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`

`
`SURFACE CHARGE AND CELL DIVISION
`
`647
`
`MATERIALS AND METHODS
`
`Cells and culture. An Escherichia coli K-12 strain, PA3092 (F’thr‘leu'lacy’
`trp"his‘thy"str"malA”xyl‘mtl'arg”suII'), was used. The cells were cultured with
`aeration in L-tubes at 37°C in L broth (1% bactotrypton, 0.5% yeast extract, 0.5%
`NaCl, 0.1% glucose) supplemented by 50 mg/liter thymine. Only cells growing
`exponentially with the shortest doubling time were used. The cell maintains a
`constant electrophoretic mobility during the logarithmic phase of growth but shows
`a reduced mobility at the stationary phase.
`Irradiation. X irradiation of the cells was carried out in culture medium in a
`3-cm-diameter plastic Petri dish (2—mm depth of cell suspension) at 3°C on ice.
`The physical factors of exposure were: 200 kVp, 20 mA, 0.5 mm Al + 0.5 mm Cu
`filter added, half-value layer 1.13 mm Cu, 25-cm target—sample distance, and
`exposure rate in air 425 R/min.
`Electrophoresis. At different times of incubation in shaking L-tubes at 37°C
`after irradiation, an aliquot of cell suspension was placed on ice, centrifuged, and
`then washed in cold 67 mM phosphate buffer supplemented with 5.4% sorbitol for
`electrophoresis. The electrophoretic mobility of individual cells was measured at
`25 : 0.5°C with a Zeiss cytopherometer as reported in (5, I0, 11). Each cell was
`allowed to move 16 pm in a scaled thin chamber under the phase microscope
`alternatively in both directions following reversal of current (4 mA) in the 67 mM
`phosphate buffer supplemented with 5.4% sorbitol. The 67 mM phosphate buffer
`(pH 7.3) contained 50.1 mM Na2HPO4 and 16.5 mM KH2PO4. The ionic strength
`and osmolarity of the buffer were usually indicated as 0.167 and 183 mosm with
`the assumption of complete dissociation of phosphates. However, the conductivity
`of the buffer supplemented with 5.4% sorbitol was 7.471 X 103 an/cm. This value
`was about one-half the conductivity of the 167 mM NaCl solution, thus indicating
`about 50% dissociation of phosphates. Since 5.4% sorbitol is isotonic (about 300
`mosm), the electrophoresis medium is hypertonic by about 183 /2 mosm due to
`phosphates. For the measurement of mobility at lower ionic strengths of solution,
`phosphate buffers diluted stepwise (6.7, 13.4, 26.8, 40.2, 53.6 mM) were supple-
`mented with 5.4% sorbitol to maintain the same viscosity. The conductivities of
`the medium were 1.076 X 103, 1.811 X 103, 3.413 X 103, 4.784 X 103, and 6.141
`X 103 all/cm, respectively. Osmolarity of the medium varied from about 309 to
`373 mosm. We chose the same concentration of sorbitol because EPM is dependent
`on the ionic strength and the viscosity of the medium, but not on its osmolarity.
`The mobility was determined from separate experiments on 10-100 cells for each
`set of conditions and calculated as um-sec"-V" -cm.
`Morphological observations. Cells were fixed in 10% formalin for 5 min, washed
`with phosphate-buffered saline, and then spread on a glass slide coated with poly-
`L-lysine using a cytocentrifuge (Shandon Elliott) for 5 min at 1500 rpm. The cells
`were dried on the slide, treated with 1 N HCl for 5 min at 60°C to digest ribonucleic
`acid, washed by running water, and then stained in freshly diluted Giemsa solution
`(Merck). Distribution of chromosomes and the size of the cells were detected using
`a microscopic photograph. The cells in the suspension were counted with a he-
`mocytometer after different incubation periods.
`
`

`
`648
`
`SATO ET AL.
`
`RESULTS
`
`Morphological change after irradiation. Figure 1 shows the nuclear staining of
`cells indicating changes in cell size and distribution of nuclear mass with time at
`37°C after irradiation with 50 kR. Nonirradiated cells (Fig. 1A) were about 2.5
`pm in mean length and contained one to four nuclei per cell. The cells with one
`nuclear mass were only 3.2% of the cells in nonirradiated culture, but were 47 and
`91% (Fig. 1B) of the cells 15 and 30 min, respectively, after irradiation with 50
`kR. As shown in Fig. 2, the mean number of nuclear masses in a cell decreased
`from 2.86 to 1.11 during the first 30 min of incubation after the exposure. During
`this 30 min, the number of cells increased about 1.6-fold, and the increase in cell
`length was slight. These results suggest that cell separation proceeded in those cells
`in which the chromosomes had segregated before irradiation. Enlargement of con-
`densed nuclear masses at the central site of cells and the elongation of the cell
`progressed after 30 min without further separation of the cells (Figs. IC, D). A
`cleavage of the cell was noticed at the central portion of some condensed nuclei.
`Morphological disintegration of cells appeared only after 2 hr of incubation when
`cell division resumed. At 3 hr of incubation, cell division was often observed in the
`
`filamentous cells in which chromosomes had been segregated to the daughter cells
`as shown by arrows in Fig. 1E. The filaments with a condensed nucleus at the
`center on occasion caused cell separation from the termini, thereby giving rise to
`anucleated cells. During the next 2 hr, normal-sized cells proliferated and became
`predominant over lysed cells and long filaments (Fig. 1F).
`
`Change in electrophoretic mobility (EPM) with time after irradiation. Figure
`3 shows the time course of change in EPM after irradiation with different doses.
`Decrease in EPM was detectable even after irradiation with 100 or 500 R, but it
`returned to normal rapidly during the subsequent incubation for 15 or 45 min,
`respectively. The EPM reduction was not different and was maximum 15 min after
`irradiation with doses ranging from 15 to 80 kR. EPM recovery, however, was
`dependent on dose; recovery began earlier and reached a higher value after smaller
`doses of irradiation. Comparison of the time course change in cell length and EPM
`indicated that EPM recovery began 2 hr after irradiation with 50 kR when the
`length of the cell was maximum.
`
`Frequency distribution of EPM and cell size. Figure 4 illustrates the frequency
`distribution of EPM of unirradiated cells and of cells exposed 30 min earlier to 50
`kR. Every irradiated cell showed reduced EPM, and the two distributions are
`clearly separated with the boundary at -1.2 um - sec“ -V" -cm. The cells irradiated
`with 50 kR progressively elongated to form filaments with incubation periods up
`to 2 hr and kept the minimum EPM. Thereafter separation of normal-sized cells
`took place from the termini of the long filaments as shown in Fig. 1. Three hours
`after irradiation with 50 kR, about 31% of the cells were of small size (below 4
`mm). Figure 5 exhibits the relationship between EPM and the length of the cell.
`The distribution of the points is separated into three groups. All the short cells
`(below 4 am) had EPM higher than -1.2 um-sec"' -V“ -cm as nonirradiated cells.
`EPM of the longer cells distributed mostly below -1.2, but 30% of them showed
`
`

`
`SURFACE CHARGE AND CELL DIVISION
`
`649
`
`-'c aria‘ ~_“'lv-we--v * 3 9'; "g:
`"8
`\:"’ gr‘
`I)
`A
`
`3-='~\§~..,‘o. 0,: I.
`
`_
`
`‘gs
`,§§\
`
`‘«,";‘4,a; '* =4-'gag(_..
`
` \ '\
`
`
`FIG. 1. Nuclear staining of Escherichia coli K- I 2 indicating cell size and distribution of chromosomes.
`Cells were fixed with 10% formalin at 30 min (B) or 1 (C), 2 (D), 3 (E), or 5 hr (F) after irradiation
`with 50 RR. treated with 1 N HCl for 5 min at 60°C, and then stained in Giemsa solution. Arrows in
`(E) indicate cleavages of elongated cells and an anuclear cell. (A) Nonirradiated cells. Bar in (A) is
`I0 um.
`
`

`
`650
`
`SATO ET AL.
`
` 0
`
`60
`
`15
`
`30
`
`
`
`
`
`Celllength,Celldensity,Nuclearnumber
`
`
`
`
`
`Incubation time after
`
`irradiation (min)
`
`FIG. 2. Changes in (O) cell length (um), (X) cell density (X107 cells/ml), and (0) number of nuclei/
`cell with incubation time after irradiation with 50 kR. Each point is the mean value of measurements
`on more than 400 cells. Results suggest early separation of cells in which the nuclei had previously
`segregated at the time of irradiation. and the later elongation of cells without nuclear segregation and
`cell division.
`
`the higher EPM. A few cleavages per cell were often noticed in those filamentous
`cells having EPM higher than that of the ordinally filamentous cells. Closed circles
`in Fig. 5 represent those filamentous cells containing visible cleavage, which always
`showed EPM greater than -1.2. Morphology of these cells is exhibited in Fig. 1E.
`About 10% of elongated cells without visible cleavage also showed EPM above
`
`mobility
`Electrophoretic
`
`01530
`
`60
`
`120
`
`180
`
`240
`
`300
`
`Time after X-irradicition(min)
`
`37°C
`
`FIG. 3. Change in electrophoretic mobility (—um-sec" -V"-cm) of E. coli with incubation time at
`37°C after irradiation with 100 R (O), 500 R (A), 15 RR (O), 30 kR (A), 50 RR (0), or 80 kR (X).
`Each point represents the mean value of measurements on more than 30 cells from three separate
`experiments.
`
`

`
`SURFACE CHARGE AND CELL DIVISION
`
`651
`
`50 kR 30min
`
`O R
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`
`
`Fractionofcells(''I..)
`
`0.9
`
`1.0
`
`1.1
`
`1.2
`
`1.3
`
`1.1.
`
`1.5
`
`1.6
`
`Eleclrophoretic mobility
`
`FIG. 4. Frequency distribution of electrophoretic mobility (-um-sec" -V" -cm) of unirradiated cells
`(shaded columns) and cells exposed to 50 RR 30 min earlier (open columns).
`
`-1.2. These results suggest that the recovery of EPM precedes or coincides with
`the separation of the daughter cells.
`Effect of ionic strength on mobility. EPM was measured in a buffer solution of
`
`Lengthofthecell
`
`1.0
`
`1.1
`
`1.2
`
`1.3
`
`1.4
`
`1.5
`
`1.6
`
`00
`
`.9
`
`Electrophoretic mobility
`
`FIG. 5. Relationship between the electrophoretic mobility (-—p.m-sec" - V"' -cm) and the length of the
`cell (pm) on 100 individual cells. Measurements were done 3 hr after irradiation with 50 kR. Closed
`
`circles are elongated cells with visible cleavage, which showed EPM above -1.2 um-sec“ -V“ -cm as
`separated small cells and unirradiated cells.
`
`

`
`652
`
`SATO ET AL.
`
`z~ b
`
`Electrophoreticmobility(-um-sec"-V'cL.m)B)‘A’
`
`
`
`
`
`'0
`
`AC0170033
`24)
`(17)
`
`0.067
`(12)
`
`0.100
`(9.7)
`
`0.134
`(5.4)
`
`0.167.
`(7.5)
`
`Ionic
`(thickness of
`
`strength
`ion atmosphere)
`
`FIG. 6. Relationship between the ionic strength of phosphate buffer used for the measurement of
`electrophoretic mobility of E. coli irradiated with 30 kR 30 min earlier (X) and of unirradiated E. coli
`(0). The vertical lines represent one standard deviation for 30 to 100 cells.
`
`various ionic strengths to determine whether a redistribution of charged groups in
`the membrane is responsible for EPM reduction. As indicated in Fig. 6, EPM
`decreased with increasing ionic strength. The difference in EPM between irradiated
`and nonirradiated cells was evident at ionic strengths of 0.100 and greater. At ionic
`strengths lower than 0.033, however, EPM was the same in irradiated and non-
`irradiated cells. Because the decay of potential with distance is less rapid in low-
`ionic-strength solution, the ionized groups embedded more deeply in the outer
`surface material exert their greater influence at lower ionic strength (1, 2,
`I 2).
`Therefore with decreasing ionic strength, the thickness of the effective ionic layer
`increases. The thickness of the ionic layer was calculated according to the Debye—
`Hiickel equation by 3.06 X (ionic strength)“/2 A as an approximate estimation
`assuming the complete dissociation of phosphates. These values are shown in pa-
`rentheses under each value of ionic strength in Fig. 6. The data can be explained
`by a vertical translocation of negatively charged groups from the outermost layer
`of 0-7.5 A into a deeper layer of 9.7—17 A, or the inverse translocation of positive
`charges from the deep layer to the outermost layer occurred in irradiated cells.
`Similar translocation of acidic sugars was suggested in our previous experiments
`using cultured mammalian cells and erythrocytes (11, 13).
`Effect of temperature and reagents on EPM change. Cells were irradiated with
`15 kR at 3°C on ice and then incubated in a water bath for 15 min with aeration
`
`

`
`SURFACE CHARGE AND CELL DIVISION
`
`653
`
`1.5
`
`1.1.
`
`> f
`
`:
`
`*3
`E 1.3
`
`E 1.2
`E’
`3 11Q .
`
`
`
`E ‘
`
`J 1.0
`
`2L
`
`L]
`
`Q9—4————+———h———H——a———+————+———
`3
`10
`15
`20
`25
`30
`35
`
`Incubation temperature ('C)
`
`FIG. 7. Relationship between the incubation temperature and electrophoretic mobility (-;.tm-sec"-
`V" -cm) 15 min after irradiation with 15 kR. The vertical lines represent one standard deviation for
`30 to 100 cells.
`
`at different temperatures. Figure 7 indicates that the decrease in EPM was max-
`imum and constant at temperatures ranging from 25 to 37°C, and smaller at
`temperatures of 10°C and lower. The slight difference of EPM between control
`and irradiated cells after incubation at the low temperature probably resulted from
`the EPM reduction during the measurement of EPM at 25°C for 5 min. The
`manifestation of the radiation effect on EPM depended markedly on temperature
`between 10 and 25°C. Incubation of unirradiated cells at different temperatures
`did not per se produce any effect on EPM.
`To estimate the factors involved in EPM reduction after irradiation, effects of
`agents which could modify the membrane were examined. Table I indicates that
`p-(chloromercuri)benzoic acid (PCMB, a sulfhydryl-blocking agent), fluorescein
`mercuric acetate (FMA, a sulfhydryl-linking agent), glycerin, and ethylene glycol
`bis(/3-aminoethyl ether)-N,N,N’N'-tetraacetic acid (EGTA, specific chelator of
`calcium ions) completely blocked EPM reduction after irradiation at the concen-
`trations at which the agent itself had no effect on EPM.
`
`DISCUSSION
`
`The present experiments demonstrate the simultaneous occurrence of reduction
`in EPM and inhibition of cell division shortly after irradiation and their concurrent
`recovery in individual cells of Escherichia coli. The advantage of cell electrophoresis
`was the simultaneous detection of EPM and cell morphology on individual cells
`seen directly under the phase microscope. Cells that were elongating due to the
`arrest of cell division after irradiation retained their reduced EPM. It should be
`noted that EPM of the cell is determined by the charge density per unit surface
`area independently of cell size or shape (3, I2). EPM reached the minimum 15
`min after irradiation, when the cells were only slightly longer than nonirradiated
`
`

`
`654
`
`SATO ET AL.
`
`TABLE 1
`
`Blocking of Mobility Change after X Irradiation by Modifying Agents
`
`El
`
`ectrop orenc
`h
`‘
`mobility
`(um-sec" - V“‘ -cm)
`
`zgni cant
`S’
`fi
`difference
`(P < 0.05)
`
`Treatment
`
`No treatment
`30 kR, 30 min
`
`PCMB 5 X 10‘° M 30 min
`PCMB 5 X l0’° M+ 30 kR
`
`FMA IO‘‘’ M
`FMA10“’ M + 30 kR
`
`Glycerin 1 M
`Glycerin 1 M + 30 kR
`
`EGTA 1 mM
`EGTA 1 mM + 30 kR
`
`-1.392 1 0.076
`-1.011 1 0.059
`
`-1.299 1 0.062
`-1.252 1 0.121
`
`-1.340 1 0.074
`-1.331 1 0.069
`
`-1.336 1 0.084
`-1.292 1 0.054
`
`-1.322 1 0.079
`-1.301 1 0.086
`
`Yes
`
`NO
`
`No
`
`No
`
`No
`
`Yes
`
`EGTA 1 mM + Ca“ 1 mM
`EGTA 1 mM + Ca“ 1 mM + 30 kR
`
`-1.329 1 0.081
`-1.042 1 0.067
`
`controls. Detailed analyses on alterations in cell number, cell length, and distri-
`bution of nuclear mass during the first 30 min of exposure were carried out. It was
`observed that the division of the cell proceeded if the daughter nuclei had already
`been segregated in the irradiated parental cell at the time of irradiation. Presumably
`septa had already been formed in these parental cells. Partition of new nuclei,
`however, was completely inhibited. After 30 min of incubation, enlargement of the
`condensed nuclear mass at the center of the cell and the elongation of the cell
`progressed without accompanying cell division but retained the reduced EPM. An
`important finding at the recovery phase was that not only the separated small cells
`but also long filamentous cells regained high EPM. The latter cells had a few
`cleavages at the cell termini, indicating the formation of dividing cross walls. This
`finding suggests that recovery of EPM in a whole elongated cell precedes or co-
`incides with the resumption of cell division at its termini. Since all small-sized cells,
`some of which lacked DNA, showed recovered EPM, cell division seemed to occur
`at the site where the surface structure had recovered irrespective of the distribution
`of DNA. This idea was supported by our recent experiment using a temperature-
`sensitive mutant which forms anucleated minicells at a high temperature (to be
`published).
`Negatively charged molecular species which are responsible for EPM in the
`bacteria have not been identified because of the absence of purified specific enzymes.
`It was shown that the anionic groups of the cell surface of E. coli begin to dissociate
`between pH 2 and 4 and are carboxylic in nature (14). A hypothetical mechanism
`which might lead to the reduction of EPM of E. coli after irradiation is considered
`to be a conformational change of membrane as suggested in the case of mammalian
`
`

`
`SURFACE CHARGE AND CELL DIVISION
`
`655
`
`cells (11, I3). Detection of reduced EPM only at low ionic strengths suggested a
`vertical translocation of charged molecules from the peripheral layer to a deeper
`layer after irradiation. The character of molecular conformational change was
`supported by the blocking of EPM loss by adding sulfhydryl-blocking agents or
`protein-linking agents or lowering the temperature of the cell culture. All these
`treatments seem"to restrict the movement and rearrangement of membrane mol-
`ecules involving SH-proteins. The requirement of calcium ions for EPM reduction
`is another important feature of the mechanism. We reported the existence of a
`calcium-dependent process as an early step of EPM change after irradiation in
`erythrocytes (15). The target of X irradiation resulting in EPM reduction in mam-
`malian cells is thought to be the membrane itself, because EPM reduction occurred
`in the erythrocyte ghosts containing neither nuclei nor cytoplasm as well as in
`whole erythrocytes and cultured mammalian cells (11).
`Involvement of membrane damage in the loss of colony-forming ability of bacteria
`after irradiation has been proposed. A type of cell killing associated with the oxygen
`effect was suggested as the result of membrane damage (16, 17). Several mem-
`brane-specific drugs such as local anesthetics and tranquilizers preferentially sen-
`sitized the hypoxic cells to the same sensitivity as that of oxic cells (18). Further
`evidence in support of the hypothesis is as follows: (a) bacterial cells are sensitized
`by iodoacetamide under conditions that prevent the drug from entering the cells
`(18), (b) A mutants of E. coli carrying pol A‘ mutation and a mutant strain
`Bs-1, both defective in a process of DNA repair, are not affected by the membrane-
`acting sensitizers (19-21). Because these membrane-acting reagents inhibit the
`repair of DNA (22), some interaction between the membrane and DNA was sug-
`gested in the process of sensitization. The DNA-membrane complex was indicated
`as a site which is particularly susceptible to enhancement of radiation damage by
`oxygen (23). Our results suggest that the inhibition and recovery of cell division
`are coordinated with the reduction and recovery of EPM, which reflects alterations
`in the structure of the cell surface after irradiation.
`
`RECEIVED: August 6, 1980; REVISED: November 12, 1980; RE-REVISED: February
`13, 1981
`
`REFERENCES
`
`I. D. H. HEARD and G. V. F. SEAMAN, The influence of pH and ionic strength on the electrokinetic
`stability of the human erythrocyte membrane. J. Gen. Physiol. 43, 635-654 (1960).
`2. A. H. MADDY, The chemical organization of the plasma membrane of animal cells. Int. Rev. Cytol.
`20, 1-65 (1966).
`3. J. N. MEHRISHI, Molecular aspects of the mammalian cell surface. In Progress in Biophysics and
`Molecular Biology (J. A. V. Butler and D. Noble, Eds.), Vol. 25, p. 1. Pergamon, Oxford/New
`York, 1972.
`
`4. M. H. REPACHOLI, Electrophoretic mobility of tumor cells exposed to ultrasound and ionizing
`radiation. Nature (London) 227, 166-167 (1970).
`5. C. SAT0 and K. KOJIMA, Change in electrophoretic mobility of cultured cells after X-irradiation
`and their modification by Sh-blocking agents and hemagglutinin. Radiat. Res. 60, 506-515 (1974).
`6. H. RINK and H.-J. MEYER-TESCHENDORF, Influence of X irradiation on the electrophoretic mobility
`of yeast cells. Radiat. Res. 72, 317-324 (1977).
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`

`
`656
`
`SATO ET AL.
`
`8. C. SATo, K. KOJIMA, M. ONozAwA, and T. MATSUZAWA, Relationship between recovery of cell
`surface charge and colony—forming ability following radiation damage in three cell-lines. Int. J.
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`9. C. SATO, K. KOJIMA, T. MATsuzAwA, and Y. HINUMA, Relationship between loss of negative
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`62, 250-257 (1975).
`10. G. F. FUHRMANN and G. RUHENSTROTH-BAUER, Cell electrophoresis employing a rectangular
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`1965.
`
`11. C. SATO, K. KOJIMA, and K. NISHIZAWA, Target of X irradiation and dislocation of sialic acid in
`decrease of cell surface charge of erythrocytes. Radiat. Res. 69, 367-374 (1977).
`12. C. C. BRINTON, JR., and M. A. LAUFFER, The electrophoresis of viruses, bacteria, and cells, and
`the microscope method of electrophoresis. In Electrophoresis. Theory, Methods, and Applications
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`13. C. SATO, K. KOJIMA, and K. NISHIZAWA, Translocation of hyaluronic acid in cell surface of cultured
`mammalian cells after X-irradiation and its recovery by added adenosine triphosphate. Biochim.
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`14. D. A. HAYDON and G. V. F. SEAMAN, An estimation of the surface ionogenic groups of the human
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`charge after X-irradiation. Int. J. Radiat. Biol. 35, 221-228 (1979).
`16. T. ALPER, Low oxygen enhancement ratios for radiosensitive bacterial strains, and the probable
`interaction of two types of primary lesion. Nature (London) 217, 862-863 (1968).
`17. M. A. SHENOY, J. C. ASQUITH, G. E. ADAMS, B. D. MICHAEL, and M. E. WATTS, Time—reso|ved
`oxygen effects in irradiated bacteria and mammalian cells: A rapid-mix study. Radiat. Res. 62,
`498-512 (1975).
`18. M. A. SH ENOY, K. C. GEORGE, B. B. SINGH, and A. R. GOPAL-AYENGAR, Modification of radiation
`effects in single-cell systems by membrane-binding agents. Int. J. Radiat. Biol. 28, 519-526
`(1975).
`19. D. K. MYERS and K. G. CHETTY, Effect of radiosensitizing agents on DNA strand breaks and their
`rapid repair during irradiation. Radiat. Res. 53, 307-314 (1973).
`20. W. A. CRAMP and P. E. BRYANT, The effects of rifampicin on electron- and neutron-irradiated E.
`coli B / r and B5_.: Survival, DNA degradation and DNA synthesis by membrane fragments. Int.
`J. Radiat. Biol. 27, 143-156 (1975).
`21. S. YONEI, Modification of radiation effects on E. coli B/r and a radiosensitive mutant B,_, by
`membrane—binding drugs. Int. J. Radiat. Biol. 36, 547-551 (1979).
`22. C. K. K. NAIR and D. S. PRADHAN, Effect of procaine hydrochloride on DNA repair in Escherichia
`coli. Chem. Biol. Interact. 11, 173-178 (1975).
`23. W. A. CRAMP, D. K. WATKINS, and J. CoLLINs, Effects of ionizing radiation on bacterial DNA-
`membrane complexes. Nature (New Biol.) 235, 76, 77 (1972).