The pulse current pattern generated by developing fucoid eggs.

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.

An ultrasensitive vibrating probe for measuring steady extracellular currents.

We describe a vibrating probe system for measuring relatively steady electrical current densities near individual living cells. It has a signal-to-noise ratio at least 100 times greater than previously available techniques. Thus it can be used to detect current densities as small as 10 nA/cm(2) in serum when a 30-microm diameter probe is vibrated at 200 Hz between two points 30 microm apart, and the amplifier's time constant is set at 10 s. Moreover, it should be generally insensitive to interference by concentration gradients. It has been first used to reveal and study 100-s long current pulses which developing fucoid embryos drive through themselves.

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation.

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.

A simple, selective method for freeze-fracturing spherical cells.

A simple and selective method for freeze-fracturing spherical cells is described. The cells are loaded into the holes of a thin nickel screen. A metal hat is applied to the cell monolayer and the whole assembly, hat-cells-screen, is frozen and then fractured by ripping the hat off. The fractured face on the screen is replicated. By varying the size of the screen holes and by applying the hat to either side of the screen, this method can selectively expose the E face (or the outer half of plasma membrane), the P face (or the inner half of the plasma membrane), or the cytoplasm of the cells. It also provides a means to produce fractures at a preselected area on the cell, if the cells can be loaded onto the screen in an oriented fashion.

Large electrical currents traverse growing pollen tubes.

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.

Slow calcium waves accompany cytokinesis in medaka fish eggs.

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.

Calcium accumulations within the growing tips of pollen tubes.

Pollen of L. longiflorum was grown in 45Ca-labeled medium and washed with nonradioactive medium. Whole, labeled pollen was then frozen and autoradiographed at -78 degrees C. The autoradiographs show striking accumulations of 45Ca in the growing tips of the pollen tubes. This result is obtained when the pollen is labeled for times as short as 1 min, or as long as 5 h. In most cases, the tip concentration is about two to four times greater than that in the bulk of the pollen tube, and extends for a length of about 20 mum. In autoradiographs of tubes longer than 1 mm, a small fraction of cells show a distinctly larger 45Ca accumulation, the tip containing more than 100 times that in the rest of the cell. The 1- to 5-h labeling experiments show that calcium is relatively concentrated within the cytoplasm of the growing tip. The 1- to 3-min labeling experiments suggest that calcium may enter the tip faster than it enters other regions. These patterns of calcium accumulation and flux may be related to the localized secretion of vesicles at the grow;ng tip.

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

Strong electrical currents leave the primitive streak of chick embryos.

The electrical fields above chick embryos were explored with a vibrating probe. These fields indicate that steady currents with exit densities of the order of 100 microamperes per square centimeter leave the whole streak and return elsewhere through the epiblast. The epicenter of these strong exit currents lies near Hensen's node. They are probably pumped into the intraembryonic space by the epiblast and then leak out of the streak because it is a zone of junctional disruption.

Polarizing fucoid eggs drive a calcium current through themselves.

Calcium ions enter the prospective growth pole of polarizing Pelvetia eggs faster than the opposite pole and leave this antipode faster than the growth pole. The calcium current is greatest when first measured at 6 hours after fertilization and decreases as the time of final commitment to growth in a particular direction approaches.

Relations between ameboid movement and membrane-controlled electrical currents.

We have studied the pattern of electrical currents through amebas (mainly Chaos chaos) with an ultrasensitive extracellular vibrating probe. Amebas drive both steady currents and current pulses through themselves. Relatively steady current with an average surface density of 0.1-0.2 muA/cm2 enters the rear quarter of an ameba and leaves its pseudopods. Streaming reversals are preceded by changes in this current pattern and the region with the largest new inward current becomes the new tail. Ion substitution studies suggest that some of the steady inward current is carried by calcium ions. Characteristic stimulated pulses of current sometimes follow the close approach of the vibrating probe to the side of an advancing pseudopod. Such a pulse enters the cytoplasm through a small patch of membrane near the probe (and seems to leave through the adjacent membrane), is usually followed by hyaline cap and then by pseudopod initiation, is calcium dependent, lasts about 5-10 s, and has a peak density of about 0.4 muA/cm2. Spontaneous pulses of similar shape and duration may enter or leave any part of an animal. They are much less localized, tend to have higher peak densities, and occur in physiological salt solutions at about 0.2-4 times per minute. Retraction of a pseudopod is always accompanied or preceded by a spontaneous pulse which leaves its sides.

Classes and mechanisms of calcium waves.

The best known calcium waves move at about 5-30 microns/s (at 20 degrees C) and will be called fast waves to distinguish them from slow (contractile) ones which move at 0.1-1 microns/s as well as electrically propagated, ultrafast ones. Fast waves move deep within cells and seem to underlie most calcium signals. Their velocity and hence mechanism has been remarkably conserved among all or almost all eukaryotic cells. In fully active (but not overstimulated) cells of all sorts, their mean speeds lie between about 15-30 microns/s at 20 degrees C. Their amplitudes usually lie between 3-30 microM and their frequencies from one per 10-300 s. They are propagated by a reaction diffusion mechanism governed by the Luther equation in which Ca2+ ions are the only diffusing propagators, and calcium induced calcium release, or CICR, the only reaction; although this reaction traverses various channels which are generally modulated by IP3 or cADPR. However, they may be generally initiated by a second, lumenal mode of CICR which occurs within the ER. Moreover, they are propagated between cells by a variety of mechanisms. Slow intracellular waves, on the other hand, may be mechanically propagated via stretch sensitive calcium channels.

On the conservation of calcium wave speeds.

Most long distance calcium signals are believed to take the form of actively propagated calcium waves. In 1991, when this proposal was first advanced, all such waves were thought to belong to one class, for which fertilization waves were the prototype. Moreover, the speeds of such waves were found to be conserved at about 10 microns/s for primary fertilization waves and 30 microns/s for waves through fully active systems at 20 degrees C. In 1993, preliminary evidence for a second class of such waves was published and the prototype for these were ones which drive cell cleavage. These move at only about 1 micron/s at 20 degrees C and were, therefore, called slow calcium waves as opposed to the fast ones first considered. Here we compile compelling evidence that slow waves comprise a second distinct class of actively propagated calcium waves. This is based on 30 papers which yield evidence of slow calcium waves in organisms ranging from Dictyostelium to mammals and phenomena ranging from the surface contraction waves seen long ago in axolotl eggs to embryonic cleavage and mitotic waves and to ones recently seen to accompany primary neural induction in axolotls. Ultraslow and ultrafast calcium waves are also considered.

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 microM in sea urchin eggs, 2 microM in Drosophila eggs, and only 0.11 microM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 microM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 microM apo-aequorin would take 50 days in zebrafish eggs, 2 days [corrected] in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 microm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.

Net calcium and acid release at fertilization in eggs of sea urchins and ascidians.

Sea urchin eggs lose about 10-30% of their total calcium content upon fertilization. We have investigated the mechanism of this calcium-loss with an ion-selective vibrating probe system. Upon fertilization of Arbacia punctulata and Lytechinus pictus eggs we could measure a calcium efflux signal with an average duration of 204 +/- 26 s and 146 +/- 46 s, respectively. Measurements of hydrogen ion signals in normal and in low sodium media showed that the release of cortical vesicle material from these eggs lasts for about 30 and 50 s, respectively. The data indicate that most of the calcium that is lost from sea urchin eggs originates from the cytosol in which it is released during fertilization and then pumped out through the plasma membrane. Calcium loss due to cortical granule release accounts for less than 14% of the total loss measured. We also measured a substantial post-fertilization calcium efflux in eggs of Phallusia mammilata, with an average duration of 265 +/- 18 s followed by smaller periodic effluxes that corresponded to oscillations in the [Ca2+]i during contractile waves in these eggs. These data, together with the lack of cortical granules in ascidian eggs, indicate that Phallusia eggs also pump out a substantial amount of calcium through the plasma membrane after fertilization.

On the dissociation constants of BAPTA-type calcium buffers.

We have determined or redetermined the calcium dissociation constants of seven BAPTA-type buffers with KD's in the range from 0.4 microM to about 20 mM in 300 mM KCl. These include four newly synthesized ones: 5-nitro BAPTA; 5,5'-dinitro BAPTA; 5-methyl-5'-nitro BAPTA; and 5-methyl-5'-formyl BAPTA. Moreover, we tabulate dissociation constants or KD's for BAPTA and eleven BAPTA-type buffers, compare most of them with an empirical curve based upon so-called Hammett values, and predict KD's for several still unsynthesized but potentially valuable buffers.

Chemiluminescence microscopy as a tool in biomedical research.

Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress has been made in techniques to measure chemiluminescence. Ultra-sensitive photometers or luminometers have become widely available and can be obtained with automatic injectors and microplate readers. In addition, imaging photon detectors have been developed that allow the imaging of chemiluminescence from gels, blots, and microplates. Imaging photon detectors have also been attached to microscopes and allow imaging of chemiluminescent probes and reporter genes in cells and tissues. Specific methods of photon collection, storage, and analysis have been developed for microscopic imaging of chemiluminescence. Two of these methods are discussed in detail. The first is a method of data storage that allows days of continuous imaging without creating oversized files. The second is a method for calibrating photon imaging microscopes using a low-light standard. Such calibration will be helpful for comparing the performance of various photon imaging systems and for comparing data obtained in different laboratories.

Calcium explosions as triggers of development.

A few years ago, Gilkey et al. showed that the development of medaka fish eggs begins with a free calcium explosion within the cytoplasm. This paper summarizes those findings; provides an interim report on the effects of injecting calcium and hydrogen ion buffers into medaka eggs; reviews recent evidence of similar calcium increases in other activating eggs, as well as sperm and oocytes (Table 1); and attempts to put these explosions in a broader context (Figures 1 and 4).