Marie-Line Géraud

Immunocytochemical localization of the DNA-binding protein HCc during the cell cycle of the histone-less dinoflagellate protoctista Crypthecodinium cohnii B

Summary— The major basic nuclear protein HCc (previously named Histone‐like) of the dinoflagellate Crypthecodinium cohnii B was immunolocalized in light and electron microscopy using an affinity‐purified polyclonal antibody. Complementary conventional and cryo‐techniques were used to study the distribution of the DNA‐binding protein in interphase cells and to follow its behaviour throughout the mitotic cycle. In non‐dividing cells, the HCc protein was found to be located on extra‐chromosomal loops and chromosomal nucleofilaments dispersed in the nucleoplasm. In mitotic cells, from prophase to early telophase, it was homogeneously distributed in the (whole) dividing chromosomes. HCc protein was also detected in two compartments of all the permanently observable nucleoli: the nucleolar organizing region and the fibrillo‐granular region. In this paper we discuss the hypothetical roles, structural and/or functional, of this DNA‐binding protein, which is specific to dinoflagellates, the only eukaryotes whose chromatin is devoid of histones and nucleosomes.

The toxic dinoflagellate Prorocentrum lima and its associated bacteria: I. An ultrastructural study

Summary Prorocentrum lima Ehrenberg (Dodge) and its associated bacteria were examined by scanning and transmission electron microscopy. The absence of trichocysts in this species is confirmed. The peculiarities of this P. lima strain (PL2V) were an abundance of intracellular reserves and extensive development of fibrous bodies, probably constituting a part of the pusule. Associated bacterial microflora was abundant. Various morphological types of free-living bacteria were found free-floating in the liquid culture medium. Numerous bacteria were directly attached to the dinoflagellate cell or agglomerated, forming large aggregates in the P. lima mucus. Bacteria-like inclusions were visible in a few percent of the P. lima cells observed. We suggest that they were intracellular bacteria, and that bacterial infection within this toxic dinoflagellate is possible, but occurs only rarely. This SEM and TEM study provides the first evidence for an association between this toxic dinoflagellate and surrounding bacteria.

Cyclic Expression of A Nuclear Protein In A Dinoflagellate

ABSTRACT. Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48‐ and 46‐kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46‐ and 48‐kDa bands. the 48‐kDa protein was detected in G1 phase but not in M phase cells. an expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinapl), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48‐kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. the second coding sequence showed a low similarity to non‐specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinapl showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained. while chromosome cores and nuclear envelopes were negative.

Nucleolar localization of rRNA coding sequences in Prorocentrum micans Ehr. (dinomastigote, kingdom protoctist) by in situ hybridization

To define the molecular mechanisms of ribosome biogenesis and to find out in which nucleolar compartment transcription of rDNA occurs, we have performed in situ hybridization (ISH) of RNase-treated cryosections using biotinylated rRNA coding sequences as a probe and the eukaryotic dinoflagellate nucleolar system as a model. Recent data from ISH of eukaryotic ribosomal genes by electron microscopy (EM) has so far failed to establish a consensus which clearly defines the function of the three compartments of the nucleolus. Dinomastigote protoctists are the only known eukaryotes whose chromatin is totally devoid of nucleosomes. Their chromosomes remain permanently condensed during the entire cell cycle and active nucleoli arise from an unwound part of some of the otherwise compact chromosomes. In this work, DNA-DNA hybrids were detected either by fluorescent avidin or by indirect immunogold staining procedures in EM; this is the first use of cryosections to detect hybrids in EM not only in the nucleolus sensu lato but also in a dinomastigote cell. Coding sequences of ribosomal genes were detected both in the periphery of the nucleolar organizer region (NOR), which corresponds to the unwound part of the nucleolar chromosome, and in the proximal part of the fibrillo-granular (FG) region. These results suggest that the rRNA gene transcription predominantly occurs at the periphery of the NOR where the coding sequences are located. A predictive model summarizes and allows discussions and comparisons with other eukaryotes in which nucleolar mechanisms were previously studied. This leads to the conclusion that dinoflagellate cells constitute an excellent model for the study of the functional structure of the eukaryotic nucleolus.(ABSTRACT TRUNCATED AT 250 WORDS)

The toxic dinoflagellate Prorocentrum lima and its associated bacteria: II. Immunolocalization of okadaic acid in axenic and non-axenic cultures

Summary An immunofluorescence method, using a monoclonal antibody against okadaic acid (OA) toxin and DNA staining, was used to visualize the toxin and extranuclear DNA which could possibly be of intracellular bacterial origin, in the dinoflagellate species Prorocentrum lima Ehrenberg (Dodge). Okadaic acid (OA) was detected within the cytoplasm of the dinoflagellate cell, frequently near the cytoplasmic membrane in the cell periphery and occasionally close to the nuclear membrane in the center of the cytoplasm. These results suggest that OA production could be related to the peripheral chloroplast and that OA accumulation may be associated with membrane lipids. OA is not co-localized with extra-nuclear DNA, implying that intracellular bacteria do not contain or accumulate the toxin. Comparison between axenic and non-axenic dinoflagellate cultures showed no differences in immunolabelling, suggesting that extracellular associated bacteria are not essential to okadaic acid production by the P. lima culture. These results tend to show that the dinoflagellate cell is able to produce OA autonomously.

Dinoflagellate centrosome: Associated proteins old and new

Summary In Dinoflagellate protists, the centrosome is localized close to the nucleus and surrounded by a complex Golgi apparatus as shown in the two phylogenetically distant species, Prorocentrum micans and Crypthecodinium cohnii . Dinoflagellate centrosomes have been identified previously by using anti-β-tubulin antibody and CTR 210, an antibody raised against human cell centrosomes. Cryofixation and cryofracture techniques enabled us to describe the ultrastructure of these centrosome regions which are devoid of classical centriolar structures. We also describe centrosome associated proteins in dinoflagellates and their behaviour during the cell cycle: p80, a nuclear and cytoplasmic protein recently characterized, myosin II antigens and γ-tubulin. We review also the previously described dinoflagellate centrosome associated proteins: β-tubulin, CTR 210 antigens, p72 (HSP 70), α-actin and p56cdc13, a homologue of Schizosaccharomyces pombe cyclin B. The functions of these proteins and their interactions in the centrosome of dinoflagellates are discussed.

Argyrophilic proteins in Dinoflagellates: An ultrastructural, cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR‐like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non‐synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo‐granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reactions temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti‐B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co‐locating with end‐products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23‐like protein is probably a DNA partner.

Cytoskeleton and mitosis in the dinoflagellate Crypthecodinium cohnii: immunolocalization of P72, an HSP70-related protein

The intracellular organization of dinoflagellate protists, particularly the cytoskeleton, the cell cycle and mitosis (“dinomitosis”), presents peculiar features governed by specific proteins. We have previously biochemically isolated and characterized a protein of 72 kDa (P72) conserved from dinoflagellates to humans. The microsequencing of the NH2-terminal sequence and the reaction with a monoclonal antibody suggested that this protein belongs to the HSP70 chaperone family (Perret et al. 1991, 1995). Using a polyclonal antibody raised against P72, we have immunolocalized this protein using standard fluorescence microscopy on cryosections, confocal laser scanning microscopy on whole mounted cells and transmission electron microscopy after fast-freeze preparation with or without high pressure during freezing. We have localized P72 in specific dense aggregates at the cortical and centrosome level, in the cytoplasmic channels passing through the nucleus during mitosis and in the cleavage furrow. We completed this study by double labelling with anti-P72/anti-β-tubulin to analyse relationships between P72 and the cytoskeleton and to discuss the possible function of P72.

P75, a novel protein in Dinoflagellates

FVll is a 54 kI>a plasma serine protease zymogen glycoprotein which may be cleaved to produce FVIIa, which binds tightly to cell-exposed tissue factor (TF). The complex is responsible for the initiation of blood coagulation (Stenflo J., Suttie J. (19771 Annu. Rev. Biochern. 46, 157-172). Although FVIIa has been cocrystallised with its cofactor TF (Banner D. et al 0996) Nature 380,41-46), no structure exists for the protease. In certain clinical situations the 56kDa plasma serine protease inhibitor (Serpin) antithrombin III (ATIII) may directly inhibit FVlIa by the formation of a FVIIa/ATIII complex (Lawson J. et al. (1993) I. &of. Chem. 268,767.770).

Contribution to the understanding of the dinoflagellate chromosome structure and its replication process: Confocal laser scanning microscope (CLSM) observations and TEM study after high pressure cryo‐fixation

Consecutive, longitudinal 5 urn-thick paraffin sections of adult rat optic nerves were dewaxed and subsequently incubated with the following monoclonal antibodies: (i) anti-MAP-2 (Clone AP-14) recognises the MAP-2 protein which is believed to be located in the somato-dendritic component of neuronal cells, (it) anti-neurofilament (Clone RT-97) immunostains the 200 kD subunit of neurofilaments (iii) anti-GFAP (Clone G-A-5) recognises the GFAP protein expressed by mature astrocytes and (iv) vimentin stains meningeal and ependymal cells