Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack

We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.     

A robust numerical algorithm for studying biomolecular transport processes

We present a numerical algorithm that is well suited for the study of biomolecular transport processes. In the algorithm a continuous Markov process is discretized as a jump process and the jump rates are derived from local solutions of the continuous system. Consequently, the algorithm has two advantages over standard numerical methods: (1) it preserves detailed balance for equilibrium processes, (2) it is able to handle discontinuous potentials. The formulation of the algorithm also allows us to calculate the effective diffusion coefficient or, equivalently, the randomness parameter. We provide several simple examples of how to implement the algorithm. All the MATLAB functions files needed to reproduce the results presented in the article are available from www.amath.unc.edu/Faculty/telston/matlab_functions.     

Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are highly selective filters that control the exchange of material between nucleus and cytoplasm. The principles that govern selective filtering by NPCs are not fully understood. Previous studies find that cellular proteins capable of fast translocation through NPCs (transport receptors) are characterized by a high proportion of hydrophobic surface regions. Our analysis finds that transport receptors and their complexes are also highly negatively charged. Moreover, NPC components that constitute the permeability barrier are positively charged. We estimate that electrostatic interactions between a transport receptor and the NPC result in an energy gain of several k _B T, which would enable significantly increased translocation rates of transport receptors relative to other cellular proteins. We suggest that negative charge is an essential criterion for selective passage through the NPC.     

Regulated transport as a mechanism for pattern generation: Capabilities for phyllotaxis and beyond

Large-scale pattern formation is a frequently occurring phenomenon in biological organisms, and several local interaction rules for generating such patterns have been suggested. A mechanism driven by feedback between the plant hormone auxin and its polarly localized transport mediator PINFORMED1 has been proposed as a model for phyllotactic patterns in plants. It has been shown to agree with current biological experiments at a molecular level as well as with respect to the resulting patterns. We present a thorough investigation of variants of models based on auxin-regulated polarized transport and use analytical and numerical tools to derive requirements for these models to drive spontaneous pattern formation. We find that auxin concentrations in neighboring cells can feed back either on exocytosis or endocytosis and still produce patterns. In agreement with mutant experiments, the active cellular efflux is shown to be more important for pattern capabilities as compared to active influx. We also find that the feedback must originate from neighboring cells rather than from neighboring walls and that intracellular competition for the transport mediator is required for patterning. The importance of model parameters is investigated, especially regarding robustness to perturbations of experimentally estimated parameter values. Finally, the regulated transport mechanism is shown to be able to generate Turing patterns of various types.     

Glucose transport in cultured animal cells: an exercise for the undergraduate cell biology laboratory

Membrane transport is a fundamental concept that undergraduate students of cell biology understand better with laboratory experience. Formal teaching exercises commonly used to illustrate this concept are unbiological, qualitative, or intricate and time consuming to prepare. We have developed an exercise that uses uptake of radiolabeled nutrient analogues by attachment-dependent animal cells cultured on multiwell trays. This system can readily be manipulated within a typical 3-h laboratory period to yield reproducible, biologically relevant, quantitative data regarding key aspects of membrane transport. Each 24-well tray of cultures allows a group of two to four students to compare eight conditions in triplicate. If different groups of students test different conditions or different types of cells, data can be shared for an even broader experience. The exercise is also readily adaptable for open-ended student projects. Here we illustrate the exercise measuring uptake of the nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose. Students successfully tested the effects of competing sugars, putative inhibitors of the GLUT1 transporter, and changes in cell physiology that might be expected to affect glucose transport in epithelial cells and fibroblasts. In this exercise students find the nutritional and medical implications of glucose transport and its regulation intriguing. They also learn to handle radioisotopes and cultured cells.     

Exciton transport in the photosynthetic unit

In this paper we present a theoretical study of the exciton transport in the photosynthetic unit and attempt to correlate the structural and organizational properties of the unit with the exciton transport efficiency. The transport mechanism is assumed to be of the Forster type. This mechanism is shown to be equivalent to the thermally assisted exciton hopping. The transport equations are solved by the Green's function technique in a more general way than has been presented previously. We thus find it possible to study the effect of the proper R^?6 coupling and random orientations of the pigment molecules. The equations are also solved for a low concentration of reaction centres when the conventional random walk picture is not valid. Various models of chlorophyll a and b mixtures are studied and by comparing their different exciton transport properties, an explanation is proposed for several observed features of the primary photochemical process.     

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila

The maternal morphogen dorsal (dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.     

Cellular iron transport

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.     

GenRGenS: software for generating random genomic sequences and structures

SUMMARY: GenRGenS is a software tool dedicated to randomly generating genomic sequences and structures. It handles several classes of models useful for sequence analysis, such as Markov chains, hidden Markov models, weighted context-free grammars, regular expressions and PROSITE expressions. GenRGenS is the only program that can handle weighted context-free grammars, thus allowing the user to model and to generate structured objects (such as RNA secondary structures) of any given desired size. GenRGenS also allows the user to combine several of these different models at the same time.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system

Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+). We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium. Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx. Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA. The compounds inhibited uptake by the bacterial S. typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain. Cation hexaammines also inhibited growth of S. typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system. In contrast, hexacyano-cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake. The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase. These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation.     

Specificity of the Na+-dependent monocarboxylic acid transport pathway in rabbit renal brush border membranes

The substrate specificity of a Na+-dependent transport pathway for L-lactate was studied in rabbit renal brush border membrane vesicles. Jmax for L-lactate transport was unaffected by the presence of a fixed concentration of two different short-chain monocarboxylic acids, while the apparent Kt(Ka) for L-lactate increased, and this is compatible with competitive inhibition. The inhibitor constants ("Ki"'s) for the transport pathway for the two solutes examined closely corresponded to the respective "Ki"'s derived from a Dixon plot. A broad range of compounds were then tested as potential inhibitors of L-lactate transport, and the "Ki"'s thereby derived yielded specific information regarding optimal substrate recognition by the carrier. A single carboxyl group is an absolute requirement for recognition, and preference is given to 3 to 6 C chain molecules. Addition of ketone, hydroxyl and, particularly, amine groups at any carbon position, diminishes substrate-carrier interaction. Intramolecular forces, notably the inductive effects of halogens, may play a role in enhancing substrate-carrier interaction; however, no correlation was found between pKa and "Ki" for the substrates examined. We conclude that a separate monocarboxylic acid transport pathway, discrete from either the D-glucose, alpha or beta neutral amino-acid, or dicarboxylic acid carriers, exists in the renal brush border, and this handles a broad range of monocarboxylates.     

c-Src control of chloride channel support for osteoclast HCl transport and bone resorption

Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b.     

Myo4p is a monomeric myosin with motility uniquely adapted to transport mRNA

The yeast Saccharomyces cerevisiae uses two class V myosins to transport cellular material into the bud: Myo2p moves secretory vesicles and organelles, whereas Myo4p transports mRNA. To understand how Myo2p and Myo4p are adapted to transport physically distinct cargos, we characterize Myo2p and Myo4p in yeast extracts, purify active Myo2p and Myo4p from yeast lysates, and analyze their motility. We find several striking differences between Myo2p and Myo4p. First, Myo2p forms a dimer, whereas Myo4p is a monomer. Second, Myo4p generates higher actin filament velocity at lower motor density. Third, single molecules of Myo2p are weakly processive, whereas individual Myo4p motors are nonprocessive. Finally, Myo4p self-assembles into multi-motor complexes capable of processive motility. We show that the unique motility of Myo4p is not due to its motor domain and that the motor domain of Myo2p can transport ASH1 mRNA in vivo. Our results suggest that the oligomeric state of Myo4p is important for its motility and ability to transport mRNA.     

Extracellular hydrophobic regions in scavenger receptor BI play a key role in mediating HDL-cholesterol transport

The binding of high density lipoprotein (HDL) to scavenger receptor BI (SR-BI) is responsible for whole-body cholesterol disposal via reverse cholesterol transport. The extracellular domain of SR-BI is required for HDL binding and selective uptake of HDL-cholesterol. We identified six highly hydrophobic regions in this domain that may be important for receptor activity and performed site-directed mutagenesis to investigate the importance of these regions in SR-BI-mediated cholesterol transport. Non-conservative mutation of the regions encompassing V67, L140/L142, V164 or V221 reduced hydrophobicity and impaired the ability of SR-BI to bind HDL, mediate selective uptake of HDL-cholesterol, promote cholesterol efflux, and enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol. In contrast, conservative mutations at V67, V164 or V221 did not affect the hydrophobicity or these cholesterol transport activities. We conclude that the hydrophobicity of N-terminal extracellular regions of SR-BI is critical for cholesterol transport, possibly by mediating receptor-ligand and/or receptor-membrane interactions.     

Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes

Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.