Calcium buffer injections delay cleavage in Xenopus laevis blastomeres.

Microinjection of calcium buffers into the two-cell Xenopus laevis embryo delays cell division in a dose-dependent manner. Four calcium buffers in the BAPTA series with different affinities for calcium were used to distinguish between a localized calcium gradient regulating cleavage and the global calcium concentration regulating this event. DibromoBAPTA (Kd = 1.5 microM) was found to delay cleavage at the lowest intracellular concentration (1.3 mM) of the four buffers tested. The effectiveness of the calcium buffers was dependent upon the buffer dissociation constant but not in a linear fashion. The concentration of buffer required to delay cleavage increased as the buffer's dissociation constant shifted above or below that of the optimum buffer, dibromoBAPTA. This relationship between a calcium buffer's effectiveness at delaying cleavage and its calcium affinity provides support for the hypothesis that a calcium concentration gradient is required for normal cell cycle progression (Speksnijder, J. E., A. L. Miller, M. H. Weisenseel, T.-H. Chen, and L. F. Jaffe. 1989. Proc. Natl. Acad. Sci. USA. 86:6607-6611). DibromoBAPTA was also injected with two different amounts of coinjected calcium to test the possibility that the free calcium concentration of the buffer solution is the important parameter for delaying cleavage. However, we found that changes in buffer concentration have a much stronger effect than changes in the free calcium concentration. This observation supports the hypothesis that BAPTA-type buffers exert their effect by shuttling calcium from regions of high concentration to those of lower concentration, reducing any calcium concentration gradients present in the Xenopus embryo.

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.

Embryonic fibroblast motility and orientation can be influenced by physiological electric fields.

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo. We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields. These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction. The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater. These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10-fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range.

Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage.

We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation. The average pHi in unfertilized eggs is 7.33 +/- 0.11 (SD; n = 21) with a resting membrane potential of -10.1 +/- 3.5 (SD; n = 38) mV. Within 2 min after the onset of the fertilization potential, there is a slight, transient pHi decrease of 0.03 +/- (SD, n = 8), followed by a distinct, permanent pHi increase of 0.31 +/- 0.11 (SD; n = 7) beginning approximately 10 min after the start of the fertilization potential and becoming complete approximately 1 h later. The pHi remains near this level of 7.67 +/- 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHi oscillations with a mean amplitude of 0.03 +/- 0.02 (SD, n = 38). Eggs perfused for at least 2 h in Na+-free solution with 1 mM amiloride exhibited all of these pHi changes, so these changes do not require extracellular Na+. Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed.

Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis.

Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)-sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER-enriched layer exhibited the largest release of Ca2+ in a dosage-dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis.

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 elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis.

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]i increased from 0.4 to 1.2 microM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]i were detected through the first cleavage division. In eggs impaled with two Ca2+ electrodes, the Ca2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of approximately 10 microns/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca2+ wave at the sperm entry site as approximately 1 min. Through these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]i during fertilization indicates that the Ca2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres.

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.

Ion currents and membrane domains in the cleaving Xenopus egg.

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.

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.

Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores.

Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4-bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction.

Simulations of intracellular calcium release dynamics in response to a high-intensity, ultrashort electric pulse.

Numerical simulations for electrically induced, intracellular calcium release from the endoplasmic reticulum are reported. A two-step model is used for self-consistency. Distributed electrical circuit representation coupled with the Smoluchowski equation yields the ER membrane nanoporation for calcium outflow based on a numerical simulation. This is combined with the continuum Li-Rinzel model and drift diffusion for calcium dynamics. Our results are shown to be in agreement with reported calcium release data. A modest increase (rough doubling) of the cellular calcium is predicted in the absence of extra-cellular calcium. In particular, the applied field of 15 kV/cm with 60 ns pulse duration makes for a strong comparison. No oscillations are predicted and the net recovery period of about 5 min are both in agreement with published experimental results. A quantitative explanation for the lack of such oscillatory behavior, based on the density dependent calcium fluxes, is also provided.

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.

Involucrin-positive keratinocytes demonstrate decreased migration speed but sustained directional migration in a DC electric field.

When skin is wounded, keratinocytes from the cut edges of the epidermis migrate over the wounded area to re-epithelialize the wound. It is not clear which cells of the epidermis have the capacity to migrate and contribute to this re-epithelialization: the less differentiated cells of the basal layer, or the more differentiated, involucrin-positive suprabasilar cells. Here we demonstrate that both involucrin-negative and involucrin-positive cells are able to respond to a directional cue for migration with sustained directional migration. When cultured keratinocytes are exposed to a physiologic DC electric field of 100 mV per mm as a cue to guide migration (galvanotaxis) they migrate toward the cathode with equivalent directionality. The involucrin-positive cells, however, display mean migration speeds approximately one half (23.6 microm per h) of the mean rate achieved by involucrin-negative cells (46.5 microm per h). Despite their decreased migration rates, involucrin-positive cells appear to possess an intact mechanism for sensing a directional signal, transducing that signal, and responding with sustained directional migration. Because electric fields are endogenous in skin wounds, it is likely that both the basal, involucrin-negative cells and the involucrin-positive suprabasilar cells respond to this cue with directional migration. The new observation that involucrin-positive cells can indeed migrate suggests that these cells may also contribute to wound re-epithelialization in vivo.

Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.

Outwardly directed ionic currents have been measured leaving skin wounds in vivo. These currents generate physiologic electric fields of approximately 100 mV/mm, which may function to direct keratinocyte migration toward the healing wound. We investigated whether the substrate on which the keratinocyte migrates modulates the galvanotactic response to an electric migratory signal. Cultured human keratinocytes were plated on different matrices; types I and IV collagen, fibronectin, laminin, and tissue culture plastic. The effect of an applied direct current (DC) electric field on directional migration was monitored by time-lapse video microscopy over a 2-h period. Directionality was quantitated by calculating the cosine of the angle of migration in relation to anodal-cathodal orientation. Migration toward the negative pole was observed on all matrices as compared with controls (no applied field), which displayed random migration. No significant increase in directional response occurred when the field strength was increased by 100 mV/mm (physiologic levels) to 400 mV/mm. The degree of directionality and the average net cell translocation however, varied significantly with the substrate. The greatest cathodal migration in response to a DC electric field was observed with keratinocytes plated on types I and IV collagens and plastic. The directional migratory response was least on a laminin substrate, whereas cells on fibronectin demonstrated a response that was intermediate between those of collagen and laminin. These results suggest that physiologic ionic currents in concert with underlying matrix may influence the rate of reepithelialization of skin wounds.

Migration of human keratinocytes in electric fields requires growth factors and extracellular calcium.

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.2%-2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media.

The galvanotaxis response mechanism of keratinocytes can be modeled as a proportional controller.

Human keratinocytes actively crawl in vitro when plated onto a collagen-coated glass substrate, and their direction of migration is totally random. In response to an imposed DC electric field, they migrate asymmetrically, moving mostly toward the negative pole of the field. The authors have analyzed experimental data reported by others to determine the basic characteristics of the cellular response machinery in these keratinocytes. This movement can be completely described mathematically using two independent variables: the speed, V, and the angle of migration, phi. The authors propose a model in which a steerer (controller without feedback) is responsible for determining the speed, and an automatic controller (controller with feedback) is responsible for determining the angle of migration. The torque to rotate is induced by a deterministic cellular signal and a stochastic cellular signal. The cellular machine characteristics are determined as follows: The angular dependence of the detection unit is sin phi; the detection unit detects the guiding field in a linear fashion; the cellular reaction unit can be described by a constant; the chemical amplifier, as well as the cellular motor work, is linear; the cellular characteristic time, which quantifies the cellular stochastic signal, is 50 min.

Chemokine receptor expression by human syncytiotrophoblast.

Despite their potential importance in placental HIV infection and placental immune function, nothing is known about the expression of chemokine receptors by human syncytiotrophoblast cells. Immunocytochemical analysis revealed that primary cultures of term syncytiotrophoblast cells express CCR1, CCR3, CXCR4, and CCR6. Immunohistochemical examination of cryosections of term placental villous tissue confirmed the expression of CCR3, CXCR4, and CCR6 by trophoblast cells. The primary syncytiotrophoblast cultures showed no reactivity with antibodies against CCR5. In the villous tissue sections, CCR5 was detected in stromal cells and blood vessel walls but was not found in trophoblast cells. RT-PCR analysis of RNA extracted from cultured syncytiotrophoblast cells confirmed that the cells express message for CCR1, CCR3, CXCR4, CCR6 and CCR10. No transcripts corresponding to CCR2b, CCR5, or CCR8 were detected. Other experiments showed that exposure of syncytiotrophoblast cells to soluble SDF-1alpha elicited a calcium mobilization response, consistent with the expression of functional CXCR4. Thus, human syncytiotrophoblast cells express CXCR4, a known co-receptor for TCL-tropic HIV-1 isolates but do not express CCR5, the major co-receptor for M-tropic isolates. In addition to implications for the maternal-fetal transmission of HIV, the expression of chemokine receptors by syncytiotrophoblast cells could be important in other aspects of placental immune function.

A computerized 2-dimensional vibrating probe for mapping extracellular current patterns.

We describe a computer-assisted 2-dimensional vibrating probe system for mapping endogenous electric current patterns in biological preparations. This system overcomes some of the main limitations of the original 1-dimensional vibrating probe design and adds several new capabilities. Two piezo-electric bender elements mounted perpendicularly are used to vibrate the probe in a circle by applying 2 sine waves (1 to each element) that are 90 degrees out-of-phase with each other. The circular rotation of the probe allows it to detect simultaneously the 2 orthogonal components of a current in the horizontal plane. The voltages measured by the probe are digitized and analyzed by a computer and are used to calculate a current vector. A graphical representation of the current vector is then superimposed on a video image of the experimental preparation. This probe system responds to known currents in the expected manner and exhibits a low inherent noise level. Also included in this paper are some preliminary measurements made with this instrument on neurulating Xenopus embryos and on transected larval sea lamprey (Petromyzon marinus) spinal cords.