The DNA‐polymerase inhibiting activity of poly(β‐<scp>l</scp>‐malic acid)  in nuclear extract during the cell cycle of <i>Physarum polycephalum</i>

Doerhoefer, Sabine; Windisch, C; Angerer, Bernhard; Lavrik, Olga I.; Lee, Bong-Seop; Holler, Eggehard

doi:10.1046/j.1432-1033.2002.02765.x

Cited by 7 publications

(7 citation statements)

References 19 publications

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“…To achieve a coordinated delivery of equilocally functioning proteins, molecular vehicles would have the advantage of stockpiling and carrying a number of different molecules jointly to defined loci, for example, nuclear proteins involved in DNA replication to nuclei. Previously, we have isolated a polyanion which could function in this regard ([5,6] and references therein). Poly(β‐ l ‐malate) is synthesized only in plasmodia and not in the other cell types of the life cycle.…”

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confidence: 99%

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Karl¹,

Gasselmaier²,

Krieg

et al. 2003

European Journal of Biochemistry

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The nuclei in the plasmodium of Physarum polycephalum, as of other myxomycetes, contain high amounts of polymalate, which has been proposed to function as a scaffold for the carriage and storage of several DNA-binding proteins [Angerer, B. and Holler, E. (1995) Biochemistry 34, 14741-14751]. By delivering fluorescence-labeled polymalate into a growing plasmodium by injection, we observed microscopic staining of nuclei in agreement with the proposed function. The fluorescence intensity was highest during the reconstruction phase of the nuclei. To examine whether the delivery was under the control of polymalatase or related proteins [Karl, M. & Holler, E. (1998) Eur. J. Biochem. 251, 405-412], the cellular distribution of these proteins was also examined by staining with antibodies against polymalatase. Double-stained plasmodia revealed a fluorescent halo around each fluorescent nucleus during the reconsititution. Fluorescent nuclei were not observed when the hydroxyl terminus of polymalate, known to be essential for the binding of polymalatase, was blocked by labeling with fluorescein-5-isothiocyanate. By immune precipitation, it was shown that polymalate and polymalatase or related proteins were in the precipitate. It is concluded that polymalate is delivered to the surface of nuclei in the complex with polymalatase or related proteins. The complex dissociates, and polymalate translocates into the nucleus, while polymalatase or related proteins remain at the surface.Keywords: Physarum polycephalum; plasmodium; polymalic acid; polymalatase; reconstituting nuclei.Physarum polycephalum is a well characterized member of the plasmodial slime molds (myxomycetes) that typically have a life cycle involving haploid (spores, amoebae) and diploid (plasmodia) cell forms [1]. Together with the cellular slime mold Dictyostelium discoideum and other Mycetozoa, P. polycephalum has been placed among the multicellular eukaryotes on the basis of molecular phylogenetic criteria [2]. The plasmodium undergoes mitosis without cytokinesis and usually develops into a multinuclear giant cell (macroplasmodium). This can contain billions of nuclei (e.g. 10 9 nuclei for a cell having a diameter of 14 cm), which divide synchronously within 3-4 min in an 8-9 h cycle. P. polycephalum, especially the plasmodium, has been chosen as a model to study biological and biochemical questions concerning differentiation and cell cycle [3,4].Considering the synchronous timing of cellular events and the giant dimension of a plasmodium, an appropriate device is necessary to achieve at any given moment an even distribution of molecular constituents. A characteristic oscillating protoplasma streaming [1] probably accounts for the travelling of material along the veins over a certain distance, although the mechanistic details are not known. To achieve a coordinated delivery of equilocally functioning proteins, molecular vehicles would have the advantage of stockpiling and carrying a number of different molecules jointly to defined loci, for example, nuclear p...

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Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Karl¹,

Gasselmaier²,

Krieg

et al. 2003

European Journal of Biochemistry

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…A recent analysis of DNA synthetic activities in extracts of plasmodia revealed a cell cycle dependent inhibition and activation of DNA polymerases. 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.…”

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Injection of poly(β‐l‐malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate

Karl

Anderson

Holler

2004

European Journal of Biochemistry

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Poly(b-L-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia 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 (M 3 CVIII) 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 dependences 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 proteins 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-malate) (PMLA) as a specific component of the plasmodium that fulfils the requirements for such a Ôdistributing activityÕ [2,3]. Its level in the nuclei is kept constant by constitutive synthesis and secretion of excess polymer from the cytoplasm 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 DNAinteracting 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][6][7][8][...

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“…Its presumed function is to coordinate transport, delivery, and activity of certain proteins (DNA polymerases, histones, etc.) to nuclei [3,7,10,19,20], and it has been suggested that it participates in the maintenance of the observed high degree of synchrony typical for plasmodia [8]. Strain M 3 CVII is one of the high PMLA producers [8].…”

Section: Discussionmentioning

confidence: 99%

“…Of the various cell types in the life cycle of P. polycephalum , only the plasmodium contains the water soluble polymer, β‐poly( l ‐malate) (PMLA) [2–4]. The polymer is concentrated in the nuclei in an amount comparable with that of DNA and histones [5].…”

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Stage specific expression of poly(malic acid)‐affiliated genes in the life cycle of Physarum polycephalum

2006

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Polymalic acid is receiving interest as a unique biopolymer of the plasmodia of mycetozoa and recently as a biogenic matrix for the synthesis of devices for drug delivery. The acellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleated amoebae and multinucleated plasmodia. In adverse conditions, plasmodia reversibly transform into spherules. Only plasmodia synthesize poly(malic acid) (PMLA) and PMLA‐hydrolase (polymalatase). We have performed suppression subtractive hybridization (SSH) of cDNA from amoebae and plasmodia to identify plasmodium‐specific genes involved in PMLA metabolism. We found cDNA encoding a plasmodium‐specific, spherulin 3a‐like polypeptide, NKA48 (spherulin 3b), but no evidence for a PMLA‐synthetase encoding transcript. Inhibitory RNA (RNAi)‐induced knockdown of NKA48‐cDNA generated a severe reduction in the level of PMLA suggesting that spherulin 3b functioned in regulating the level of PMLA. Unexpectedly, cDNA of polymalatase was not SSH‐selected, suggesting its presence also in amoebae. Quantitative PCR then revealed low levels of mRNA in amoebae, high levels in plasmodia, and also low levels in spherules, in agreement with the expression under transcriptional regulation in these cells.

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The DNA‐polymerase inhibiting activity of poly(β‐l‐malic acid) in nuclear extract during the cell cycle of Physarum polycephalum

Cited by 7 publications

References 19 publications

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum

Injection of poly(β‐l‐malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate

Stage specific expression of poly(malic acid)‐affiliated genes in the life cycle of Physarum polycephalum

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