We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA.. By comparing two strains of Physarum polycephalum tha
Trang 1Injection of poly(b- L -malate) into the plasmodium of Physarum
Michael Karl1, Roger Anderson2and Eggehard Holler1
1
Institut fu¨r Biophysik und Physikalische Biochemie der Universita¨t Regensburg, Germany;2Molecular Biology and Biotechnology, University of Sheffield, UK
Poly(b-L-malate) (PMLA) has been reported as an
uncon-ventional, physiologically important biopolymer in
plasmo-dia of myxomycetes, and has been proposed to function in
the storage and transport of nuclear proteins by mimicking
the phospho(deoxy)ribose backbone of nucleic acids It is
distributed in the cytoplasm and especially in the nuclei of
these giant, multinucleate cells We report here for the first
time an increase in growth rate and a shortening of the cell
cycle after the injection of purified PMLA By comparing
two strains of Physarum polycephalum that differed in their
production levels of PMLA, it was found that growth
activation and cell cycle shortening correlated with the
relative increases of PMLA levels in the cytoplasm or the
nuclei Growth rates of a low PMLA producer strain
(LU897· LU898) were increased by 40–50% while those of
a high producer strain (M3CVIII) were increased by only
0–17% in comparison with controls In both strains,
shortening of the cell cycle occurred to a similar extent (7.2– 9.5%), and this was associated with similar increases in nuclear PMLA levels The effects showed saturation de-pendences with regard to the amount of injected PMLA A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to pro-teins that control translation and/or transcription Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium
Keywords
1 : cell cycle; growth rate; Physarum polycephalum; plasmodium; polymalic acid
In the absence of cytokinesis, repeated nuclear divisions give
rise to giant multinucleate cells (plasmodia) in Physarum
polycephalum [1], a well studied representative of the
myxomycete family One of the notable features of
plasmodia is the high synchrony of events during the cell
cycle The maintenance of this synchrony over large cellular
distances must require an activity that accounts for the rapid
and ubiquitous distribution and coordination of protein
activities in the periodical cell cycle events We have
previously identified the unusual polyanion poly(b-L
-ma-late) (PMLA) as a specific component of the plasmodium
that fulfils the requirements for such a distributing activity
[2,3] Its level in the nuclei is kept constant by constitutive
synthesis and secretion of excess polymer from the
cyto-plasm to the culture medium, and the levels in the nuclei for
different strains are of the same magnitude [4] PMLA binds
reversibly to histones, DNA polymerases, and other
DNA-interacting proteins, thus favouring the formation of large
complexes consisting of a variety of proteins The binding involves specifically the array of negative carboxylates on the PMLA chain that is isosteric with the array of phosphates in nucleic acids [2,5–9] This complex-forming property and the high mobility seen for the fluorescently labelled polymer in plasmodia [10] suggest that PMLA could function as a constituent of the postulated distributing activity
In addition to functioning as a distributing activity, PMLA might act as a synchronizing agent by competing with nucleic acids for the binding of structural proteins, enzymes, and regulatory proteins A recent analysis of DNA synthetic activities in extracts of plasmodia revealed a cell cycle dependent inhibition and activation of DNA poly-merases This could be explained by the binding of DNA polymerases to endogenous PMLA in competition with periodically synthesized histones or certain other proteins [11] Competition of this kind is likely to inhibit various kinds of activities involving the binding of proteins to nucleic acids, and it could affect cell growth and cell cycle duration
The distributing activity of PMLA and the efficiency of competition between PMLA and nucleic acids would both be influenced by the concentration of PMLA An abnormal increase in PMLA level would therefore be expected to modulate growth properties To test this prediction, we injected purified PMLA into plasmodia and measured the cytoplasmic and nuclear levels of the polymer in parallel with changes in growth rate and cell cycle duration The
Correspondence toE Holler, Institute fu¨r Biophysik und Physikalische
Biochemie der Universita¨t Regensburg, D-93040 Regensburg,
Germany Fax: +49 941943 2813, Tel.: +49 941943 3030,
E-mail: eggehard.holler@biologie.uni-regensburg.de
Abbreviations: PMLA, poly(b- L -malic acid) and poly(b- L -malate).
Enzyme: phosphatase (EC 3.1.3.16).
Note: A website is available at http://www.biologie.uni-regensburg.
de/biophysik
(Received 25 May 2004, revised 5 July 2004, accepted 23 July 2004)
Trang 2investigation included wild-type and mutant strains of
P polycephalumwith distinctly different levels of PMLA in
the cytoplasm, but more or less comparable levels in the
nuclei We found that increased cytoplasmic and nuclear
levels of PMLA induced a strain specific enhancement of cell
growth and an equal (between strains) shortening of the
cell cycle
Materials and methods
Strains and materials
The following strains of P polycephalum were used
(Table 1): the high PMLA producing strains M3CVIII
ATCC 96951 (yellow, wild-type), M3CVII ATCC 204388,
CH813· LU861 (yellow), LU688 (yellow); the medium
PMLA producing strain OX 110· RA271 (yellow); and
the low PMLA producing strains LU897· LU898 (white,
mutant) [12], and LU887 (white, mutant) Plasmodia were
routinely grown for 1.5–2 days at 24C, except M3CVIII,
which was grown at 27C The axenic growth medium has
been described [13] Macroplasmodia were started from the
fusion of microplasmodia (15–20 mg) on the surface of 2%
(w/v) agar in 8-cm plastic Petri-dishes [14] PMLA,
potas-sium salt, was purified as described [15], having Mr¼
50 kDa and a polydispersity¼ 2.0 [16] Anti-(histone H1)
Ig (bovine) raised in sheep (diluted 1 : 500 ELISA),
(histone H2B) (bovine) raised in sheep (1 : 2000),
anti-(histone H3) Ig (bovine, subgroup f3) raised in sheep
(1 : 2000), anti-(histone H2A + H4) (calf thymus) raised in
sheep (1 : 1000) were all from BioTrend (Cologne,
Ger-many)
2 Peroxidase-coupled anti-IgG (sheep) raised in rabbit
was from Calbiochem All anti-histone immunoglobulins
bound to histones purified from P polycephalum [17] in
ELISA and Western blotting The specificity was
charac-terized by Western blotting and ELISA of purified total
histones and nuclear and chromatin extracts of P
poly-cephalum A low degree of cross reaction of anti-H1 Ig with
the core histones was observed that was constant and
negligible during ELISA under the conditions used;
other-wise, the immunochemical responses of the antibodies were
specific Lambda protein phosphatase (400 000 unitsÆmL)1;
> 300 000 UÆmg protein)1) was from Calbiochem All
other reagents were from Merck or Sigma and were of the highest purity available
Microinjection, growth rate Before each macroplasmodium received a microinjection, a small piece was cut out and the stage of the cell cycle determined as described previously [18] The stages could be fairly well predicted by calculation, taking into account the time elapsed after the fusion and the known length of the cell cycle The injection solution of 1–4 lL contained 15–200 mgÆmL)1 PMLA, potassium salt, or a reference solution containing either KCl,L-malate (potassium salt), poly(L-glutamate) (potassium salt) or distilled water Solu-tions were injected into veins in parallel to the protoplasmic streaming at five different points distributed over the plasmodium Fluorescently labelled PMLA [10] showed
an even distribution over > 97% of the plasmodium within less than 20 min A Leitz
Germany) equipped with a laboratory course binocular (Wild Heerbrugg, Heerbrugg, Switzerland)
borosilicate capillaries (World Precision Instruments, Sarasota, FL, USA)
either early S-phase following the third mitosis or early
G2-phase following the second mitosis Sizes of plasmodia were then of the order of 4–7 cm2 The third metaphases were observed at 25.6 ± 0.5 h (mean ± SD, 10 replicates) after the fusion of microplasmodia for the yellow M3CVII strain, at 25 ± 0.5 h for the white mutant strain LU897· LU898, and at 21 ± 0.4 h for the white mutant strain LU887 Cell growth was measured at various times and interpolated for 5 h after the injection The duration of the nuclear division cycle was measured microscopically [18] between the third and the fourth mitosis, when at least 60%
of the nuclei were in metaphase
To follow the growth of a plasmodium in a noninvasive manner, its surface area was measured at successive times [19] The plasmodia did not contact the walls of the agar plates at any time The surface areas correlated significantly with the weight of (wet) plasmodia measured after their removal from the agar plates One square centimeter corresponded to 18.3 ± 0.5 mg plasmodium (mean ± SD, six independent measurements)
Table 1 PMLA contents and numbers of nuclei in various strains of P polycephalum All results are given in means and standard deviations of at least three independent measurements.
Strain Colour (genotype) a
PMLA contents (lgÆg plasmodium)1) in extracts of:
Number of nuclei (10 8 Æg plasmodium)1) Nuclei Cytoplasm Culture medium
M 3 CVII Yellow (whiA+/whiA+)a 200 ± 75b 60 ± 15b High 2.5 ± 0.4c
450 ± 45 c 350 ± 25 c
M 3 CVIII Yellow (whiA + /whiA + ) 189 ± 20 b 63 ± 14 b High 2.0 ± 0.5 c
340 ± 28c 350 ± 25c CH813 · LU861 Yellow (whiA + /whiA + ) 550 ± 60 b 460 ± 90 b Very high 5 ± 1 b
LU688 Yellow (whiA + ) 1730 ± 150 b 1000 ± 60 b High 4.8 ± 1 b
OX110 · RA271 Yellow (whiA1/whiA+) 18 ± 3b 47 ± 15b Low 0.2 ± 0.05b LU897 · LU898 White (whiA1/whiA1) 270 ± 25b Not detectableb Not detectable 1.8 ± 0.2b
LU887 White (whiA1) 130 ± 16 b Not detectable b Not detectable 2.0 ± 0.5 c
260 ± 15c 60 ± 15c
a Presumed genotype b Microplamodia c Macroplasmodia: G -phase.
Trang 3Quantitative ELISA
Extracts of cytoplasm, nuclei, and chromatin were diluted
104)108-fold in buffer (1.59 g Na2CO3, 2.93 g NaHCO3in
1 L H2O, pH 9.6) and coated in varying amounts onto
microwell plates for 3 h at 37C The plates were washed
with phosphate/saline (2 mM KH2PO4, 8 mM Na2HPO4,
137 mMNaCl, 4 mMKCl pH 7.4), blocked overnight in a
solution of 2.5 mgÆmL)1milk powder in phosphate/saline
and 0.05% (v/v) Tween 20 at 4C, followed by four
changes of washing solution, containing 0.9% (w/v) NaCl,
0.05% (v/v) Tween 20, before incubation with 50 lL per
well anti-histone Igs for 1.5 h at 37C After three changes
of washing buffer, the second antibody (peroxidase
conju-gated) was administered at 50 lL per well for 1 h at 37C
Following four changes of washing buffer, plates were
incubated with 50 lL per well of a solution of
(o-phenylene-diamine dihydrochloride) Fast TabletsTM (Sigma) for
25 min at room temperature in the dark After the addition
of 16 lL per well of 3M HCl, the extinction was read at
490 nm and reference 630 nm wavelengths in a
Micro-Reader Dynatech MR700
Chantilly, VA, USA) The readings were plotted as a
function of A595units (protein assay according to Bradford
[20]) Increments of the linear fits through the origin were
used to calculate the relative antigen concentrations in units
of 105· A490/A595 The ELISA for H1 depended on the
degree of phosphorylation, and no attempts were made to
standardize the ELISA readings on an H1 mass basis Series
of measurements were compared after standardization with
a reference sample prepared by the same method PMLA
in the extracts did not affect ELISA readings
Other methods
Cytoplasmic, nucleoplasmic and chromatin extracts were
prepared according to a modified method of Angerer and
Holler [6] One gram of plasmodia was lysed in 11 mL of
homogenization buffer and centrifuged for 10 min at
2000 g The supernatant was removed and centrifuged
again at 20 000 g; the resulting supernatant was the
cytoplasmic extract The pellet was treated with nuclei
extraction buffer [6] and centrifuged at 20 000 g; the
supernatant was referred to as the nuclear extract The
residual pellet was incubated with an equal volume
containing sodium carbonate buffer and 10M NaCl,
pH 11.4; the supernatant after 20 000 g centrifugation
was the chromatin extract This fractionation system was
optimal with regard to the preservation of nuclei during the
preparation of the cytoplasmic extract, and the same results
were obtained with the homogenization buffer of Loidl and
Gro¨bner [21] Nuclei were counted in the pellets using a
Neubauer hemocytometer before extraction for calculation
of the PMLA content The purification of nuclei over a
Percoll gradient was not used, as nuclei prepared in this way
were devoid of PMLA [8] For whole plasmodia, nuclei were
counted in alcohol-fixed smears under the phase contrast
microscope or after staining with
4¢,6¢-diamino-2-phenyl-indole Plasmodium mass was assessed after removal of
adhering liquid with tissue paper Values are given as
means ± SD for measurements performed at least in
triplicate
To dephosphorylate histone H1, extracts were incubated for 30 min at 30C in the presence of 800 U Lambda protein phosphatase, 50 mM Tris/HCl pH 7.5, 5 mM
dithiothreitol, 2 mMMnCl2, and 100 lgÆmL)1BSA Poly-malate was quantitated as described by Karl et al (2003) [10] DNA was measured by the method described by Gold and Shochat (1980) [22]
Results and Discussion Growth rate and duration of cell cycle
In the intial experiments, PMLA was injected into macro-plasmodia, and growth was measured in comparison to control plasmodia injected with water (mock-injected plasmodia) The yellow strain M3CVIII produces and secretes high amounts of PMLA, while the white strain LU897· LU898 produces considerably less PMLA (Table 1) Both strains grew very slowly during the first few hours following fusion of microplasmodia, before they assumed an approximately constant or slightly exponential growth rate Eighteen hours after fusion, each plasmodium was injected with 200 lg PMLA (mock injected plasmodia received an equal volume of water), and growth was allowed
to continue until 45 h after fusion (Fig 1) Sizes (corres-ponding to masses, see Materials and methods) were measured 5, 20, and 27 h after injection The yellow plasmodia grew faster than the white ones, but PMLA-injected plasmodia of both strains grew at comparable rates After 45 h, the sizes of the PMLA-injected yellow plasmo-dia were larger by 20% (P < 0.007) and the PMLA-injected white plasmodia were larger by 44% (P < 0.004) than their water-injected control counterparts Noninjected plasmodia grew at the same rates as the water-injected controls (data not shown) Thus, the effect of PMLA on growth was greater on the low PMLA producing white plasmodia, which contained less PMLA, than on the high PMLA producing yellow plasmodia (Table 1) The greater sizes persisted until the end of the experiment
We then tested whether the plasmodial size (growth rate, Fig 2A,C,E) and the duration of the cell cycle (Fig 2B,D,F) depended on the dose of injected PMLA, over the range 0–400 lg PMLA (A–D) The times of the injections were early G2-phase before mitosis III (Fig 2C,D) or early S-phase, following mitosis III (Fig 2A,B,E,F), in order to see whether mitosis influenced the efficacy of the PMLA-induced phenomena Plasmodial sizes were measured at 5 h after injection (Fig 2A,C,E) and cell cycle duration between the third and fourth mitosis (Fig 2B,D,F) The ratio of cell sizes in the figures is expressed relative to control (mock¼ water-injected) plasmodia and reflects the growth rate To test for specificity, 0–300 lg poly(L-glutamate) were injected
in control experiments (Fig 2E,F) Poly(L-glutamate) was chosen as a negative control because it is not isosteric with the (deoxy)ribosephosphate backbone of nucleic acids [5] Control experiments to test for osmotic effects were carried out with KCl and L-malate (potassium salt) at equimolar amounts with the malyl residues of injected PMLA The PMLA-injected plasmodia grew to be larger than the mock-injected ones, with a dose–response relationship indicating that the system was approaching saturation at high doses of PMLA (Fig 2A,C) The low PMLA producer
Trang 4(mutant white strain LU897· LU898, solid lines in Fig 2)
showed a greater increase in size than the high PMLA
producer (yellow strain M3CVIII, broken line in Fig 2)
Thus, after injection of 400 lg PMLA in S-phase, the yellow
plasmodia were larger by 17% (P < 0.015) that their
mock-injected controls (Fig 2A, dashed line) and not
detectably larger after injection in G2-phase (Fig 2C,
dashed line) In contrast, the white plasmodia were larger
by 50% (P < 0.0003) than the mock-injected controls after
injection in S-phase (Fig 2A, continuous line), and larger
by 40% (P < 0.0006) after injection in G2-phase Fig 2C,
continuous line) The differences between the strains are
highly significant and are in agreement with those in Fig 1
The cell cycle duration decreased significantly in
com-parison with the mock-injected plasmodia, the overall
changes being similar for the two strains and independent
of the injection times Following injection of 400 lg PMLA,
the decrease was 8.2% (P < 0.0012) for yellow plasmodia
injected in S-phase or G2-phase (Fig 2B,D, dashed lines),
and 9.5% (P < 0.0003) and 7.2% (P < 0.0005) for white
plasmodia injected in S-phase or G-phase, respectively
(Fig 2B,D, continuous lines) The saturation behaviour of the dose dependence was less pronounced than that of the plasmodium size The effects of injecting at different times (S- and G2-phase) were similar and did not give evidence for
a control point, except for the failure of yellow plasmodia to increase in size after injection in early G2-phase This could indicate that during the 5 h after injection in early G2-phase, growth stimulation was low because plasmodia already contained high levels of PMLA
To examine the effect on cellular protein and DNA concentrations, 200 lg PMLA was injected in early S-phase (after mitosis III) and the contents compared with those of mock-injected plasmodia After 7 h, the protein contents (mgÆg plasmodia)1) were: 10.9 ± 1.2 (mock-injected) and 11.1 ± 1.2 (PMLA-injected) for the yellow strain M3CVIII; and 14.6 ± 1.5 (mock) and 14.9 ± 1.7 (PMLA) for the white strain LU897· LU898 The DNA contents (lgÆg plasmodia)1) were: 522 ± 18 (mock) and 540 ± 18 (PMLA) for the yellow strain; and 690 ± 12 (mock) and
744 ± 30 (PMLA) for the white strain Thus, the concen-trations for PMLA- and mock-injected plasmodia were not significantly different (P > 0.05 at 95% confidence levels) Also injections of eitherL-malate, KCl or poly(L-glutamate), potassium salt (Fig 2E,F) at relevant concentrations had
no significant effect on size or cell cycle duration (P > 0.05
at 95% confidence levels) These results showed that the effects were specific and not caused by an increased osmolarity or availability of malate as a metabolite Distribution kinetics of injected PMLA
A comparison of the results in Figs 1 and 2 (A,C) reveals that the low PMLA-producing white strain manifests a larger increase in the growth rate than the high PMLA-producing yellow strain, while almost no difference between the strains is seen for the cell cycle duration In the following discussion, these phenomena will be related to the changes
in the amounts of PMLA in cytoplasmic and nuclear extracts As shown in Table 1, the PMLA contents of nuclei extracted from the high PMLA producing (M3CVIII) and the low PMLA producing (LU897· LU898) strains are comparable, while the PMLA concentrations in cytoplasmic extracts differ considerably A similar relationship is seen for strains M3CVII and LU887, which also represent high and low producers of PMLA (Table 1) In order to study changes in the levels of PMLA in cytoplasm and nuclei, we injected 400 lg of the polymer into M3CVII and LU887 plasmodia (weights of 150 mg) and measured the PMLA contents of nuclear and cytoplasmic extracts The results in Fig 3A,B for the low PMLA-producing strain LU887 show
an immediate increase after injection, peaking at approxi-mately 2.8 mgÆg plasmodium)1 in the nuclear extract (Fig 3A) and at 350 lgÆg plasmodium)1in the cytoplasmic extract (Fig 3B) The PMLA contents in mock-injected or noninjected plasmodia remain constant (Fig 3B and [3]) Similar increases were found for the yellow strain M3CVII (data not shown) Thus, the absolute increases in PMLA levels in the extracts were the same for the white and the yellow strains Nevertheless, because the level in the cytoplasm of white plasmodia before injection was very low (Table 1), the relative increase in these strains was considerably higher than for the yellow strains, which
Fig 1 Effect of injected PMLA on growth Volumes of 2 lL PMLA
solution (200 lg PMLA, dashed lines) or distilled water
(mock-injec-ted, continuous lines) were injected at the time indicated by the arrows.
The size of plasmodia was measured in terms of the surface area
covered by each plasmodium (1 cm2corresponding to 18.3 ± 0.5 mg
plasmodium, see Materials and methods) (A) Yellow wild-type strain
M 3 CVIII (B) White mutant strain LU897 · LU898 Standard
devi-ations of three independent measurements are indicated.
Trang 5already had high levels of cytoplasmic PMLA before
injection Assuming similarity between the two white strains
on one hand and the two yellow strains on the other the
different relative increases of PMLA in the cytoplasmic
extracts from yellow and white plasmodia can be seen to
correlate qualitatively with the different increases in growth
rates (Fig 2A,C) In contrast, the PMLA contents of
nuclear extracts from yellow and white plasmodia are
similar (Table 1), and this correlates with the almost equal
degree of shortening of the cell cycle (Fig 2B,D)
The kinetics of PMLA distribution were remarkable (a)
PMLA contents increased rapidly after injection, in
agree-ment with previous findings [10] A calculation shows that
the injected 400 lg PMLA (into 150 mg plasmodium)
was recovered in the extracts (b) The amounts of free
L-malate in the cytoplasmic/nuclear extracts were
260 ± 30 lg/45 ± 8 lg and constant over the period of
the investigation (data not shown) PMLA was not detectably degraded, but was instead secreted into the culture medium [3,10] (c) Clearance from the plasmodium
in 4 h (Fig 3A,B) corresponded to a clearance activity of
600 lg PMLAÆh)1Æg plasmodium)1and compares with the rate of 920 lgÆh)1Æg plasmodium)1 secretion by micro-plasmodia (60 h after inoculation) [23] These results are in agreement with the homeostatic model described previously [3] and show that plasmodia do not tolerate artificially increased levels of PMLA The data in Table 1 allow calculation and comparison of the PMLA contents of nuclei
in a variety of strains These nuclear contents vary relatively little, between 0.65 pmol for LU887 and 3.6 pmol for LU688, whereas the variation in cytoplasmic contents is much greater (> 1000-fold) This suggests that the nuclear concentration of PMLA is regulated to maintain a relatively constant, high level in all strains
Fig 2 Effects of polymalate injection on growth and duration of the cell cycle Variable amounts of PMLA (A–D) or poly( L -glutamate) (E,F) were injected in early S-phase (40 ± 5 min after the third metaphase; A, B and E) or in G 2 -phase (180 ± 10 min after the second metaphase; C,D,F).
M 3 CVIII (yellow, high PMLA producer; j) and LU897 · LU898 (white, low PMLA producer; d) The sizes of plasmodia were measured after
5 h and the cell cycle duration between the third and fourth mitoses The sizes are given relative to those of mock-injected plasmodia, which were grown in parallel, and indicate the ratio of growth rates (Fig 1) Standard deviations refer to at least three independent measurements.
Trang 6Transient appearance of histone H1 in the cytoplasm
We suspected that the mechanism(s) underlying the increase
in the growth rate and the shortening of the cell cycle might be
related to the ability of PMLA to form complexes with
nucleic acid binding proteins and thus to compete with
nucleic acids Known examples are histones, especially H1
[5,6] Therefore we tested whether free H1 and core histones
(probably in complexes with PMLA) could be detected as a
consequence of the injection of PMLA Using quantitative
ELISA on cytoplasmic extracts (Fig 3C), increased levels of
H1 were indeed detected The increase followed the injection
of PMLA with a delay of 1–2 h As epitopes had been
masked by phosphorylation in vivo, a higher amount of H1
was detected after dephosphorylation with Lambda protein
phosphatase (Fig 3C, j) Injection of -malate did not
provoke H1 appearance (Fig 3, h) ELISA with specific antibodies did not detect an increase of the core histones (data not shown) Also, the nuclear extract and chromatin extract did not indicate a PMLA-dependent variation in histone content
Conclusion Our main finding was that plasmodia responded to an artificial, unprogrammed increase in the cellular content of PMLA by an increase in size (growth rate) and by a shortening of the cell cycle duration This conclusion is based on the study of more than 120 plasmodia, which gave relatively high experimental reproducibility and results that are statistically highly significant The effect was structure specific for PMLA Swelling of plasmodia or an accumu-lation of slime after injection could be ruled out as explanations, because concentrations of protein and DNA remained unaffected The possibility that PMLA might be hydrolysed to L-malate and then used as a carbon and energy source was excluded on the basis that not only was PMLA hydrolysis absent (this work and [3,10]) but that injection ofL-malate did not reproduce the effect It was concluded that acceleration of the cell cycle and enhanced growth were functional effects of injected PMLA
We propose that the underlying mechanism by which PMLA increases the growth rate and shortens the duration
of the cell cycle is the polymer-inherent isosterism of the carboxylates with the array of phosphates in nucleic acids and, consequently, the competition with nucleic acids in the (reversible) binding of proteins, such as histones and DNA polymerases [5,6,10] An example of competition between DNA polymerases and histones in the binding of PMLA has been demonstrated in a purified system [2] and was suggested as an explanation for the periodic activation of DNA polymerases in the cell cycle [11] The degree of competition depends on the concentration of free PMLA, DNA, and the binding affinity and follows saturation functions It is speculated that the levels of protein complexes of intrinsic PMLA and nucleic acids are tuned for optimal function in the plasmodium and that an unscheduled increase in PMLA by injection perturbs this tuning and causes the observed effects Different proteins and nucleic acids gave rise to the different dose depend-encies in Fig 2 For instance, in competing with mRNA for the binding of regulatory proteins in the cytoplasm, PMLA might derepress translation activity Examples of translational regulation are known for higher eukaryotes: mRNAs are masked during gametogenesis until embryonic development [24] In another example, the expression of maternal proteins is suppressed in mouse oocytes by the binding of MSY2 protein to mRNA [25] Also, PMLA might bind to histones and enhance the rates of transcrip-tion by facilitating chromatin remodelling Our finding of high levels of free (probably PMLA-bound) H1 in the cytoplasm after injection supports this assumption High concentrations of (phosphorylated) linker histone H1 in the cytoplasm during S-phase and G2-phase of the cell cycle but not in G1-phase have been reported for HeLa cells [26]
In some other mammalian cells, examples of cytoplasmic accumulation of H1, but not of core histones have been reported [27]
Fig 3 Levels of polymalate and histone H1 during the cell cycle after
injection of 400 lg PMLA per macroplasmodium (white mutant strain
LU887, 150 mg) in early S-phase (arrow) (A) PMLA in the nuclear
extracts (B) PMLA in the cytoplasmic extracts; j, noninjected
macroplasmodia (C) ELISA of histone H1 in cytoplasmic extracts
without (m) and after incubation with Lambda protein phosphatase
(j); one relative unit ¼ 10 5
· A 490 /A 595 (A 490 ELISA readings, A 595
protein readings according to the assay described by Bradford [20], see
Materials and methods) Controls refer to macroplasmodia having
received 400 lg L -malate (h) and to noninjected macroplasmodia (s).
The symbol M3 refers to the third mitosis after the fusion of
micro-plasmodia Mean values and SDs of at least three independent
meas-urements are indicated.
Trang 7One may wonder why the large increase in PMLA
content after injection was paralleled by only modest
changes in growth and cell cycle duration However, we
do not know how great an effect should be expected If
we assume that certain PMLA–protein complexes are
involved in growth and cell cycle timing, then the size of
the observed effect will depend on the amount of such
newly formed PMLA–protein complexes If the proteins
are already almost completely saturated by intrinsic
PMLA before the injection, even large amounts of
injected PMLA will not give rise to significant increases
in the levels of these complexes and thus will not induce
large effects On the basis of these considerations, the
greater effect on growth in Fig 2A,C can be explained by
assuming lower levels of certain PMLA–protein complexes
in white plasmodia than in yellow plasmodia before the
injection (because there is much less PMLA in the
cytoplasm of white plasmodia; Table 1) By the same
token, the equal (between-strains) effects on the cell cycle
duration (Fig 2B,D) might reflect almost identical levels
of certain PMLA–protein complexes in the nuclei of white
and yellow strains (containing similar amounts of PMLA;
Table 1)
The results provide evidence not only that PMLA
functions as a storage and carrier molecule for certain
proteins [6], but also that it may be involved in molecular
events concerned with growth and cell cycle duration in
plasmodia Because these events are synchronized, PMLA
may also play a role in synchronization The fundamental
mechanism of its function is mimicry of the charge array on
the nucleic acid backbone and competition with nucleic
acids for binding to specific proteins A full understanding
of this mechanism remains to be established
References
1 Burland, T.G., Solnica, K.L., Bailey, J., Cunningham, D.B &
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