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NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS

II. Electrophysiological Observations

SIEGFRIED W. DE LAAT and JOHN G . BLUEMINK From the Hubrecht Laboratory, International Embryological Institute, Utrecht, The Netherlands

ABSTRACT The electrical membrane potential (Em) and electrical membrane resistance (Rm) were measured continuously during the first cleavage of Xenopus eggs, using intracellular micro- electrodes . A sharp hyperpolarization of Em and decrease in Rm can be observed from 6 to 8 min after the onset of cleavage . This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the inter- blastomeric membrane during normal cleavage . Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the mem- brane newly formed during cleavage . These conditions allow for a direct measurement of the permeability properties of the new membrane . It was found that under -these con- ditions Em reaches values about 3 times more negative and Rm reaches values about 1 .5-3 times smaller than during normal cleavage. The extent of reduction of Rm can be cor- related with the surface area of the newly formed membrane . We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K+ ions) . Its mean specific resistance is 1-2 kQ- cm2, as against 74 k2 . cm2 for the pre-existing membrane . No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected .

INTRODUCTION It is possible to distinguish two phases in the proc- 1937 ; Selman and Perry, 1970 ; Selman and ess of furrow formation in the amphibian cleaving Waddington, 1955 ; Zotin, 1964) . However, direct zygote. The cleavage process starts with a local morphological evidence regarding the process of surface constriction due to the action of a con- membrane formation during cytokinesis is scarce tractile microfilament system underlying the cell (see Bluemink and de Laat, 1973), and data on the membrane (Bluemink, 1970, 1971 a, 1971 b ; physiological properties of this membrane material Kalt, 1971 ; de Laat et al ., 1973 ; Selman and are even more scanty. Woodward (1968) made an Perry, 1970). Surface constriction is followed by a analysis of the changes in electrical membrane process of membrane insertion in the plane of the characteristics in Rana pipiens eggs dividing nor- furrow to complete the separation of the blasto- mally and in eggs undergoing furrow regression meres (Bluemink, 1970, 1971 b; Schechtman, due to exposure to hypertonic salt solutions .

THE JOURNAL OF CELL BIOLOGY • VOLUME 60, 1974 . pages 5129-540 529

Mainly on the basis of the increase in membrane Hammer et al ., 1971 ; de Laat et al., 1973) was capacitance during furrow regression, he sug- studied and calculations were made of the specific gested that during cleavage new membrane is membrane resistance of the pre-existing and newly introduced between the blastomeres, and that this formed membrane . membrane has a low resistance compared to the MATERIALS AND METHODS pre-existing outer membrane . The finding that in Xenopus eggs electrical Eggs of Xenopus laesis were derived from hormonally membrane characteristics start to change at 6-8 stimulated couples, chemically decapsulated, and handled as described before (de Laat et al ., 1973) . min after the onset of first cleavage (de Laat et al ., Eggs were kept in Steinberg solution (Steinberg, 1973), and the hypothesis that these changes are 1957) unless otherwise indicated . Experiments were brought about by the incorporation of new mem- carried out at room temperature (20-24°C) . For brane in the bottom of the cleavage furrow have some experiments the vitelline membrane was re- formed the basis of our recent work . The foregoing moved with forceps. study (Bluemink and de Laat, 1973) was devoted to the investigation of the ultrastructural changes Electrical Measurements connected with new membrane formation, to the Em and Rm were measured continuously during rating of new membrane formation by surface egg cleavage, using microelectrode techniques . measurements under different conditions, and to Current-passing and voltage-recording glass micro- the direct demonstration of de novo surface forma- electrodes were filled with 3 M KCI and had 5-10 tion by surface-marking experiments. A tem- MSZ DC resistance. Tip potentials were smaller than poral correlation was found between the morpho- I mV. Microelectrodes were connected to Pt-Ag- logical signs of new membrane formation and AgCl electrodes via Steinberg solution bridges . When changes in the electrical membrane properties . the total drift of electrode potentials was greater than This provides evidence for the above hypothesis . 1 mV during one experiment (usually 30-60 min), Measurements of the geometrical surface area of the results were not used . Generally, two voltage- the newly formed surface showed that the rate of recording microelectrodes were used as a control for isopotentiality of the cytoplasm. Intracellular volt- new membrane formation is influenced by en- ages were measured with respect to an indifferent vironmental conditions . When the vitelline mem- Pt-Ag-AgCl electrode in the bath by means of differ- brane is removed the entire new surface is exposed ential preamplifiers (Transidyne MPA-6, Transidyne to the medium, then visible as unpigmented sur- General Corp ., Ann Arbor, Mich.) . The bath was face material (Fig . 7) . This area of newly formed grounded via a separate Pt-Ag-AgCl electrode . Con- surface is smaller than the interblastomeric surface stant current pulses of 1 .2-s duration and 3 X 10-s-A formed in normally cleaving eggs . The exposure amplitude were delivered every 10 s by a constant of unpigmented surface area to the medium occurs current stimulator . The polarity of the pulses was also after cytochalasin B (CCB)-induced furrow alternated to prevent net current flow through the regression . Both treatments enable one to investi- electrodes and the cell membrane . The current was gate directly the properties of the unpigmented measured by means of a current-to-voltage converter in the ground return of the circuit (Fig. 1) . surface, which in normally cleaving eggs will form In most cases the microelectrodes were introduced the intercellular membrane, and as such, is rela- into the egg about 10 min before the onset of first tively inaccessible to the investigation of perme- cleavage . This stage can be determined from pig- ability properties. ment movements in the animal egg cortex resulting In the present study an analysis was made of the in a more "granular" pigmentation pattern (Rzehak, relation between the changes in membrane proper- 1972) . The voltage-recording microelectrodes were ties and the insertion of new membrane material inserted first . When the measured Em reached a during the first cytokinetic cycle . Using micro- stable value (after ca. 3 min), the current-passing electrode techniques, the electrical membrane microelectrode was inserted. Both the intracellular voltages and the applied current were monitored on potential (Em) and electrical membrane resistance an oscilloscope (Tektronix 565, Tektronix, Inc ., (R m) were measured in eggs cleaving inside and in Beaverton, Ore .) and recorded on a pen-recorder the absence of the vitelline membrane, and in eggs (Hellige 19, Fritz Hellige & Co., GMBH; Freiburg undergoing CCB-induced furrow regression . The in Breisgau, B .R.D.). Every second, in phase with possible influence of CCB on the ion-permeability the front edges of the current pulses, the outputs properties of the membrane (Bluemink, 1971 b ; of the preamplifiers and of the current-to-voltage

5 3 0 THE JOURNAL OF CELL BIOLOGY . VOLUME 60, 1974

-80 1 .6

i -60 1.2 { C

E e 40 0 .8 c Ô L e C s a e -20 0.4 C mc E EC AgCI E E 0 0 0 +5 10 15 20 25 30 35 let time (min) 2nd FIGURE 1 Schematic diagram of the Perspex measur- ing chamber and the electrode arrangement used in the FIGURE 2 Changes in Em and Rm during the first present experiments . Not to scale . cleavage of a Xenopus egg within the vitelline mem- brane . The arrows indicate the onset of the first and second cleavage, respectively . converter were simultaneously digitally measured and recorded on punch tape (Facit 4070, Facit A. B., Atvidaberg, Sweden) . Calculations and plots of tively small increase in Em (not shown : the process Em and Rm were made on a Philips X-8 computer of wound closure precedes the time span shown (Philips Nederland N . V., Eindhoven, The Nether- in the figure), Em remains nearly constant during lands) at the Academic Computer Centre, Utrecht . the first 6-8 min of cleavage . In the next 10 min it For further details of the method used, see de becomes about 10 mV more negative, while during Laat et al . (1973) . the second half of cleavage it often shows a small decrease (1-3 mV) preceding the onset of the Replacement of Bathing Medium second cleavage. Rm shows a pattern almost in- For some experiments it was necessary to replace verse to that of Em. Being constant initially, it the bathing medium during a measurement by a drops drastically from 7 till 17 min after the onset medium of different composition. The egg was placed of first cleavage . Often a small increase precedes in a small hemispherical hole (diameter 1 .5 mm, the onset of second cleavage . The absolute values depth 0.5 mm) on top of a cylindrical elevation in of both Em and Rm vary from egg to egg, the varia- the middle of the Perspex measuring chamber (Fig . tion in resistance being relatively great (Table I) . 1). The measuring chamber contained about 20 ml However, the pattern of changes is well defined . of medium . A ring could be turned up and down the The change in Em and Rm invariably starts be- elevation . When it was turned up, about 950/0 of the tween 6-8 min after the onset of first cleavage . The bathing medium could be removed while the egg small drop in Em coincides with the moment at remained in the original medium within the ring . After the introduction of a new solution the ring was which the blastomeres become apposed more turned down, resulting in a rapid exchange of the closely. The potentials recorded by two micro- medium surrounding the egg . This method was pre- electrodes inserted in different regions of the ani- ferred over the reciprocal infusion-withdrawal pump mal part of the egg never showed significant system (see e.g. Tupper, 1972), since it requires less differences, indicating isopotentiality of the cyto- time to obtain the same dilution and gives a rapid plasm and complete electrical coupling during exchange of solutions in the egg environment . the first cleavage . From these experiments it can be concluded that RESULTS the electrical membrane properties change from a well-defined moment onwards during the cleavage Normal Cleavage cycle . This may be due to a change in perme- During first cleavage of Xenopus eggs within the ability properties of the pre-existing cell mem- vitelline membrane, Em and Rm invariably change brane, to the insertion of new membrane material according to a characteristic pattern (Fig. 2) . with permeability properties different from those (See also de Laat et al ., 1973 .) After initial wound of the pre-existing membrane, to a rapid change closure around the inserted microelectrodes, in intracellular ion activities, or to any combina- which is reflected in an increase in Rm and a rela- tion of these factors.

SIEGFRIED W. DE LAAT AND JOHN C. BLUEMINK New Membrane Permeability 531

TABLE I The Membrane Potential and Membrane Resistance at the Onset of the First and Second Cleavages under Various Conditions

Onset of first cleavage Onset of second cleavage

Condition Membrane potential Membrane resistance Membrane potential Membrane resistance

mV kn m V kO + Vitelline mem- -9 .7 t 0 .6 (25) 852 f 48 (22) -20 .3 t 0.8 (20) 488 f 28 (17) brane - CCB

- Vitelline mem- -9 .8 t 0.7 (8) 901 dr 62 (8) -60 .0 t 2 .5 (7) 336 t 31 (7) brane - CCB

+ Vitelline mem- -10 .8 f 0 .5 (7) 779 f 43 (6) -60 .9 t 3.4 (6) 165 f 10 (6) brane + CCB

The results are presented as mean f standard error (number of observations in brackets) .

Intracellular Na+, K+, and Cl- activities have contraction . Beginning approximately 7 min after been measured during cleavage, using ion-selec- the onset of cleavage new membrane material tive microelectrodes .1 Although minor but signifi- is inserted into the outer surface along the original cant changes can be observed, they cannot account plane of cleavage . (See also Bluemink, 1971 a, for the change in Em. Morphologically it can be 1971 b ; Bluemink and de Laat, 1973 ; Hammer et seen that new membrane material is inserted into al., 1971 ; Selman and Perry, 1970 .) This results in the pre-existing membrane (Bluemink and de a meridional band of unpigmented surface sur- Laat, 1973) from approximately 7 min after the rounding the egg (Fig . 8) . onset of first cleavage onwards . During normal cleavage, only a minor part of this material is Cleavage in the Absence of the exposed to the outer medium, as can be judged Vitelline Membrane from the surface area of the visible, unpigmented surface in the furrow (Fig . 6) . The major part As pointed out above, the membrane forming forms the intercellular membrane of the two the interblastomeric surface in normally cleaving blastomeres. eggs is exposed to the outer medium in eggs It might be possible to distinguish between the cleaving in the absence of the vitelline membrane . various possibilities mentioned above if one could The pattern of changes in Em and Rm is qualita- force the new membrane material to expose its tively similar to that of normally cleaving eggs, entire surface to the surrounding medium . If the but the changes themselves show great quantita- altered membrane properties are due to the newly tive differences (Fig . 3) . By comparing the mean formed membrane only, exposure of more of its Em and mean R. in eggs cleaving normally and in surface should lead to a greater alteration of the eggs cleaving in the absence of the vitelline mem- measured Em. and Rm . In cleaving eggs devoid of brane using the Student's t test, it can be shown their vitelline membrane the two blastomeres that removal of the vitelline membrane does not tend to separate under gravity and expose com- significantly alter these parameters at the onset of pletely their newly formed membrane to the outer first cleavage (P > 0 .50) . Being constant initially, medium (Fig . 7) . (See also Bluemink and de Em and R m change drastically from approximately Laat, 1973 ; de Laat et al ., 1973 .) Eggs exposed to 7 min after the onset of cleavage onwards . Within CCB show furrow regression after the initial 10 min Em reaches a value about three times more negative than in eggs cleaving insider the vitelline 1 de Laat, S. W., and R. F. D. Buwalda. Manu- membrane, while Rm becomes about 1 .5 times script in preparation . smaller. During the second half of cleavage Em

532 THE JOURNAL OF CELL BIOLOGY . VOLUME 60, 1974

and Rm remain more or less constant, although in properties, and to examine the permeability some cases minor fluctuations can be observed properties of the new membrane formed during (Fig. 3). CCB-induced furrow regression, Em and R m were At the onset of second cleavage both E m and measured in eggs dividing within their vitelline Rm differ significantly from the values obtained in membrane in the presence of CCB . In most meas- normally cleaving eggs (P < 0.001 and P < 0 .005, urements the voltage-recording and current- respectively ; see also Table I) . Removal of the passing microelectrodes were inserted into the egg vitelline membrane does not significantly alter the 20-30 min before the onset of cleavage, the bath- electrical properties of the pre-existing membrane ing medium being Steinberg solution. After before cleavage . It seems unlikely that removal as stabilization of the observed Em and Rm, the such would alter the pre-existing membrane medium was replaced by Steinberg solution con- during the process of cleavage . Thus, it can be taining 10 ,ug CCB/ml and 1 % dimethylsulfoxide concluded that exposure to the medium of a (DMSO) . In some experiments the microelec- greater area of inserted new membrane material trodes were inserted while the egg was in the CCB- results in an enhanced alteration of the electrical Steinberg solution. CCB affects the process of properties of the outer membrane. Apparently wound closure around the microelectrode tip. the new membrane has ion-permeability proper- Normally, closed wounds do not reopen upon ties different from those of the pre-existing mem- addition of CCB to the medium . However, when brane . the microelectrodes are inserted into eggs already exposed to CCB, wounds sometimes fail to close, Cleavage in the Presence of CCB resulting in leakage between the cytoplasm and the medium. The chances for a successful meas- It has been suggested previously (Bluemink, urement are improved when penetration of the 1971 b, Manasek et al ., 1972) that CCB might cell membrane is perpendicular to the cell surface, interfere with ion-permeability properties of the resulting in a tight, small, circular wound . Wound cell membrane . No evidence supporting this closure is then reflected in a contraction (Blue- possibility has been found so far . CCB injected into mink, 1972) around the microelectrode tip and an Xenopus eggs immediately beneath the middle of increase in measured Rm (de Laat et al., 1973) . the furrow 3-4 min after the onset of cleavage An example of a successful experiment is given in leads to local furrow regression and the appear- Fig. 4. ance of an unpigmented surface area, but does not The striking result is that Em and Rm change alter Em or Rm during cleavage (de Laat et al ., according to a pattern essentially the same as ob- 1973). served in eggs cleaving without the vitelline mem- To investigate whether exposure to externally brane in the absence of CCB . Again, Em and Rm applied CCB influences the electrical membrane remain constant during approximately the first

-80 1.6 -80 1.6

-60 1.2 8 -60 1.2 8 G /Io f 0 E E u m 40 0.8 -40 0.8 c a mc c 4 0 n m -20 0.4 20 0.4 P m C m a L E E E E E E 0 0 0 0 0 5 10 15 20 25 30 35 0 + 5 10 15 20 25 30 4 35 1st time (min) 2nd let time (min) 2nd FIGURE 3 Changes in E m and R m during the first FIGURE 4 Changes in Em and R, during the first cleavage of a Xenopus egg devoid of the vitelline mem- cleavage of a Xenopus egg within the vitelline mem- brane. The arrows indicate the onset of the first and brane, exposed to 10 ug CCB/ml . The arrows indicate second cleavage, respectively. the onset of the first and second cleavage, respectively .

SIEGFRIED W. DE LAAT AND JOHN C . BLUEMINK New Membrane Permeability 533

7 min of cleavage. Comparison of Em. and Rm at (see Figs . 4-6 of Bluemink and de Laat, 1973) . the onset of first cleavage in CCB-treated eggs with Measurements made upon such eggs are techni- those in untreated eggs cleaving inside the vitelline cally relatively difficult . Small surface injuries are membrane (Table I) yields no statistical differ- easily provoked during handling of eggs outside ences (P > 0 .20 and P > 0.40, respectively) . the vitelline membrane, and these often do not The same holds for a comparison with eggs devoid heal completely in the presence of CCB . Only of their vitelline membrane (P > 0.20 and three successful experiments' were carried out. P > 0 .10, respectively) . Apparently CCB does not Although this is not enough for a reliable statistical influence the ionic permeability of the pre-existing comparison, a qualitative analysis can be made . membrane . This is in accordance with our pre- Fig. 5 shows the results of one such experiment . vious findings (de Laat et al ., 1973) . The changes in Em and R m are similar to those in From approximately 7 min after the onset of eggs inside the vitelline membrane exposed to cleavage onwards, the observed changes are similar CCB. However, the drop in Rm seems to be rela- to those in eggs cleaving in the absence of the tively greater. vitelline membrane with one exception. Rm de- creases over a longer period and reaches a lower Specific Membrane Resistance of the level at the onset of second cleavage than it does Uncleaved Egg in eggs devoid of their vitelline membrane in the absence of CCB (P < 0 .001), but no statistical By combining surface area measurements of the difference is seen in Em at the onset of second uncleaved egg with measurements of Rm in such cleavage (P > 0 .20) (see also Table I) . As a eggs, one can obtain an estimate of the specific control, Em and Rm were measured in a series of membrane resistance . It had been found that the eggs exposed to 1 % DMSO in Steinberg solution . mean geometrical surface area of uncleaved Such eggs show a completely normal cleavage and Xenopus eggs inside the vitelline membrane is the measured parameters change as in normally 5.11 mm 2 (Bluemink and de Laat, 1973) . The cleaving eggs. mean Rm is 852 kS2 (Table I) . Without making In a final series of experiments Em and Rm were corrections for surface foldings, the specific mem- measured in eggs cleaving outside the vitelline brane resistance would be 43 .5 kS2'cm2. However, membrane while exposed to Steinberg solution scanning electron microscopy and thin-section containing 10 •g CCB/ml and 170 DMSO. Such transmission show the cell membrane to be very an egg shows a furrow formation during the first rough (Figs . 9 and 10) . Measurements of the 6-8 min of cleavage followed by complete furrow distance between two points on the surface follow- regression . The egg flattens strongly against the ing the contour of the membrane yield a length substrate, and a wide band of unpigmented new of about 3070 in excess of the linear distance surface appears in the original cleavage plane (Bluemink and de Laat, 1973) . This means that the real surface area is about 1 .7 times the meas- 1 .6 ured geometrical surface area and equals 8.69 mm2. Using this number, the specific membrane resistance of the uncleaved egg can be put at / %1,/- 1 .2 I . 8 C 74 kSU cm2. f E -40 cu s 0.8 Specific Membrane Resistance of the a 0 New Membrane 0 m Y 0.4 W It is assumed that the change in Em and Rm a E during cleavage arises from the insertion of new 0 8E E E 0 0 membrane material having different ionic perme- 4 5 10 15 20 25 30 } 35 ability properties than the pre-existing membrane . let time (min) 2nd The quantitative differences in these changes due FIGURE 5 Changes in E m and R m during the first to exposure to CCB or to removal of the vitelline cleavage of a Xenopus egg devoid of the vitelline mem- brane, exposed . t o 10 •g CCB/ml . The arrows indicate membrane could originate from the differences in the onset of the first and second cleavage, respectively . surface area of inserted new membrane exposed to

534 THE JOURNAL OF CELL BIOLOGY . VOLUME 60, 1974

the medium. If this is so, then the specific mem- cleaved egg and 1070 of the total geometrical brane resistance of the new membrane formed surface area of new membrane formed during first during cleavage in the absence of the vitelline cleavage (Bluemink and de Laat, 1973) . The membrane or in the presence of CCB should be remaining 90% will form the interblastomeric equal . Neither treatment alters R.m. of the un- surface . cleaved egg . By assuming that Rm of the pre- existing membrane remains constant throughout DISCUSSION cleavage, it is possible to calculate the resistance The course of Em and R.m during the first cleavage of the new membrane inserted into the outer of Xenopus eggs has been measured in eggs under surface, because the new membrane will form three different conditions : inside the vitelline a resistance parallel to the resistance of the mem- membrane, in the absence and in the presence of brane of the uncleaved egg. CCB; and without vitelline membrane, in the For those eggs in which Rm was measured con- absence of CCB . The last two conditions per- tinuously during cleavage, the resistance of the mitted a direct study of the properties of the new membrane at the onset of the second cleavage newly formed membrane, since this membrane is was calculated. In eggs cleaving in the absence of inserted as part of the outer surface. The previous the vitelline membrane the mean f SE of the study (Bluemink and de Laat, 1973) had shown resistance of the new membrane is 553 t 66 that this new surface is formed de novo and not at kQ (n = 7), while in eggs cleaving inside the the expense of the pre-existing surface . Further- vitelline membrane in the presence of CCB it is more, ultrastructural observations had indicated 210 t 15 kQ (n = 6) . The geometrical surface that the insertion of the membranous material area of the new membrane had been found to be starts between 4 and 8 min after the onset of the 0.33 f 0.02 mm2 (n = 11) and 0 .65 ± 0 .04 mm2 first cleavage . (n = 15), respectively (Bluemink and de Laat, Our present results show that the electrical 1973). membrane properties start to change at approxi- Multiplication of the mean resistance with the mately 7 min after the onset of cleavage under all mean surface area gives an estimate of the specific conditions used . This moment coincides with the resistance of the new membrane . In eggs cleaving structural changes which accompany the appear- outside the vitelline membrane this value is ance of new membrane . Removal of the vitelline 1 .82 kSl •cm2, while in eggs exposed to CCB it is membrane or exposure to CCB does not lead to an 1 .36 kbl . cm2. No corrections for surface foldings alteration in the electrical properties of the pre- have to be made, since the new membrane has an existing membrane . For the former treatment this almost smooth appearance (Figs . 9 and 11). A would, anyhow, not have been expected. Never- direct statistical comparison of these values is not theless, the hyperpolarization of Em and decrease possible, since different eggs were used for resist- in Rm during cleavage are significantly greater ance and surface area measurements . However, under these conditions than in normally cleaving knowing the variances of both the resistances and eggs. At least in the case of vitelline membrane the surface areas, it seems unlikely that the differ- removal, the most obvious explanation for this ence between the two values is significant . As an difference is the exposure of a greater area of newly estimate their average can be used : 1 .59 kSl .cm2. formed membrane having ionic permeability Using this calculated specific resistance, it is properties different from those of the pre-existing possible to estimate the surface area of new mem- membrane . On the basis of this assumption and brane exposed to the outer medium after normal the measurements of the geometrical surface area first cleavage . The resistance of the new surface of the new membrane (Bluemink and de Laat, area in the cleavage furrow that is exposed to the 1973), the mean specific membrane resistance of outer medium can be calculated as indicated the new membrane is calculated to be 1 .82 kQ above. Its mean f SE is 1148 f 104 kQ(n = 17) . cm2. This is comparable to the value of 950 Sl . cm2 Using the previously estimated specific resistance computed for the specific membrane resistance of of the new membrane of 1 .59 kQ •cm2, the surface the new membrane in Rana pipiens eggs (Wood- area of new membrane formed in the normal ward, 1968) and to the specific resistance of the cleavage furrow would be 0 .14 mm2. This is 2 .7 % cell membrane of many tissue cells (Plonsey and of the total geometrical surface area of the un- Fleming, 1969). The general appearance of the

SIEGFRIED W . DE LAAT AND JOHN C . BLUEMINK New Membrane Permeability 535

new membrane is that of a smooth surface (Fig . specific resistance of the new membrane to be 11) . The lumina of the open cisternae of the constant throughout the process of insertion, it is endoplasmic reticulum (ER) reported previously possible to use the change in Rm for calculating the (Bluemink and de Laat, 1973) can be considered growth of new membrane area as a function of as extracellular, and consequently, the membrane time. If this is done for eggs cleaving without the of this part of the ER system is an integral part of vitelline membrane in the absence of CCB (Fig . 3) the newly formed membrane . In calculating the and for eggs cleaving inside the vitelline membrane specific membrane resistance of the new mem- in the presence of CCB (Fig . 4), it is found that brane, no correction has been made for a possible the growth rate is constant in both cases and has a increase in surface area due to these open cis- value of about 0 .04 mm2 per min. However, the ternae. No estimates are available for the number duration of growth is 7-10 min for the former of cisternae open at any given moment, but it treatment and 15-17 min for the latter . We have seems to be rather small . If the open necks of the no explanation for this difference . On the basis of cisternae would be large enough to constitute a the same assumption, eggs cleaving without the low-resistance pathway, and if the membrane of vitelline membrane in the presence of CCB show the ER would have a low specific resistance, then a different membrane growth pattern . During the the specific resistance of the new membrane would first 15-17 min the growth rate is constant and of be higher than calculated . The membrane of the the same magnitude as under the conditions men- uncleaved egg is relatively impermeable to ions as tioned earlier . This period is followed by a period judged by its specific resistance of 74 kS2 .cm2 of exponential increase in growth rate during the (corrected for surface foldings) . latter part of cleavage, resulting in a much larger With respect to their membrane properties, new membrane area (Bluemink and de Laat, CCB-treated eggs behave roughly like eggs cleav- 1973) . The cause of this prolonged and increased ing in the absence of the vitelline membrane . The growth is unknown . main difference is the prolonged decrease in Rm Our experiments do not provide evidence that and its lower level at the onset of second cleavage . CCB does alter the ionic permeability properties These effects become more pronounced when of the membrane, a possibility advanced by Blue- CCB is applied to eggs cleaving outside the vitel- mink (1971 b) and by Manasek et al. (1972) . line membrane. There is good agreement between CCB interferes with the contractile system re- the calculated specific membrane resistances of sponsible for furrow formation (de Laat et al., the new membrane after CCB treatment and after 1973) and deranges the rate and the direction of removal of the vitelline membrane. Assuming the growth of new membrane (Bluemink and de

FIGURE 6 End of first cleavage, egg inside the vitelline membrane . The two blastomeres are closely apposed ; hardly any unpigmented surface (arrow) is visible . X 35 .

FIGURE 7 End of first cleavage, egg without vitelline membrane. The two blastomeres have moved apart ; unpigmented surface (arrows) is exposed to the medium . X 35 .

FIGURE 8 End of first cleavage, egg inside the vitelline membrane showing complete CCB-induced furrow regression . Unpigmented surface has been inserted in the former furrow area . The edge of this area is shown in the next figure . X 35 .

FIGURE 9 Scanning electron micrograph . Borderline between pigmented rough (left) and unpigmented smooth surface (right) is marked by a ridge . The smooth surface extends across the ridge into the rough surface area . Dislocated fragment of the ridge (arrow) . X 900 . FIGURE 10 Pigmented rough surface of the animal hemisphere outside the furrow region at the onset of first cleavage . X 6600 . FIGURE 11 Egg exposed to 10µg CCB/ml at the onset of first cleavage, fixed 11 min later . Unpigmented smooth surface along the regressed furrow, i .e ., new membrane area. X 6600 . The Xenopus eggs were prepared for electron microscopy following the method described previously (Bluemink and de Laat, 1973) .

53 6 THE JOURNAL OF CELL BIOLOGY • VOLUME 60, 1974

SIEGFRIED W . DE LAAT AND JOHN C . BLUEMINK New Membrane Permeability 537

Laat, 1973) . Whether both effects are due to a Our results may have some important conse- common cause, or whether one effect causes the quences for the concept of intercellular communi- other is unknown . cation in early amphibian development. It is a Knowing that the altered permeability proper- well-established fact that the blastomeres in early ties are due to the new membrane, the sequence of cleavage stages are electrically coupled (Ito and changes in E. and R. during normal cleavage Hori, 1966 ; Palmer and Slack 1970 ; Woodward, (Fig . 2) becomes understandable. During approxi- 1968). In our measurements the coupling ratio mately 7 min of cleavage the furrow is established, V2/V1 (Loewenstein, 1966) was always greater most probably by the action of a microfilamentous than 0.95 during first cleavage . Nothing else can contractile system underlying the cell membrane be expected since the intercellular membrane is (Bluemink, 1970, 1971 a, 1971 b ; Kalt, 1971 ; not yet completed at the onset of second cleavage, Selman and Perry, 1970) . The increase in total and a cytoplasmic bridge still connects the two geometrical surface area necessary for furrow blastomeres (Bluemink and de Laat, 1973) formation results from smoothing out of the pre- However, after the completion of the intercellular existing rough surface, as can be judged by the membrane, its relatively low specific resistance, unâltered Em and R. during this phase . From that due to high potassium permeability, will result in moment on, the interblastomeric surface begins an appreciable coupling ratio without it being to be formed, a process not yet completed at the necessary to assume that specialized membrane onset of the second cleavage (Bluemink and de junctions are involved in the transport of ions Laat, 1973) . A small fraction of this newly formed across the intercellular membrane . According to membrane is inserted into the walls of the cleavage Sanders and Zalik (1972) such junctions are furrow itself during the first 10 min of new mem- indeed absent at these stages . brane formation . It is visible as unpigmented sur- The hyperpolarization of the membrane accom- face at the bottom of the furrow . This fraction is panied by an increased conductance, as we ob- responsible for the rapid hyperpolarization of E. served, seems to be a general phenomenon during and decrease in R,„ . Also, in scanning electron cleavage stages of amphibian embryos . Palmer and micrographs, patches of smooth surface can be Slack (1970) reported similar changes during seen to be inserted into the pre-existing, rough early development of Xenopus embryos. However, surface (Fig . 9) . During the latter part of the no detailed analysis of the course of changes cleavage process the distance between the walls of during a single cleavage cycle was presented by the cleavage furrow is reduced, and the two blasto- them, and as a consequence, no direct relation meres tend to flatten against and adhere to each between the observed changes and new membrane other. The degree of adhesion is rather variable formation could be detected . Their suggestion among different eggs . This process results in the that the hyperpolarization could be due to a fall observed small depolarization of E„ and small in intracellular sodium concentration in combina- increase in R„, towards the onset of the second tion with a change in the relative permeability cleavage, which is due to a reduction of the ex- of the surface membrane to sodium and potassium posed area of new membrane . It is important to now appears to be unrealistic, since no such realize that changes in E„ and R,,, similar to changes in sodium activity have been observed those described for the first cleavage can be ob- during first cleavage .' Ito and Hori (1966) per- served during the succeeding cleavage cycles . formed a similar but less detailed study on Triturus To determine which ion permeabilities are pre- eggs, but no attempt was made to explain the ob- dominant in the new membrane, E., R., and served increase in membrane potential . intracellular Na+, K+, and Cl- activities were The situation during first cleavage of Rana measured simultaneously in dividing eggs bathed pipiens eggs has been studied in more detail by in media of different ionic composition .' The ion Woodward (1968) . In eggs dividing normally, the activities were measured with ion-selective micro- membrane capacitance remains relatively con- electrodes . From this study it was concluded that stant while Em and R,,, change according to a the new membrane has a relatively high perme- pattern roughly similar to that described here for ability for potassium ions . During cleavage the Xenopus eggs. However, the pattern is obscured by membrane potential shifts towards the equi- many rather large fluctuations, which could be librium potential for potassium (in Steinberg artifactual (incomplete wound healing) . In eggs solution : EK + = - 115 mV) . undergoing furrow regression due to exposure to

538 THE JOURNAL OF CELL BIOLOGY • VOLUME 60, 1974

hypertonic salt solutions, he observed an increase surface contractility and cell junction formation in membrane capacitance and a greater altera- during egg cleavage in Xenopus laevis. Cytobiologie. 3 :176 . tion in Em and Rm. Mainly on the basis of the BLUEMINK, J . G . 1971 b. change in membrane capacitance, Woodward Cytokinesis and cytochalasin- induced furrow regression in the first cleavage suggested that new membrane is introduced be- zygote of Xenopus laevis . Z . Zellforsch. Mikrosk. tween the blastomeres, and that this membrane Anat. 121 :102 . has a low resistance compared to the pre-existing BLUEMINK, J . G. 1972 . Cortical wound healing in the membrane . Em was found to be sensitive to the amphibian egg . J. Ultrastruct. Res. 41 :95 . potassium concentration in the bathing medium . BLUEMINK, J . G ., and S . W . DE LAAT 1973 . New mem- He concluded that the new membrane has selective brane formation during cytokinesis in normal and permeability to potassium ions, but no intracellu- cytochalasin B-treated eggs of Xenopus laevis . I . lar ion activities were determined . Electron-microscopical observations . J. Cell Biol. In nonamphibian embryos, early cleavage is 59 :89. . M ., B also associated with a hyperpolarization of the BURGER, M . M . BOMBIK, B . MCL . BRECKEN- RIDGE, and J. R . SHEPPARD . 1972 . Growth control membrane (e .g . Fundulus, Bennett and Trinkaus, and cyclic alterations of cyclic AMP in the cell 1970 ; Asterias, Tupper, 1972 ; Fucus, Bentrup, cycle . Nat. New Biol . 239 :161 . 1970 ; Weisenseel and Jaffe, 1972) . In the latter HAMMER, N . G ., J . D . SHERIDAN, and R . D. ESTEN- Em two species, it was shown that the change in SEN . 1971 . Cytochalasin B . II . Selective inhibition is related to an increase in potassium perme- of cytokinesis in Xenopus laevis eggs. Proc . Soc . Exp. ability . However, no direct relation with the Biol . Med . 136 :1158 . process of egg cleavage was reported . ITo, S., and N . HORI . 1966 . Electrical characteris- Whether these changes in membrane properties tics of Triturus egg cells during cleavage . J. Gen . have any significance for embryonic development Physiol. 49 :1019 . still has to be shown, and at this time one can only KALT, M . R . 1971 . The relationship between cleav- age and blastocoel formation in speculate . The finding that cyclic cell surface Xenopus laevis . II . Electron microscopic observations . J. Embryol. alterations (Burger et al ., 1972) as well as changes Exp . Morphol. 26 :51 . in Em as such (McDonald et al ., 1972) can influ- DE LAAT, S. W ., D. LUCHTEL, and J . G . BLUEMINK . ence the rate of DNA synthesis suggests that post- 1973 . The action of cytochalasin B during egg mitotic new membrane formation may be instru- cleavage in Xenopus laevis : dependence on cell mental in development . membrane permeability. Dev . Biol. 31 :163 . LOEWENSTEIN, W . R . 1966 . Permeability of membrane We thank Dr . S . B. Carter, Imperial Chemical In- junctions . Ann . N. Y. Acad . Sci . 137 :441 . dustries, England for a gift of cytochalasin B, Miss MANASEK, F . J ., B. BURNSIDE, and J . STROMAN . 1972 . E . S. Marcus and Mr . E . van Voorst for technical The sensitivity of developing cardiac myofibrils assistance, and Miss E . Bartova, Mrs. I . Aleven, to cytochalasin B. Proc . Natl. Acad. Sci . U.S.A . and Mr . L. Boom for preparing the drawings and 69 :308 . prints. We are grateful to Dr. J . Faber and Prof. P. MCDONALD, T. F ., H . G . SACHS, C. W. ORR, and D. Nieuwkoop for reading the manuscript . J. D . EBERT . 1972 . External potassium and baby Received for publication 7 May 1973, and in revised form hamster kidney cells : intracellular ions, ATP, 9 October 1973. growth, DNA synthesis, and membrane potential . Dev . Biol . 28 :290. PALMER, J . F ., and C . SLACK . 1970 . Some bio-electric REFERENCES parameters of early Xenopus embryos . J. Embryol. Exp . Morphol . 24 :535 . M . V . L . P . . BENNETT, ., and J TRINKAUS 1970 . PLONSEY, R ., and D. G . FLEMING . 1969 . Bioelectric Electrical coupling between embryonic cells by Phenomena. McGraw-Hill Book Company, New way of extracellular space and specialized junc- York . tions. Cell Biol . 44 :592. J. RZEHAK, K . 1972 . Changes in the pigment pattern of BENTRUP, F . W . 1970 . Elektrophysiologische Unter- eggs of Xenopus lraevis following fertilization . Folia suchungen am Ei von Fucus serratus : Das Biol . (Krakow) 20 :409. Membranpotential . Planta . (Berl.) . 94 :319. SANDERS, E . J ., and S . E . ZALIK . 1972 . The blasto- BLUEMINK, J . G. 1970 . The first cleavage of the amphibian egg . An electron microscope study of mere periphery of Xenopus laevis, with special the onset of cytokinesis in the egg of Ambystoma reference to intercellular relationships . Wilhelm mexicanum . J. Ultrastruct . Res . 32 :142 . Roux' Arch . Entwicklungsmech . Org . 171 :181 . BLUEMINK, J. G . 1971 a. Effect of cytochalasin B on SCHECHTMAN, A . M . 1937 . Localized cortical growth

SIEGFRIED W. DE LAAT AND JOHN C. BLUEMINK New Membrane Permeability 539 as the immediate cause of cell division . Science TOPPER, J . T . 1972 . The ionic basis of the membrane (Wash. D. C.) . 85:222 . potential in the early Asterias embryo. Dev . Biol. SELMAN, G . G., and C. H . WADDINGTON . 1955. The 29:273 . mechanism of cell division in the cleavage of the WEISENSEEL, M, H ., and L. F. JAFFE . 1972. Mem- newt's egg . J. Exp. Biol . 32 :700 . brane potential and impedance of developing SELMAN, G . G., and M . M. PERRY. 1970. Ultra- Fucoid eggs. Dev . Biol. 27 :555. structural changes in the surface layers of the WOODWARD, D . J . 1968. Electrical signs of new newt's egg in relation to the mechanism of its membrane production during cleavage of Rana cleavage. J. Cell Sci. 6 :207 . pipiens eggs. J. Gen . Physiol . 52 :509 . STEINBERG, M. 1957. A nonnutrient culture me- ZOTIN, A . I . 1964. The mechanism of cleavage in dium for Amphibian embryonic tissues . Carnegie amphibian and sturgeon eggs. J. Embryol. Exp. Inst. Wash. Year Book. 56:347 . Morphol. 12 :247 .

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