Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Modulation of N-methyl-d-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants1

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd2+ uptake as a model system. Radiotracer techniques were used to quantify unidirectional 109Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for 109Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd2+ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd2+ influx across the root-cell plasma membrane. The Cd2+ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd2+ influx, whereas the maximum initial velocity for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cd2+ uptake. Expression studies with the Fe2+ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd2+ and Zn2+, in addition to Fe2+.

Nucleotide Triphosphates Are Required for the Transport of Glycolate Oxidase into Peroxisomes1

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.

Proton transport by a bacteriorhodopsin mutant, aspartic acid-85-->asparagine, initiated in the unprotonated Schiff base state.

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form (lambda max approximately 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type--i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

Characterization of spatial–temporal patterns in dynamic speckle sequences using principal component analysis

Abstract. Speckle is being used as a characterization tool for the analysis of the dynamics of slow-varying phenomena occurring in biological and industrial samples at the surface or near-surface regions. The retrieved data take the form of a sequence of speckle images. These images contain information about the inner dynamics of the biological or physical process taking place in the sample. Principal component analysis (PCA) is able to split the original data set into a collection of classes. These classes are related to processes showing different dynamics. In addition, statistical descriptors of speckle images are used to retrieve information on the characteristics of the sample. These statistical descriptors can be calculated in almost real time and provide a fast monitoring of the sample. On the other hand, PCA requires a longer computation time, but the results contain more information related to spatial–temporal patterns associated to the process under analysis. This contribution merges both descriptions and uses PCA as a preprocessing tool to obtain a collection of filtered images, where statistical descriptors are evaluated on each of them. The method applies to slow-varying biological and industrial processes.

A software tool for accurate assessment of costs and CO2 emissions in wood transport using OpenStreetMap©

Costs and environmental impacts are key elements in forest logistics and they must be integrated in forest decision-making. The evaluation of transportation fuel costs and carbon emissions depend on spatial and non-spatial data but in many cases the former type of data are dicult to obtain. On the other hand, the availability of software tools to evaluate transportation fuel consumption as well as costs and emissions of carbon dioxide is limited. We developed a software tool that combines two empirically validated models of truck transportation using Digital Elevation Model (DEM) data and an open spatial data tool, specically OpenStreetMap©. The tool generates tabular data and spatial outputs (maps) with information regarding fuel consumption, cost and CO2 emissions for four types of trucks. It also generates maps of the distribution of transport performance indicators (relation between beeline and real road distances). These outputs can be easily included in forest decision-making support systems. Finally, in this work we applied the tool in a particular case of forest logistics in north-eastern Portugal

Historic records of organic compounds from a high Alpine glacier: influences of biomass burning, anthropogenic emissions, and dust transport

Historic records of α-dicarbonyls (glyoxal, methylglyoxal), carboxylic acids (C6–C12 dicarboxylic acids, pinic acid, p-hydroxybenzoic acid, phthalic acid, 4-methylphthalic acid), and ions (oxalate, formate, calcium) were determined with annual resolution in an ice core from Grenzgletscher in the southern Swiss Alps, covering the time period from 1942 to 1993. Chemical analysis of the organic compounds was conducted using ultra-high-performance liquid chromatography (UHPLC) coupled to electrospray ionization high-resolution mass spectrometry (ESI-HRMS) for dicarbonyls and long-chain carboxylic acids and ion chromatography for short-chain carboxylates. Long-term records of the carboxylic acids and dicarbonyls, as well as their source apportionment, are reported for western Europe. This is the first study comprising long-term trends of dicarbonyls and long-chain dicarboxylic acids (C6–C12) in Alpine precipitation. Source assignment of the organic species present in the ice core was performed using principal component analysis. Our results suggest biomass burning, anthropogenic emissions, and transport of mineral dust to be the main parameters influencing the concentration of organic compounds. Ice core records of several highly correlated compounds (e.g., p-hydroxybenzoic acid, pinic acid, pimelic, and suberic acids) can be related to the forest fire history in southern Switzerland. P-hydroxybenzoic acid was found to be the best organic fire tracer in the study area, revealing the highest correlation with the burned area from fires. Historical records of methylglyoxal, phthalic acid, and dicarboxylic acids adipic acid, sebacic acid, and dodecanedioic acid are comparable with that of anthropogenic emissions of volatile organic compounds (VOCs). The small organic acids, oxalic acid and formic acid, are both highly correlated with calcium, suggesting their records to be affected by changing mineral dust transport to the drilling site.

Component NRM directions for sediment core HLY0503-06JPC

The Arctic oceans have been fertile ground for the recording of apparent excursions of the geomagnetic field, implying that the high latitude field had unusual characteristics at least over the last 1-2 Myrs. Alternating field demagnetization of the natural remanent magnetization (NRM) of Core HLY0503-6JPC from the Mendeleev Ridge (Arctic Ocean) implies the presence of primary magnetizations with negative inclination apparently recording excursions in sediments deposited during the Brunhes Chron. Thermal demagnetization, on the other hand, indicates the presence of multiple (often anti-parallel) magnetization components with negative inclination components having blocking temperatures predominantly, but not entirely, below ~ 350 °C. Thermo-magnetic tests, X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicate that the negative inclination components are carried by titanomaghemite, presumably formed by seafloor oxidation of titanomagnetite. The titanomaghemite apparently carries a chemical remanent magnetization (CRM) that is partially self-reversed relative to the detrital remanent magnetization (DRM) carried by the host titanomagnetite. The partial self-reversal could have been accomplished by ionic ordering during oxidation, thereby changing the balance of the magnetic moments in the ferrimagnetic sublattices.

A review of the sediment delivery ratio techniques component of the Highland Silver Lake watershed project /

Prepared in cooperation with the Soil Conservation Service, USDA.

Sources and transport of surface sediment and metals in B.C. fjord, Tahsis and Zeballos Inlets

Senior thesis written for Oceanography 445

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.