Photomorphogenesis in Physarum polycephalum

Eur. J. Biochem. 179, 141 - 146 (1989) g3 FEBS 1989

Photomorphogenesis in Physarum polycephalum Temporal expression pattern of actin, a- and P-tubulin Bettina POETSCH l , Thomas SCHRECKENBACH' and Anne K. WERENSKIOLD'

*

Max-Planck-Institut fur Biochemie, Martinsried Merck, Central Chemical Research Department, Darmstadt

(Received April 18/July 18, 1988) - EJB 88 0451

Photo-induced fruiting-body formation of the slime mold Physarum polycephalum can be divided into two stages. The first stage (I) starts with the beginning of illumination (0 h) and ends with the formation of nodules (12 h). The second stage (11) is characterized by culmination, sporangiophore formation and melanization of sporangiophore heads (13 - 17 h). We investigated the expression of actin, E- and P-tubulin during this differentiation process using Northern blot analysis and run-off transcription in isolated nuclei. Whereas actin mRNA is irreversibly lost during stage I, we observed two peaks in mRNA concentration for a-tubulin and one peak for that of P-tubulin during stage I and a coordinated alp-tubulin mRNA induction during stage 11. Stage I1 induction appears to be related to a presporangial mitosis. Transcriptional activity of the three genes studied shows two maxima, namely one in the middle of stage I and the other at the end of stage 11. Our data suggest that the expression of the three cytoskeletal proteins investigated follows a distinct temporal pattern comprising changes in both mRNA synthesis and decay. We also propose a novel function of tubulin in Physarum which is not related to mitosis.

In lower eukaryotes several light-induced developmental and biochemical events have been described. These effects are almost exclusively blue - ultraviolet light-dependent processes. Thus, conidiation in Trichoderma [l], sporangiophore formation in Phycomyces [2] the carotinogenesis and morphogenesis in Neuvospora crassa [3] are examples for blue-lightcontrolled processes. A light regulation of distinct genes has not been shown so far for any of these systems. A lightdependent transcription has been suggested for some genes in N . crassa, the assumption being based on differential regulation of yet unidentified translatable mRNAs [4]. The fruiting-body formation of Physarum polycephalum plasmodia is most effectively induced by blue - ultraviolet and red light [5, 61. Morphogenesis proceeds in two distinct stages [7]. Stage 1 comprises a post-induction period without significant changes of the plasmodial strands and ends with cleavage of the strands into nodular structures. During stage I1 the processes of culmination, sporangiophore formation and melanization of sporangiophore heads are observed. Mitosis and meiotic divisions complete the maturation of the spores P, 91. We have recently shown that photomorphogenesis in Physurum is accompanied by striking changes in the concentration of distinct translatable mRNA species [7]. In a highly synchronous developmental program, sporulation-specific mRNAs are sequentially induced and various translatable Correspondence to B. Poetsch, Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, D-8033 Martinsried, Federal Republic of Germany Abbreviations. DNase I, deoxyribonuclease I; SSC, 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0; Tris/EDTA, 10 mM Tris/CI, 1 mM EDTA pH 7.5. Enzymes. Deoxyribonuclease I (EC 3.1.21.I); restriction endonucleases BglII, EcoRI, HindIII, PstT, Suc.1, SalI, StuI (EC 3.1.21.4).

mRNAs are lost, the most prominent of which codes for a 42-kDa protein. Two of the photo-induced proteins have been identified as a- and P-tubulin. Actin is the major cytoskeletal protein of Physarum and serves for a variety of functions in cell structure, cytoplasmic shuttle streaming and plasmodial migration [lo, 111. ETubulin and D-tubulin are the major subunits of microtubules. In most other eukaryotes they are involved in complex structures like the mitotic spindle, centrioles, cilia, flagella and the cytoskeleton [12] but, although four isotypes of tubulins (al, a2,PI, Pz) are expressed in Physarum plasmodia, microtubules are found only in the mitotic spindle fiber [13, 141. As in other eukaryotes, actins and tubulins in Physarum are encoded by niultigene families. Four unlinked actin loci, four unlinked atubulin and three unlinked P-tubulin loci have been characterized [15,16]. Expression of actin and tubulin isotypes has been studied in some detail [13, 171. The regulation of tubulins and their rnRNAs during the synchronous Physarum cell cycle have been well documented. During the 2 h preceding mitosis, the rate of tubulin biosynthesis increases by 30-fold and returns to the premitotic level 1 h after mitosis [18]. Quantitative analysis of a-tubulin mRNA demonstrated an increase in the mRNA level of more than 40-fold preceding mitosis; no changes during the cell cycle in actin mRNA are detectable [19]. Here, we document expression of cytoskeletal proteins during light-induced differentiation. We also show that the down-regulated 42-kDa protein is plasmodial actin. These data are based on the measurement of mRNA concentration and transcriptional activity in isolated nuclei. Our data suggest that changes both in mRNA life-time and transcriptional activity of all three genes occur and that they reflect a temporal program of differential gene expression during photomorphogenesis.

142 MATERIALS AND METHODS Culture conditions and induction of sporulation

Microplasmodia of the strain CL were cultured according to [20]. Generation of macroplasmodia and induction of sporulation were performed as described [7].

urea. and the absorbance at 260 nm was measured (~-A 7 6 n = I z l o 7 nuclei/ml). R N A synthesis in isolated nuclei

Isolation of actin by DNase I affinity chromatography was essentially performed as described by Zechel [23]. An appropriate excess of unlabelled plasmodial extract was added as carrier material to the [35S]methionine-labelled material. Equal amounts of radiolabelled protein from control and induced samples (in vivo l o 7 cpm, in vitro lo4 cpm) were applied to the DNase I column. Aliquots of the eluates were subjected to SDS/PAGE.

For in vitro transcription assays, nuclei were added at a concentration of 2 x 107/ml to a cocktail containing 0.5 M hexylene glycol, 2 mM Pipes pH 7.5, 1 mM MnC12, 5 mM MgCI2, 250 mM KCl, 0.5 mM dithiothreitol, 30 U human placenta ribonuclease inhibitor (BRL, Eggenstein, FRG), 0.15 mM each of ATP, GTP and CTP and 200 pCi/ml [a32P]UTP( % 400 Ci/mmol). The reaction volume was 0.2 ml and was incubated for 40 min at 30°C. Reactions were terminated by addition of 2% SDS, extracted once with Tris/ EDTA-saturated phenol and once with chloroform. Incorporation of radioactivity was measured using absorption of nucleic acids to DE-81 paper (Whatman, Maidstone, UK) as described by Maniatis et al. [32]. A typical incorporation of radioactivity was lo6 cpm per 5 x lo6 nuclei per assay. Nucleic acids were precipitated by addition of 0.1 vol. 10 M LiCl and 2.5 vol. ethanol at - 20°C overnight. After centrifugation, pellets were redissolved in 100 p1 Tris/EDTA and chromatographed on a Sephadex G-50 column equilibrated in Tris/EDTA (1 ml packed volume) to remove unincorporated nucleotides.

R N A preparation and electrophoresis

Hybridization of nuclear R N A synthesized in vitro

Total RNA was extracted following the procedure described by Chirgwin et al. [24] and translated in vitro employing the rabbit reticulocyte lysate system (NEN, Dreieich, FRG). RNA was separated under denaturating conditions in 1.5% agarose gels containing 6% formaldehyde, blotted to Genescreen (NEN) by electrophoretic transfer and baked for 2 h at 90°C. For slot-blot assays RNA was denatured by heating (10 min, 65°C) diluted onefold with icecold 20 x SSC, spotted onto Genescreen and heated for 2 h at 90°C.

Single-stranded M13mplS/mp19 phages containing the antisense sequences specific for actin, a- and 8-tubulin described under 'Hybridization probes', were denaturated by heating (10 min, 95"C), cooled on ice and diluted twofold with 20 x SSC. Dilution series of the cloned DNAs (0.5, 1.O, 2.0, 4.0 pg) were spotted onto Genescreen membrane and heated at 90°C. For hybridization and washing condition see above. Calf thymus DNA was replaced by yeast tRNA (200 pg/ml) and 5 x lo5 cpm of labelled RNA were used per assay.

Hybridizution probes

Quantfication of DNA and R N A

The Physarum actin probe was a 1.4-kb PstI fragment of cDNA clone PpA35 [25]. The Physurum a-tubulin probe was a 930-bp BglII - SucI fragment of the cDNA clone pPccrl25 [26]. The Physarum 8-tubulin probe was a 880-bp genomic EcoRI - StuI fragment from ~ 8 4 0 - 2[27]. The histone probe was a genomic 600-bp Hind111 fragment from p4121 of Physarum histone H4 [28].

Hybridization signals in Northern and slot blots were quantified by densitometric scanning of autoradiographs using an LKB 2202 Ultroscan laser densitometer (LKB, Freiburg, FRG). The linear range of the X-ray film was checked. Four amounts of DNA (0.5, 1.0, 2.0, 4.0 pg) were tested.

Hybridization of' Northern blots

RESULTS

The restriction fragments were labelled by nick translation according to a standard procedure [29]. Genescreen filters were prehybridized and hybridized according to manufacturer's instructions (NEN). Filters were extensively washed with 2 x SSC/l% SDS at 65"C, dried and exposed at -70°C to preflashed X-ray film [30] using a Quanta 111 intensifying screen.

Inhibition of actin protein synthesis during sporulation

Labelling and separation of proteins

Radioactive labelling and extraction of cellular protein were carried out as described [7]. Proteins were separated by SDS/polyacrylamide-gel electrophoresis according to [21] in 5 - 20% gradient gels. Fluorography was performed according to [22]. lden t if icution of act in

Isolation of nuclei

Isolation of nuclei was performed according to (311. For determination of nuclear concentration, aliquots of the preparation were lysed in a solution containing 2 M KCl and 5 M

Actin is the most abundant cellular protein in starving Physarum polycephalum plasmodia and is probably identical to the 42-kDa protein which is down-regulated during photoinduced fructification [7]. In order to prove this assumption, we isolated plasmodial actin from total cellular protein using affinity chromatography, based on the highly specific binding of actin to DNase-I - Sepharose [17, 23, 331. Non-induced, starving plasmodia were labelled with [35S]methionine and the soluble proteins extracted were applied to a DNase-I Sepharose column. Tightly bound material that could be eluted with 40% formamide, consisted of a single 42-kDa protein band as determined by SDS-gel electrophoresis

143

Fig. 1 , Inhibition of uctin synthesis during sporulation. Control plasmodia (dark grown, lanes 1) and induced plasmodia (lanes 2) were labelled in vivo (a) and by in vitro translation (b). (a) The labelling of the induced plasmodia was carried out between 11 - 17 h after light induction. Radioactive protein was subjected to affinity chromatography on DNase-I - Sepharose. Equal amounts of radiolabelled protein were applied to the column. Tightly bound material was eluted with 40% formamide and analyzed by SDSjPAGE in a 5-20% gradient gel. The 42-kDa protein band shown represented the only band on the fluorogrdph. (b) In vitro translation was directed by RNA (0.5 pg) from control plasmodia (lane 1) and induced plasmodia (1 2 h of development, lane 2)

(Fig. l a , lane 1). Based on its apparent molecular mass, DNase-binding properties and isoelectric point (around 5.3, results not shown), this protein showed the characteristics of plasmodial actin. The same protein was obtained when in vitro translation products directed by RNA from non-induced plasmodia were subjected to affinity chromatography and subsequent SDS-gel separation (Fig. 1b, lane 1). To monitor differentiation-dependent changes in the actin synthesis in vivo, we also labelled starving and induced plasmodia (1 1 - 17 h after induction) with [35S]methionine. This period is characterized by a reduced synthesis of this gene product 171. The labelled extract was subjected to DNase I affinity chromatography and SDS-gel electrophoresis. The results are presented in Fig. l a (lane 2) and clearly document that the down-regulated 42-kDa protein is identical to plasinodial actin. The amount of radiolabelled actin isolated from differentiating plasmodia (Fig. 1a, lane 2) was strongly decreased in comparison to control plasmodial extracts (Fig. 1a, lane 1). Densitometric scanning of electrophoretically separated in-vivo-labelled proteins revealed a 95% reduction of actin during photomorphogenesis relative to the total radioactivity incorporated into cellular protein. A similar result was obtained when proteins synthesized in vitro from total RNA of induced (12 h after induction) plasmodia were subjected to identical analysis (Fig. 1b). Here, we found a 92% reduction of actin synthesis suggesting that the inhibition of actin synthesis in vivo is mediated by a decrease in the level of translatable mRNA.

Expression of actin, a- and P-tuhulin mRNAs Protein labelling in vivo and cell-free translation assays had provided evidence that the regulation of actin and tubulins during stage I1 of development was based on changes in the amount of translatable RNA [7]. We monitored the expression of actin and tubulin mRNA through stages I and I1 (Fig. 2b) of differentiation by Northern blot analysis. To this end, 10 pg total RNA isolated from different morphogenetic stages was separated by denaturing agarose gel electrophoresis and, after transfer onto Genescreen membrane, subjected to hybridization with homologous actin, aand P-tubulin gene sequences (Fig. 2a). When probed with actin DNA we found hybridization to a 1.6-kb mRNA in the range 0 - 8 h after light induction. N o signal for actin mRNA was detected beyond 8 h of development. This decline in the

Fig. 2. Regulation of actin, a-tubulin, p-tubulin and histone H4 mRNA. (a) Total RNA (10 pg) isolated from different stages ofmorphogenesis (0-17 h after light induction) was fractionated on a 1.5% agarose/ formaldehyde gel and blotted onto a Genescreen membrane. Replicate filters were prehybridized for 16 h at 42°C and hybridized for 2 days at 42°C in 10 ml buffer containing 5 x lo6 cpm nick-translated actin (PpA35), a-tubulin (pPca125), 8-tubulin (~840-2)and histone H4 (p4121), respectively. Filters were washed in 2 x SSC at 65°C for 1 h with one change of buffer. Blots were exposed at - 70°C Tor 24 h using an intensifying screen. The molecular sizes in bases are given and were calculated from an RNA ladder (BRL) and 19-S and 26-S Physarurn rRNA. (b) Scanning electron micrographs of morphogenetic stages taken from [7]. Bar represents 500 pm. The morphogenetic periods are given in hours after light induction; stage I and stage I1 are indicated

actin mRNA concentration was consistent with the observed inhibition of actin protein synthesis (Fig. l a , lane 2). Densitometric scanning of the autoradiograph (Fig. 2 a) revealed a greater than 100-fold decrease in actin mRNA between 0 and 12 h after light induction (Fig. 4 below). Tubulin mRNAs were investigated in Northern blot assays using DNA fragments of the coding regions of the genes specific for a- and a-tubulin as hybridization probes. Both probes recognized a 1.7-kb mRNA (Fig. 2a). During stage I (0 - 12 h), a- and P-tubulin mRNA exhibited two peaks which are temporally shifted. During stage I1 (13 - 17 h), however, a coordinated and striking induction of a- and /3-tubulin mRNA was observed. We then quantified tubulin mRNA concentrations by densitometric analysis of the Northern blots (Fig. 4). The coordinated increase in expression of a- and 8tubulin during stage I1 (13-17 h) was greater than 80-fold relative to the 12-h level.

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Fig. 3. Transcription ofsequences specific,for uctin, a- andb-tubulin in isolatednuclei. Nuclei were isolated from different stages of sporangiophore development (0, 4, 6, 8, 12, 17 h). In vitro transcription ( 5 x lo6 nuclei per assay) was performed in the presence of [E-~’P]UTPand in the presence or absence of a-amanitin (1 pg/ml). The RNA was cxtracted and hybridized to samples of dilution series (1 = 0.5 pg, 2 = 1.0 pg, 3 = 2.0 pg, 4 = 4 pg) of filter-bound single-stranded DNA from M 1 3 clones of Physarum actin (a), a-tubulin (b), and /I-tubulin (c). Lane d shows the vcctor control. 5 x l o 5 cpm of labelled transcript were used for each hybridization. The exposure time was 5 days

In growing Physarum plasmodia, the synthesis of tubulins and their mRNAs was shown to be coupled to the cell cycle and to be restricted to the late Gz phase [19]. To determine whether the induction of tubulins in morphogenesis reflected mitotic division, a histone-H4-specific probe was hybridized to total RNA isolated from Physarum at different morphogenetic time points. Histone H4 has been shown to be strongly expressed during the late Gz phase preceding mitosis, and thus could be used as an indicator for mitosis in Physarum [28]. As seen in Fig. 2a, histone expression accompanied the second period of tubulin expression (13- 17 h), which strongly suggested that a presporangial mitosis took place at about 17 h of photomorphogenesis. Since histone was not expressed during stage I, the first synthesis period of tubulin obviously was not correlated with a mitotic event. Actin and tuhulin transcr@tion in isolated nuclei

To check for transcriptional control of actin and tubulin expression, in vitro transcription studies using isolated Physarum nuclei were performed. The nuclear system used exhibited RNA-polymerase-11-dependent transcription of actin-specific sequences 1341. The rate of actin and tubulin transcription was measured in nuclei isolated at five selected times of sporangiophore formation under conditions favouring the elongation of transcripts already initiated in vivo [31, 341. After incubation of nuclei with [a-32P]UTP, radioactive RNA was isolated and then hybridized to filterbound single-strand DNA sequences coding for actin, atubulin and P-tubulin (Fig. 3). Addition of a-amanitin (1 pg/ ml) to the transcription assay reduced the incorporation of radioactivity into total RNA to 50% of the untreated samples (at 17 h). This treatment suppressed the transcription of actin, E- and P-tubulin as expected for RNA-polymerase-II-dependent genes (Fig. 3). The relative transcription rate was calculated by densitometric evaluation of the autoradiographs. The data obtained (Fig. 4) revealed a qualitatively similar transcription pattern for all three genes. Transcription of these

genes was induced from the non-detectable level in non-illuminated cells to a high activity at 4 h after light induction, although the total incorporation of radioactivity into RNA was only twofold higher in 4-h, 8-h, 12-h and 17-h induced nuclei compared to the zero-time nuclei (not shown). For actin and P-tubulin genes, transcription rates reached a maximum at this time point and then decreased almost linearly to a minimum at 12 h of differentiation. By the end of stage I1 (at 17 h), a second maximum of transcriptional activity was observed. For the a-tubulin gene, the pattern was shifted temporally. The first peak in transcriptional activity at 4 h demonstrated a half-maximal transcription rate. After a slight transient reduction (at 8 h), maximal transcriptional activity was reached at 12 h after light induction and was continuously present at 17 h when actin and j-tubulin genes were also transcribed at maximal rates. Stimulation of transcription during stage I1 between 12- 17 h was 5-fold and 10fold for actin and P-tubulin genes, respectively. Evaluation of the quantitative data obtained from Fig. 3 revealed that 3fold more /Ithan a-tubulin RNA was synthesized in vitro (17 h).

DISCUSSION In this study we have presented results on actin, a-and Stubulin regulation during light-dependent photomorphogenesis. The Physarum polycephalum hybridization probes used in this study were derived from the highly homologous coding regions of the respective genes and, therefore, do not discriminate between different isotypes expressed. The specificity of the actin hybridization probe has been demonstrated by other authors [25]. The homologous a-tubulin DNA probe has been sequenced [35] and has been used recently in two studies on cell-cycle-dependent tubulin regulation in Physarum [19, 361. The specificity of the homologous Ptubulin sequence has been proved by Southern blot experiments and sequence analysis [27].

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Fig. 4. Comparison uf the relative mRNA concentrutions to the transcriptional activity. Steady-state mRNA levels at 0, 4, 8, 12 and 17 h of photomorphogenesis were quantified by densitometric analysis of the Northern blots given in Fig. 2a using a laser densitomcter (LKB Ultroscan). The quantification of the in-vitro-synthesized RNA was carricd out by densitometric analysis of slot-blot experiments such as that shown in Fig. 3. The maximum level of the in-vitro-synthesized RNA and the steady-state RNA is given as 100%. Experiments were carried out three times; the result from a typical experiment is given

Part of our data is based on run-off transcription experiments in isolated nuclei. This method has been employed in the analysis of specific mRNA species in various systems [37 391 including Physarum [34,36]. A comparison of steady-state mRNA and in vitro synthesized RNA has been previously used to demonstrate transcriptional control [37, 391. To analyze the mode of actin and tubulin expression, we compared the data on the relative mRNA concentration with the data on the relative transcriptional activity (Fig. 4). For all three genes, we found high levels of mRNA in the absence of measurable transcription in starving cells. A similar result was also obtained in the analysis of proliferating plasmodia (not shown) and thus seemed to indicate a high stability of the actin and tubulin mRNAs during growth and starvation. Light induced a dramatic transient stimulation of the transcription rate of actin and tubulin genes (at 4 h), whereas it exhibited differing effects on the accumulation of the corresponding mRNA species. In spite of a high transcription rate,

actin mRNA was rapidly lost during stage I, implying an increased degradation of actin transcripts. The maximal transcription rate of actin at the end of stage I1 (17 h) probably reflected the re-accumulation of actin mRNA during spore maturation. We have shown earlier that translatable actin mRNA was lost during sporulation and that it accumulated again in maturing spores [7]. Likewise, the decrease in tubulin mRNA between 8 - 12 h of photomorphogenesis, in spite of continuing transcription, indicated destabilization of the mRNA. The strong increase in transcription rate/mRNA accumulation in stage I1 was evidently related to a presporangial mitosis. This presporangial mitosis has been described earlier by cytological methods [40]. We conclude from our data that both transcriptional and post-transcriptional mechanisms are involved in the regulation of Physarum tubulins. Our findings in sporulation are in accord with results obtained in analysis of the cell cycle. Transcriptional regulation of Physurum tubulin synthesis has been suggested as the cause for the increase of cr-tubulin mRNA at the late Gz phase of the cell cycle [36]. After mitosis, post-transcriptional regulation at the level of RNA stability was assumed to cause the rapid decline of tubulin mRNA [19]. In contrast to the Physarum system, tubulin expression in most eukaryotic systems, such as in mouse cells, seems to be regulated mainly at the post-transcriptional level [41- 431. We cannot explain the non-coordinated expression of crand P-tubulin during stage I (Figs 2a and 4). We believe that tubulin function during this time period is related to a new, differentiation-specific function. Cytoplasmic microtubules are well known to participate in important cellular processes such as organelle transport [44]. The transformation of plasmodial strands to fruiting bodies represents a rearrangement of the cellular architecture and requires vertical transport of cellular organelles (especially of nuclei) into the sporangiophore heads. Preliminary data obtained by immunocytochemical methods using tubulin-specific antibodies indicated that the expression of tubulin mRNA during stage I of photomorphogenesis is related to this process. We thank Dr D. Pallotta for providing the actin clone PpA35, Dr T. Schedl for providing the a-tubulin clone pPca125, and Dr X.W. Wilhelm for supplying the histone clone ~ 4 1 2 1 We . particularly wish to thank Drs J. Shiozawa and D. Ernst for critical comments on the manuscript. This work was supported by thc Deutsche Forschungsgemeinschuft (Schr. 24111-3).

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