Kenneth R. Robinson

Asymmetries in H+/K+-ATPase and Cell Membrane Potentials Comprise a Very Early Step in Left-Right Patterning

A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.

The direction of growth of differentiating neurones and myoblasts from frog embryos in an applied electric field.

1. Disaggregated single neurones and myoblasts obtained from the neural tube and somites of Xenopus laevis embryos (stages 17‐21) were cultured in the presence of steady small electric fields. 2. Neurites grew preferentially towards the negative pole, or cathode, in field strengths of 7‐190 mV/mm. Many turned through considerable angles to do so. This effect disappeared below a threshold level of about 7 mV/mm. 3. Greater numbers of neurones sprouted neurites in cultures exposed to an electric field compared to control cultures. The difference could be as much as tenfold. The threshold level of this phenomenon was about 6‐8 mV/mm. Other cell types such as pigment cells and fibroblasts were also stimulated to differentiate in culture by an electric field, although to a lesser extent than neurones. 4. Applied electric fields had no effect on the location of the origin of neurites on the cell body. 5. Spherical myoblasts cultured in applied electric fields (36‐170 mV/mm) elongated with a bipolar axis of growth which was perpendicular to the electric field. The response was graded and disappeared at field strengths below 36 mV/mm. 6. It is suggested that in vivo, the direction of neural outgrowth from the neural tube and the strict spatial organization of somites might be under the control, in part, of endogenous electric fields. Possible sources of these are discussed.

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H+ flux. A pharmacological screen implicated the H+-pump H+-V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H+-V-ATPase subunits during the first three cleavages. H+-flux across plasma membranes is also asymmetric at the four- and eight-cell stages, and this asymmetry requires H+-V-ATPase activity. Abolishing the asymmetry in H+ flux, using a dominant-negative subunit of the H+-V-ATPase or an ectopic H+ pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H+-V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (Vmem) regulation. Varying either pH or Vmem, independently of direct manipulation of H+-V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H+-V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H+-V-ATPase induces heterotaxia in both species; in chick, H+-V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffers vesicle and Spaw expression. Our data implicate H+-V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH- and Vmem-dependent model of the early physiology of LR patterning.

A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes☆

We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.

Identification and Characterization of Stretch-Activated Ion Channels in Pollen Protoplasts

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradients magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.

LOCAL CATION ENTRY AND SELF‐ELECTROPHORESIS AS AN INTRACELLULAR LOCALIZATION MECHANISM*

We are concerned with the mechanisms of intracellular localization that contribute to development. How, for example, does an ameboid cell form a protrusion at one point and not a t another? How does a neuron initiate an outgrowth a t one point and not a t another? How is a neurite’s continued growth oriented? How does a plant egg or spore initiate an outgrowth at one point and not a t another? How are “vegetal” materials localized in one end of an animal egg so that it develops into gut, not skin? Genetic mechanisms have proven to have considerable generality; much of what is true of the genetics of bacteria is likewise true of man. Similarly, we expect morphogenetic mechanisms, in particular those of intracellular localization, to have much generality. Therefore, we have focused our study upon the early development of the fucoid egg. Unlike animal eggs, this common seaweed egg has no preformed animal-vegetal axis. The fucoid zygote is essentially apolar. Then, in the course of a day or less, it initiates growth at one pole, visibly polarizes, and divides into two quite different cells: a rhizoid, or attachment, cell a t the growth pole and a thallus cell a t its antipode (FIGURE 1). This first day of the fucoid egg’s development is a prototype of the localization process. We are further focusing our study upon an essentially electrical hypothesis of localization. According to this hypothesis, the plasma membrane in a growth region, or presumptive growth region, becomes relatively leaky to certain cations that are normally a t a much higher electrochemical potential outside of the cell than within it. These cations could include Caz+, MgZ+, Na+, and H+. The resultant movement of these cations into this region constitutes entry of an electrical current. Movement of this cation flux or current through the resistance of the cytoplasm under the leak will necessarily generate a cytoplasmicjeld that is relatively positive under the leaky portion of the membrane. This field will generate movement. It will tend to pull vesicles and other cytoplasmic constituents with a negative surface charge toward the leaky membrane region. This movement, in turn, may act to make the local membrane leakier, which thus provides the last link in apositive feedback loop. This loop would serve to establish and maintain localized growth, expansion, segregation. and other functions. Specifically, this movement could give such feedback by causing fusion of certain vesicles with the plasma membrane if these vesicles were themselves relatively leaky to particular cations or if they thus released substances that made preexisting parts of the membrane leaky. In our view, the mature nerve synapse may serve as a model of this hypothesis,

Endogenous electrical currents and the resultant voltage gradients in the chick embryo

We have studied some of the electrophysiological properties of 2 1/2- to 4-day-old (stage 14-22) chick embryos. Using a recently developed two-dimensional vibrating probe, large currents were found to exit the posterior intestinal portal (p.i.p.) during the period of tail gut reduction. During this period, epithelial cells lining cloacal regions of the hindgut are dying, thus creating a low-resistance pathway for current flow out of the embryo. Currents entered the intact epithelium over other regions of the embryo. The outward currents at the p.i.p. were first detected at stage 15 and reached their average maximum current density of 112 +/- 10 microA/cm2 at stage 17. After stage 17, the magnitude of the currents decreased, dropping to 16 +/- 0.3 microA/cm2 by stage 22. The currents were reversibly reduced by about 50% when Na+ was replaced by choline in the bathing solution. The magnitude of the currents leaving the p.i.p. suggested the existence of a measurable intraembryonic voltage gradient. The transepithelial potential (TEP) of stage 14-21 embryos was measured lateral to the neural tube through the dorsal ectoderm. For all stages, the combined average TEP was 16 +/- 0.5 mV. Differences in the TEP between various regions of the embryo were used to calculate an intraembryonic voltage gradient. At stage 14, before outward current was found at the p.i.p., no significant intraembryonic voltage gradient was detected. At stage 17, when the outward current at the p.i.p. was maximum, a voltage gradient of 21 +/- 5 mV/mm (mean +/- SEM; N = 6) was measured in the caudal end of the embryo. This gradient in some cases was as steep as 33 mV/mm. This is well above the minimum level needed to affect the direction of embryonic cell migration in vitro. We hypothesize that this endogenous electrical field acts as a directional cue for neural crest cell movements in the developing chick embryo.

Membrane potential of the unfertilized sea urchin egg

Abstract The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm2· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (−70 mV) and specific resistance (420 kΩ · cm2). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were −75 ± 3 mV and 380 ± kΩ · cm2.

Calcium, potassium, and sodium exchange by full-grown and maturing Xenopus laevis oocytes.

Abstract The kinetics of calcium, potassium, and sodium exchange by Xenopus laevis oocytes were monitored with radioactive tracers both before and during progesterone-induced maturation. The rate of 45 Ca release steadily elevates for several hours during maturation, beginning within 40 min after progesterone exposure. About an hour later, the rate of 45 Ca uptake also increases. The rate of 45 Ca release begins to decline 1–2 hr before germinal vesicle breakdown (GVBD); the rate of calcium uptake declines only after GVBD. Similar changes are seen after maturation is induced with other steroids, but not when maturation is blocked by inhibitors. The passive potassium flux initially increases after progesterone treatment to be followed later by a decrease. These observed changes occur coincidently with those of 45 Ca efflux. The passive sodium flux, on the other hand, steadily increases from the time of progesterone treatment until GVBD.

Localization of steady currents in the lens

Steady currents around the rat lens were measured with a vibrating probe. Inward currents were found at the anterior pole and to a lesser degree at the posterior pole. Outward currents were localized at the equator. These currents were inhibited and even reversed by high concentrations of potassium. Ouabain blocked currents after 24 hrs but not after 6 hrs of exposure. The composition of the medium was not critical. For example, currents were observed in NaCl and in MgSO4 solutions.