.4%) in the Bolster group. CONCLUSION: The incidence of occult cervical injury in patients with boney cervical pain despite normal plain films and CT scans in this study was 3.4%. The AROM technique has better total range of motion than the Bolster, although results of the Bolster technique remain within acceptable standards. The present care pathway appears to be an effective screening tool for evaluation of this population of patients. Additional evaluation of the obtunded patient is necessary before broad implementation of this technique.
Adult↗
Genetic analysis of the cytoplasmic dynein subunit families.
Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.
Animals↗
Cytoplasmic dynein nomenclature.
A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.
Animals↗
Differential light chain assembly influences outer arm dynein motor function.
Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.
Amino Acid Sequence↗
Identification and analysis of axonemal dynein light chain 1 in primary ciliary dyskinesia patients.
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic infections of the upper and lower airways, randomization of left/right body asymmetry, and reduced fertility. The phenotype results from dysfunction of motile cilia of the respiratory epithelium, at the embryonic node and of sperm flagella. Ultrastructural defects often involve outer dynein arms (ODAs), that are composed of several light (LCs), intermediate, and heavy (HCs) dynein chains. We recently showed that recessive mutations of DNAH5, the human ortholog of the biflagellate Chlamydomonas ODA gamma-HC, cause PCD. In Chlamydomonas, motor protein activity of the gamma-ODA-HC is regulated by binding of the axonemal LC1. We report the identification of the human (DNAL1) and murine (Dnal1) orthologs of the Chlamydomonas LC1-gene. Northern blot and in situ hybridization analyses revealed specific expression in testis, embryonic node, respiratory epithelium, and ependyma, resembling the DNAH5 expression pattern. In silico protein analysis showed complete conservation of the LC1/gamma-HC binding motif in DNAL1. Protein interaction studies demonstrated binding of DNAL1 and DNAH5. Based on these findings, we considered DNAL1 a candidate for PCD and sequenced all exons of DNAL1 in 86 patients. Mutational analysis was negative, excluding a major role of DNAL1 in the pathogenesis of PCD.
Amino Acid Motifs↗
Solution structure of the Tctex1 dimer reveals a mechanism for dynein-cargo interactions.
Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.
Amino Acid Sequence↗
The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation.
Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.
Amino Acid Sequence↗
Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase.
The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.
Amino Acid Sequence↗
A novel Tctex2-related light chain is required for stability of inner dynein arm I1 and motor function in the Chlamydomonas flagellum.
Tctex1 and Tctex2 were originally described in mice as putative distorters/sterility factors involved in the non-Mendelian transmission of t haplotypes. Subsequently, these proteins were found to be light chains of both cytoplasmic and axonemal dyneins. We have now identified a novel Tctex2-related protein (Tctex2b) within the Chlamydomonas flagellum. Tctex2b copurifies with inner arm I1 after both sucrose gradient centrifugation and anion exchange chromatography. Unlike the Tctex2 homologue within the outer dynein arm, analysis of a Tctex2b-null strain indicates that this protein is not essential for assembly of inner arm I1. However, a lack of Tctex2b results in an unstable dynein particle that disassembles after high salt extraction from the axoneme. Cells lacking Tctex2b swim more slowly than wild type and exhibit a reduced flagellar beat frequency. Furthermore, using a microtubule sliding assay we observed that dynein motor function is reduced in vitro. These data indicate that Tctex2b is required for the stability of inner dynein arm I1 and wild-type axonemal dynein function.
Amino Acid Sequence↗
The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.
The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.
Amino Acid Sequence↗
Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1.
Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.
Biomarkers, Tumor↗