Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum.

BACKGROUND: Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. RESULTS: We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Omega-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. CONCLUSION: This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Omega-loop structure could play a crucial role in the NLS function of PSRPK.

Progress in plasmodial differentiation improves regularity of oscillating contractions in Physarum polycephalum.

Based on the knowledge about subcellular morphogenetic processes in the acellular slime mold Physarum polycephalum, we hypothesized that during differentiation of undifferentiated endoplasm to the highly differentiated complex structure of the contractile apparatus of this organism, the regularity of oscillating contractions must improve. We measured the endogenous contraction automaticity starting from the de novo generation within minutes after sampling small portions of undifferentiated endoplasm. The standard deviation of the normalized period duration of these samples was compared to the respective values of radial contractions of differentiated protoplasmic plasmodial strands. The mean normalized standard deviation in endoplasmic drops was 28.3+/-12.2%. Respective values in protoplasmic strands were 10.0+/-3.7%. The difference between the experimental groups was highly significant (p<<0.0001). We interpret the verification of our hypothesis as an indication that the very regular oscillating contractions in fully differentiated stages of Physarum require the complex structure of the sophisticated contractile apparatus, represented by the circular plasmalemma invagination system of protoplasmic strands, while the regularity is lower in stages, where the differentiation is still in progress. We believe that this is due to deficits in coordination capabilities, which need a directional and spatially oriented protoplasmic streaming as a precondition.

Use of the giant multinucleate plasmodium of Physarum polycephalum to study RNA interference in the myxomycete.

The plasmodium of Physarum polycephalum harbors billions of synchronized nuclei in a single cell of complex structure. Due to its synchrony and extreme size, it is used as a model to study events on a single cell level, such as cell cycle and differentiation. We show here for the first time that this model, despite its enormous size and structural complexity, is accessible to RNA interference by simple injection of dsRNA or siRNA. The targeted gene is that of polymalatase, an intracellular adapter of poly(beta-l-malate) involved in the maintenance of the synchrony and functioning as an extracellular hydrolase of this polymer. Real-time reverse transcriptase polymerase chain reaction analysis revealed that the specific mRNA was knocked down to about 10% of the original level. The suppression of a single injection lasted for approximately 14 cell cycles (144 h) and could be prolonged for any time by repeated dsRNA injections. Western blots indicated that the knockdown of RNA was paralleled by a strong reduction in polymalatase synthesis. However, a change in the phenotype of the plasmodium could not be clearly observed. In principle, the plasmodium offers an easy system for studying gene knockdown by RNA interference.

[A SC35-like protein is localized in the nucleus of Physarum polycephalum].

After being labeled with an anti-SC35 antibody, the specimens of Physarum polycephalum at S, G2, prophase, metaphase and ana-telophase were observed with an Hitachi electron microscope and gold particles marking the location of the SC35-like protein were mainly found in the nucleus,indicating the existence of a SC35-like protein in it. Judging from the densities of the gold particles in the individual domains of the nucleus, the SC35-like protein was principally located in the nucleolar domain and interchromatin domain during G2 and prophase, and the protein was distributed in the interchromosome domain at metaphase and ana-telophase when the nucleus was disintegrated, suggesting that the nucleolus and interchromatin (interchromosome) domain are the two main locations of the SC35-like protein in the nucleus. Further observations upon the nucleolus revealed that the density of the gold particles in the dense fibrillar component (DFC) of the nucleolus was much higher than that of the fibrillar center (FC), demonstrating that the protein was largely situated in the DFC rather than FC.

Rapid, selective digestion of mitochondrial DNA in accordance with the matA hierarchy of multiallelic mating types in the mitochondrial inheritance of Physarum polycephalum.

Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then disappeared completely approximately 1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10(-4). Nevertheless, the matA hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.

Behavior of phosphatase isoforms during sclerotium formation in Physarum polycephalum.

The behavior of phosphatase isoforms under dark-starvation from plasmodium of Physarum polycephalum were investigated to determine their possible roles in sclerotium formation. Two and a half days after dark-starvation, approximately 95% of plasmodia plates formed sclerotia. Specific phosphatase activity increased markedly up to ca. two-fold within the first day of starvation, after which the enzymatic activity decreased rapidly to a level less than the initial level within 2 days of the starvation period. Among the two isoforms of enzyme detected just before sclerotization under dark-starvation conditions, the enzymatic activity of the major isoform (Rm value of 0.6) decreased gradually within 1.5 days of starvation, then linearly to less than 20% of that at the beginning of the observation. Those of other major isoform (Rm value of 0.7) increased up to ca. two-fold within the first day of starvation, then decreased linearly to levels less than that of the first 2 days of the starvation period. Behavior of this isoform strongly suggests that it initiates the formation of sclerotium under dark-starvation conditions.

Editing site recognition and nucleotide insertion are separable processes in Physarum mitochondria.

Insertional RNA editing in Physarum polycephalum is a complex process involving the specific addition of non-templated nucleotides to nascent mitochondrial transcripts. Since all four ribonucleotides are substrates for the editing activity(s), both the site of insertion and the identity of the nucleotide to be added at a particular position must be specified, but the signals for these events have yet to be elucidated. Here we report the occurrence of sporadic errors in RNAs synthesized in vitro. These mistakes, which include omission of encoded nucleotides as well as misinsertions, occur only on templates that support editing. The pattern of these misediting events indicates that editing site recognition and nucleotide addition are separable events, and that the recognition step involves features of the mitochondrial template that are required for editing. The larger deletions lack all templated nucleotides between editing sites, suggesting that the transcription/editing apparatus can "jump" from one insertion site to another, perhaps mediated by interactions with editing determinants, while smaller omissions most likely reflect misalignment of the transcript upon resumption of templated RNA synthesis.

Path finding by tube morphogenesis in an amoeboid organism.

We have studied how the plasmodium of Physarum polycephalum, a large amoeboid cell, is able to track the shortest path between two selected points in a labyrinth. When nutrients are supplied at these points to a sheet-like plasmodium extended fully in a maze, the organism forms a single tube which connects the two sites via the shortest route. During the path finding, plasmodial parts in dead ends of the maze shrink and finally the tube with the minimum-length is selected from the existing possibilities. A simple cellular mechanism based on interacting cellular rhythms may describe the experimental observations.

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.

[Immunocytochemical identification of tropomyosin in nucleoli and nucleolar matrix of Physarum polycephalum].

Nucleoli were isolated from physarum polycephalum, and nucleolar matrix was prepared by digesting the nucleoli respectively with DNase 1, 0.25 mol/L (NH4)2SO4 and 2 mol/L NaCl to remove DNA and most proteins. SDS-PAGE analysis indicated that there were about 20 polypeptides in nucleolar matrix component, including the 37 kD polypeptide which was similar to tropomyosin in molecular weight. The result of indirect immunofluorescence treated with anti-tropomyosin antibody and sheep anti-rabbit IgG antibody labelled with FITC showed that bright fluorescence was observed in the nucleoli and nucleolar matrix, but no bright fluorescence in the controls. Indirect Immunoblotting detection further verified that tropomyosin existed in nucleolar matrix. Protein A-colloidal gold immunoelectron microscopic study showed that there were many gold particles in the specimens labelled with tropomyosin antibody, and there were few gold particles found in the controls. Tropomyosin distributed dispersedly in nucleoli.

Rearrangements of mitochondrial DNA and the mitochondrial fusion-promoting plasmid (mF) are associated with defective mitochondrial fusion in Physarum polycephalum.

A specific linear mitochondrial plasmid (mF) is genetically associated with the fusion of mitochondria in the true slime mould, Physarum polycephalum. In matings between mF+ and mF- strains, which respectively carry and do not carry the mF plasmid, mitochondrial fusion occurs in the zygote. Mitochondrial fusion induces recombination between specific sites in the mitochondrial DNA (mtDNA) and in the mF plasmid. To detect a region which is associated with the mitochondrial fusion in the mF plasmid, we isolated, by fluorescence microscopy, strains which showed defective mitochondrial fusion (delta mif-) from those which showed normal mitochondrial fusion (mif+). Analysis of the mitochondrial genomes of delta mif- strains showed only mtDNA which recombined with the mF plasmid in mitochondria. Comparison of this recombinant mtDNA in one delta mif- strain (NG 15) with that of a mif+ strain showed that a 2.2-kbp region, which included the integration site of the mF plasmid, was deleted in the delta mif- strain by recombination between the main mtDNA and the mF plasmid. In other strains, in addition to this deletion, a 6-kbp region which included both termini was deleted by recombination at six repeats of AAT sequences in the mF plasmid. Moreover, transcripts of the mF plasmid were not detected in NG15 by slot hybridization.

Molecular and cellular mechanisms of mitochondrial nuclear division and mitochondriokinesis.

Our present understanding of mitochondrial division can be summarized as follows: Mitochondria contain a specific genome, synthesize their own DNA, and multiply semi-autonomously. Strands of mitochondrial DNA (mt-DNA) in the in vivo organelles of all eukaryotes are organized to form mitochondrial nuclei (nucleoids) (mt-nuclei) with specific proteins including a histone-like protein and transcription factors at the central region of the mitochondrion. We can easily observe the mt-nucleus in vivo mitochondria in various organisms such as fungi, algae, plants, and animals by using high-resolution epifluorescence microscopy. Therefore, the process of mitochondrial division can be clearly separated into two main events: division of the mt-nuclei and mitochondriokinesis analogous to cytokinesis. Mitochondria undergo binary division which is accompanied by the division of the mt-nucleus. A remarkable characteristic of mitochondrial multiplication during the mitochondrial life cycle is that mitochondria can multiply the mt-chromosome by endoduplication until 50-100 copies are present. Mitochondria can then divide without mitochondrial DNA synthesis to eventually contain 1-5 copies of the mt-chromosome. This characteristic phenomenon can be observed during cell differentiation, such as during the formation of plasmodia and sclerotia of Physarum polycephalum and during embryogenesis and the formation of meristematic tissues in plants. The mitochondrial chromosome has a mitochondrial "kinetochore (centromere)" which is A-T rich and contains specific sequences such as topoisomerase binding sites, tandem repeats, and inverted repeats. A bridge of proteins may exist between the kinetochore DNA and membrane systems. Mitochondrial chromosomes can divide according to the growth of a membrane system between the kinetochores. Mitochondriokinesis progresses steadily along with mitochondrial nuclear division. As the membrane at the equatorial region of a mitochondrion contracts, the neck of the cleavage furrow narrows, and eventually the daughter mitochondria are separated. An actin-like protein may power mitochondriokinesis by separating the daughter mitochondria. In general, mitochondriokinesis occurs by contraction rather than by partition of the inner membrane.

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum.

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

Insertional editing in mitochondria of Physarum.

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.

Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC-PK1 cell line. A comprehensive investigation of different preparation procedures.

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.

Fine structure observations of phagotrophic activity by plasmodia of Physarum polycephalum.

Scanning electron microscopic observations of feeding plasmodia show three characteristic features: 1) extension of multilobed pseudopodia protruding from the leading edge of the plasmodium as it advances onto the surface of a food particle, 2) confluence of the lobes to form a sheath-like pseudopodium attached to the surface of the food particle, and 3) protrusion of small nodules with thin lamellar projections from the leading edge of the plasmodium. Sections through freeze-dried preparations of the feeding plasmodium exhibit a highly convoluted under surface in contact with loosened starch grains that appear to be released by extracellular digestion. The cytoplasm, viewed by transmission electron microscopy, contains branched, internally penetrating canals (ca. 2 microns wide) enclosing engulfed starch grains. Starch grains in the deeper part of the canals are more electron dense and appear to be digested. Micropseudopodia (70-80 nm dia.), projecting from the surface of the canals, protrude toward and into the ingested starch grains. Digestive marker enzyme (acid phosphatase) activity was detected cytochemically in food particles penetrated by micropseudopodia indicating a digestive role for these structures not reported previously.

Effects of tubulozole on the amoeboflagellate transformation in Physarum polycephalum.

The Amoeboflagellate Transformation (AFT) of Physarum polycephalum involves rapid changes in the cytoskeleton, cell shape and cell motility. Use of pharmacologic agents to probe the role of cytoskeletal elements in the AFT are impeded because the transforming cells are very sensitive to such commonly-used drug solvents as DMSO. The anti-microtubule agent tubulozole is found to disrupt, rapidly and transiently, the AFT, inhibiting flagella formation, cell elongation and the arrangement of microtubules and microfilaments. Cells recover quickly, possibly due to precipitation of the drug; the reappearance of normal arrays of microfilaments and cytoplasmic microtubules lags behind flagella formation.

Cell cycle-dependent assembly and disassembly of cytoplasmic microtubules in the plasmodium of the myxomycete Physarum polycephalum.

The giant syncytium of Physarum plasmodia possesses a complex cytoplasmic microtubule network except during the occurrence of the intranuclear mitosis. In early prophase stages, intranuclear spindles assemble concomitantly as the cytoplasmic microtubule network disassembles. No cytoplasmic microtubules are present in metaphase. They begin to reassemble in telophase. The complex cytoplasmic microtubule network reappears in early reconstruction stages. The assembly of cytoplasmic microtubules occurs on cytoplasmic foci, both in telophase stage and during rewarming after cold microtubule disassembly. These foci, independent of the nuclei, correspond to the foci observed in the cytoplasm during interphase, both by immunofluorescence and electron microscopy. As cytoplasmic and intranuclear microtubule-organizing centers are spatially distinct, plasmodial syncytia offer the possibility to study the effects of cell regulatory pathways on two types of microtubule-organizing centers that differ in their nucleating activity during the cell cycle.

Reversible histone modifications and the chromosome cell cycle.

During the eukaryotic cell cycle, chromosomes undergo large structural transitions and spatial rearrangements that are associated with the major cell functions of genome replication, transcription and chromosome condensation to metaphase chromosomes. Eukaryotic cells have evolved cell cycle dependent processes that modulate histone:DNA interactions in chromosomes. These are; i) acetylations of lysines; ii) phosphorylations of serines and threonines and iii) ubiquitinations of lysines. All of these reversible modifications are contained in the well-defined very basic N- and C-terminal domains of histones. Acetylations and phosphorylations markedly affect the charge densities of these domains whereas ubiquitination adds a bulky globular protein, ubiquitin, to lysines in the C-terminal tails of H2A and H2B. Histone acetylations are strictly associated with genome replication and transcription; histone H1 and H3 phosphorylations correlate with the process of chromosome condensation. The subunits of histone H1 kinase have now been shown to be cyclins and the p34CDC2 kinase product of the cell cycle control gene CDC2. It is probable that all of the processes that control chromosome structure:function relationships are also involved in the control of the cell cycle.