Transport of electrolytes in muscle

Summary The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells.In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

Studies of translocation catalysis

There is a symbiotic relationship between the evolution of fundamental theory and the winning of experimentally-based knowledge. The impact of the General Chemiosmotic Theory on our understanding of the nature of membrane transport processes is described and discussed. The history of experimental studies on transport catalysed by ionophore antibiotics and the membrane proteins of mitochondria and bacteria are used to illustrate the evolution of knowledge and theory. Recent experimental approaches to understanding the lactose-H⁺ symport protein ofEscherichia coli and other sugar porters are described to show that the lack of experimental knowledge of the three-dimensional structures of the proteins currently limits the development of theories about their molecular mechanism of translocation catalysis.     

Stem cell biology and neurodegenerative disease

The fundamental basis of our work is that organs are generated by multipotent stem cells, whose properties we must understand to control tissue assembly or repair. Central nervous system (CNS) stem cells are now recognized as a well-defined population of precursors that differentiate into cells that are indisputably neurons and glial cells. Work from our group played an important role in defining stem cells of the CNS. Embryonic stem (ES) cells also differentiate to specific neuron and glial types through defined intermediates that are similar to the cellular precursors that normally occur in brain development. There is convincing evidence that the differentiated progeny of ES cells and CNS stem cells show expected functions of neurons and glia. Recent progress has been made on three fundamental developmental processes: (i) cell cycle control; (ii) the control of cell fate; and (iii) early steps in neural differentiation. In addition, our work on CNS stem cells has developed to a stage where there are clinical implications for Parkinson's and other degenerative disorders. These advances establish that stem cell biology contributes to our understanding of brain development and has great clinical promise.     

Phosphate transport processes in eukaryotic cells

Conclusion Much more work has been done on P_i transport processes, even in the last five years, than we have been able to mention in the space available. We have restricted our discussion to studies on mechanisms of transport or transport regulation, identification of transport proteins and their essential amino acids, and isolation, purification, and reconstitution of P_i transport systems. Many valuable studies on the physiology of P_i transport and its regulation and P_i transport in nonepithelial cells have also been conducted. Transport of P_i into and out of organelles other than the mitochondrion is gaining well-deserved attention, as are transport processes in fungi and plants. It is hoped that in another five years many P_i transport processes will be understood in true molecular terms and that this will increase our knowledge of cellular bioenergetics and metabolism.     

Symbioses between salamander embryos and green algae

The symbiosis between Ambystoma maculatum (spotted salamander) embryos and green algae was initially described over 120 years ago. Algae populate the egg capsules that surround individual A. maculatum embryos, giving the intracapsular fluid a characteristic green hue. Early work established this symbiosis to be a mutualism, while subsequent studies sought to identify the material benefits of this association to both symbiont and host. These studies have shown that salamander embryos benefit from increased oxygen concentrations provided by their symbiotic algae. The algae, in turn, may benefit from ammonia excreted by the embryos. All of these early studies considered the association to be an ectosymbiotic mutualism. However our recent work has shown that algae invade both embryonic salamander cells and tissues during development. The unexpected invasion of algal cells into a salamander host changes our understanding of this symbiosis. This review will summarize the earlier research on this association in the context of these recent findings. It will also emphasize gaps in our understanding of this and other amphibian embryo-algal interactions and suggest various research avenues to address these unanswered questions.     

Contributions of secondary active transport processes to membrane potentials

Summary Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.     

Ricardo Miledi and the calcium hypothesis of neurotransmitter release

Ricardo Miledi has made significant contributions to our basic understanding of how synapses work. Here I discuss aspects of Miledi's research that helped to establish the requirement of presynaptic calcium for neurotransmitter release, from his earliest scientific studies to his classic experiments in the squid giant synapse.     

Subcellular trafficking of PIN auxin efflux carriers in auxin transport

The directional transport of the plant hormone auxin is a unique process mediating a wide variety of developmental processes. Auxin movement between cells depends on AUX1/LAX, PGP and PIN protein families that mediate auxin transport across the plasma membrane. The directionality of auxin flow within tissues is largely determined by polar, subcellular localization of PIN auxin efflux carriers. PIN proteins undergo rapid subcellular dynamics that is important for the process of auxin transport and its directionality. Furthermore, various environmental and endogenous signals can modulate trafficking and polarity of PIN proteins and by this mechanism change auxin distribution. Thus, the subcellular dynamics of auxin transport proteins represents an important interface between cellular processes and development of the whole plant. This review summarizes our recent contributions to the field of PIN trafficking and auxin transport regulation.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Ion-selective Microelectrodes: Their Use and Importance in Modern Plant Cell Biology

The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists.     

A two-component high-affinity nitrate uptake system in barley

The analysis of genome databases for many different plants has identified a group of genes that are related to one part of a two-component nitrate transport system found in algae. Earlier work using mutants and heterologous expression has shown that a high-affinity nitrate transport system from the unicellular green algae, Chlamydomonas reinhardtii required two gene products for function. One gene encoded a typical carrier-type structure with 12 putative trans-membrane (TM) domains and the other gene, nar2 encoded a much smaller protein that had only one TM domain. As both gene families occur in plants we investigated whether this transport model has more general relevance among plants. The screening for nitrate transporter activity was greatly helped by a novel assay using (15)N-enriched nitrate uptake into Xenopus oocytes expressing the proteins. This assay enables many oocytes to be rapidly screened for nitrate transport activity. The functional activity of a barley nitrate transporter, HvNRT2.1, in oocytes required co-injection of a second mRNA. Although three very closely related nar2-like genes were cloned from barley, only one of these was able to give functional nitrate transport when co-injected into oocytes. The nitrate transport performed by this two-gene system was inhibited at more acidic external pH and by acidification of the cytoplasm. This specific requirement for two-gene products to give nitrate transport function has important implications for attempts to genetically manipulate this fundamental process in plants.     

Polar auxin transport: an early invention

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4-5 cm/h) of auxin transport through the giant (3-5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.     

How Does the Plant Plasma Membrane H+-ATPase Pump Protons?

The plasma membrane H⁺-ATPase couples ATP hydrolysis to protontransport thereby establishing the driving force for solutetransport into and out of plant cells. As such, this enzymeparticipates in a number of cellular processes important tothe overall physiology of plants. From biochemical studies andthe recent application of molecular approaches, the enzyme reactionmechanism and structure of this protein have been characterized.However, our basic understanding of how this enzyme links theendergonic reaction of ATP hydrolysis to proton translocationis far from complete. In this review, several significant questionsregarding the energy coupling mechanism will be addressed interms of information available on the plant plasma membraneH⁺-ATPase and from studies on other P-type transport ATPases.These questions focus on the chemical nature of proton translocation,how this is linked or driven by the ATP hydrolysis reactionand what role, if any, K⁺ has in the transport process. Key words: Energy coupling, membranes, bioenergetics, ion transport, P-type transport ATPase     

Uptake of cadmium and zinc by the alga Chlorella vulgaris: part 1. Individual ion species

The ability of algae and bacteria to accumulate heavy metals from the surrounding environment is a widely recognized phenomenon that has a number of important implications. This work reports on the development of a quantitative model that addresses the basic mechanisms inherent in many uptake processes. The model postulates two mechanisms: an initial rapid metal ion uptake due to attachment onto the cell wall followed by a relatively slow uptake due to membrane transport of the metal into the cell. The mathematical model has been tested using the alga Chlorella vulgaris in the presence of cadmium and zinc in solution under various experimental conditions.     

Effect of solution environment on the permeability of red blood cells

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties. (c) 1994 John Wiley & Sons, Inc.     

Imaging in vivo neuronal transport in genetic model organisms using microfluidic devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.     

Ion transport, membrane traffic and cellular volume control

Throughout their development, plants balance cell surface area and volume with ion transport and turgor. This balance lies at the core of cellular homeostatic networks and is central to the capacity to withstand abiotic as well as biotic stress. Remarkably, very little is known of its mechanics, notably how membrane traffic is coupled with osmotic solute transport and its control. Here we outline recent developments in the understanding of so-called SNARE proteins that form part of the machinery for membrane vesicle traffic in all eukaryotes. We focus on SNAREs active at the plasma membrane and the evidence for specialisation in enhanced, homeostatic and stress-related traffic. Recent studies have placed a canonical SNARE complex associated with the plasma membrane in pathogen defense, and the discovery of the first SNARE as a binding partner with ion channels has demonstrated a fundamental link to inorganic osmotic solute uptake. Work localising the channel binding site has now identified a new and previously uncharacterised motif, yielding important clues to a plausible mechanism coupling traffic and transport. We examine the evidence that this physical interaction serves to balance enhanced osmotic solute uptake with membrane expansion through mutual control of the two processes. We calculate that even during rapid cell expansion only a minute fraction of SNAREs present at the membrane need be engaged in vesicle traffic at any one time, a number surprisingly close to the known density of ion channels at the plant plasma membrane. Finally, we suggest a framework of alternative models coupling transport and traffic, and approachable through direct, experimental testing.     

Genetic and genomic prospects for Xenopus tropicalis research

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cell cycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cell biological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review the resources that will make genetic studies using X. tropicalis a reality.