Functionally distinct isoforms of dynactin are expressed in human neurons.

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.     

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2- aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.      

Design and implementation of a qualitative simulation model of lambda phage infection

MOTIVATION: Molecular biology databases hold a large number of empirical facts about many different aspects of biological entities. That data is static in the sense that one cannot ask a database 'What effect has protein A on gene B?' or 'Do gene A and gene B interact, and if so, how?'. Those questions require an explicit model of the target organism. Traditionally, biochemical systems are modelled using kinetics and differential equations in a quantitative simulator. For many biological processes however, detailed quantitative information is not available, only qualitative or fuzzy statements about the nature of interactions. RESULTS: We designed and implemented a qualitative simulation model of lambda phage growth control in Escherichia coli based on the existing simulation environment QSim. Qualitative reasoning can serve as the basis for automatic transformation of contents of genomic databases into interactive modelling systems that can reason about the relations and interactions of biological entities.      

ADP release is rate limiting in steady-state turnover by the dynein adenosinetriphosphatase

The kinetics of the product release steps in the pathway of ATP hydrolysis by dynein were investigated by examining the rate and partition coefficient of phosphate-water 18O exchange under equilibrium and steady-state conditions. Dynein catalyzed both medium and intermediate phosphate-water oxygen exchange with a partition coefficient of 0.30. The dependence of the rate of loss of the fully labeled phosphate species on the concentration of ADP was hyperbolic, with an apparent Kd for the binding of ADP to dynein of 0.085 mM. The apparent second-order rate constant for phosphate binding to the dynein-ADP complex was 8000 M-1 s-1. The time course of medium phosphate-water oxygen exchange during net ATP hydrolysis was examined in the presence of an ATP regeneration system. The observed rate of loss of P18O4 was comparable to the rate observed at saturating ADP which implies that ADP release is rate limiting for dynein in the steady state. Product inhibition of the dynein ATPase was also examined. ADP inhibited the enzyme competitively with a Ki of 0.4 mM. Phosphate was a linear noncompetitive mixed-type inhibitor with a Ki of 11 mM. These data were fit to a model in which phosphate release is fast and is followed by rate-limiting release of ADP, allowing us to define each rate constant in the pathway. A discrepancy between the total free energy calculated compared to the known free energy of ATP hydrolysis suggests that there is an additional step in the pathway, perhaps involving a change in conformation of the enzyme-ADP state preceding ADP release.     

Inhibitory effects of some esters of 2- and 3-substituted alkoxyphenylcarbamic acids on photosynthetic characteristics

The inhibitory effect of 23N-alkyl-4-piperidylesters (alkyl = ethyl-butyl) (APEA) and 8N-ethyl-2-pyrrolidinylmethylesters (EPMEA) of 2- and 3-substituted alkoxyphenylcarbamic acids (alkoxy = butoxy-heptyloxy-) on photosynthetic Hill reaction activity of spinach chloroplasts and on chlorophyll (Chl) synthesis in green algaeChlorella vulgaris was investigated. Inhibitory activities of these compounds were strongly connected with the lipophilicity of the whole molecule. A lower inhibitory activity of 2-alkoxy-substituted derivatives in relation to the corresponding 3-substituted ones was confirmed. Electron spin resonance (ESR) spectra of spinach chloroplasts demonstrated that the studied compounds affected the structure of photosystem (PS) 2 with the release of Mn²⁺ ions into interior of thylakoid membranes.     

When dispersal fails: unexpected genetic separation in Gibraltar macaques (Macaca sylvanus)

Barbary macaques (Macaca sylvanus), now restricted in the wild to a few isolated forested areas of Morocco and Algeria, are present in a free‐ranging colony on Gibraltar. For many decades, the Gibraltar colony was exposed to multiple bottlenecks due to highly nonrandom removal of animals, followed by repeated introductions of animals from North Africa. Moreover, because of complete isolation, Gibraltar's several social groups of macaques provide an ideal system to study the genetic consequences of dispersal in cercopithecines in situ. Predictions of genetic consequences due to male‐biased dispersal in cercopithecines will be different for autosomal and maternally inherited genetic markers, such as the control region of the mitochondrial DNA. We used a panel of 14 highly polymorphic microsatellite loci and part of the hypervariable region I of the mitochondrial control region to estimate genetic structure between five social groups in Gibraltar. Surprisingly, for autosomal markers, both classical summary statistics and an individual‐based method using a Bayesian framework detected significant genetic structure between social groups in Gibraltar, despite much closer proximity than wild Algerian and Moroccan populations. Mitochondrial data support this finding, as a very substantial portion of the total genetic variation (70.2%) was found between social groups. Using two Bayesian approaches, we likewise identified not only a small number of male first‐generation immigrants (albeit less than expected for cercopithecines) but also unexpectedly a few females. We hypothesize that the culling of males that are more likely to disperse might slow down genetic homogenization among neighbouring groups, but may also and more perversely produce selection on certain behavioural traits. This may have important repercussions for conservation, as it could lead to evolutionary changes that are not due to inbreeding or genetic drift.     

Regulation of dynactin through the differential expression of p150Glued isoforms

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.     

Motor neurons rely on motor proteins

The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot-Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases.     

Dynein binds to beta-catenin and may tether microtubules at adherens junctions

Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.