The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing.

DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing.

Nuclear envelope organization in Dictyostelium discoideum.

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.

Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81.

The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src1(1-646), a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.

Dictyostelium centrin B localization during cell cycle progression.

Recently, we have reported the initial characterization of a novel centrin from Dictyostelium discoideum (DdCenB).1 Sequence and phylogenetic analyses clearly establish DdCenB as a centrin, yet further characterization revealed some interesting peculiarities about this novel centrin. Figure 1 depicts the localization of DdCenB at three points in the cell cycle: interphase, mitosis and cytokinesis. In interphase DdCenB primarily localizes to the nuclear envelope (NE). Although the NE remains intact during mitosis and cytokinesis in Dictyostelium, DdCenB disappears from the NE at these two stages of the cell cycle. In addition to localization at the NE, we also see weak localization in the nucleoplasm and cytoplasm (weakest). Although the nucleoplasmic concentration appears constant throughout the cell cycle, the very faint localization in the cytoplasm does appear to increase to the level of the nucleoplasm during mitosis and cytokinesis. Unlike most centrins characterized to date, we found no evidence of DdCenB at the centrosome at any point in the cell cycle. Here we examine the importance of DdCenB localization in cell cycle progression, as well as several other roles.

Dictyostelium discoideum CenB is a bona fide centrin essential for nuclear architecture and centrosome stability.

Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.

Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability.

The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatin-binding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosome-nucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.

Case report: epidural blood patch in the treatment of abducens palsy after a dural puncture.

PURPOSE: To describe a case of iatrogenically induced abducens nerve palsy following a diagnostic lumbar puncture, and to review the evidence for blood patching in the management of sixth cranial nerve palsy after dural puncture. CLINICAL FEATURES: A 45-yr-old woman developed post-dural puncture headache with bilateral abducens palsy following a diagnostic lumbar puncture. Magnetic resonance imaging showed findings compatible with intracranial hypotension. An epidural blood patch was performed five days after the onset of diplopia and ten days following the dural puncture. After blood patching, the patient reported relief of the headache, but still complained of diplopia. The palsies recovered spontaneously 21 months after the dural puncture. CONCLUSION: Experience from this case as well as other case report evidence suggest that an epidural blood patch performed more than 24 hr after the onset of a sixth cranial nerve palsy consistently fails to relieve diplopia. An epidural blood patch executed within 24 hr from the onset of diplopia could possibly lead to partial improvement and/or earlier resolution of symptoms.

Chimeric analysis of the small GTPase RacE in cytokinesis signaling in Dictyostelium discoideum.

RacE is a small GTPase required for cytokinesis in Dictyostelium discoideum. To investigate RacE's potential binding and signaling interfaces that allow its function in cytokinesis, 10 different chimeras were created between RacE and the closely related small GTPase, RacC. RacE/RacC chimeras, containing various combinations of four RacE regions, E I-IV: E-I (aa 1-67), E-II (aa 68-124), E-III (aa 125-184), and E-IV (aa 185-223), were tested for their ability to rescue the multinucleated, cytokinesis-defective phenotype of RacE null cells grown in suspension. Regions E-II and E-IV were essential but not sufficient for the rescue of RacE null cells. These two regions, in combination with either region E-1 or E-III, resulted in rescue. Results presented here suggest that region E-II contains a crucial, yet incomplete, binding site. Regions E-I or E-III separately provide additional, necessary elements for RacE's function. The extended E tail of RacE (E-IV) may act as a 'sensor' of the bound nucleotide state of RacE and facilitate GDP to GTP exchange (possibly through interactions with a GEF molecule), thereby resulting in activation of RacE. This study provides new evidence for small GTPases engaging several distinct protein interfaces to mediate signaling in various cellular processes.

The identification of pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum.

Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1). that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.

The three-dimensional model of Dictyostelium discoideum racE based on the human rhoA-GDP crystal structure.

The three-dimensional structure of racE was modeled using several homologous small G proteins, and the best model obtained using the human rhoA as modeling template is reported. The three-dimensional fold of the racE model is remarkably similar to the cellular form of human ras p21 crystal structure. Its secondary structure consists of six alpha-helices, six beta-strands and three 3(10) helices. The model retains its secondary structure after a 300 K, 300 ps molecular dynamics (MD) simulation. Important domains of the protein include its effector loop (residues 34-46), the insertion domain (residues 121-136), and the polybasic motif (between 210 and 220) not modeled in the current structure. The effector loop is inherently flexible and the structure docked with GDP exhibits the effector loop moving significantly closer to the nucleotide binding pocket, forming a tighter complex with the bound GDP. The mobility of the effector loop is conferred by a single residue 'hinge' point at residue 34Asp, also allowing the Switch I region, immediately preceding the effector loop, to be equally mobile. In comparison, the Switch II region shows average mobility. The insertion domain is highly flexible, with the insertion taking the form of a helical domain, with several charged residues forming a complex charged interface over the entire insertion region. While the GDP moiety is loosely held in the active site, the metal cation is extensively co-ordinated. The critical residue 38Thr exhibits high mobility, and is seen interacting directly with the metal ion at a distance of 2.64 A, and indirectly via an intervening water molecule. 64Gln, a key residue involved in GTP hydrolysis in ras, is seen facing the beta-phosphate group and the metal ion. Certain residues (i.e. 51Asn, 38Thr and 65Glu) exhibit unique characteristics and these residues, together with 158Val, may play important roles in the maintenance of the protein's integrity and function. There is strong consensus of secondary structural elements between models generated using various templates, such as h-rac1, h-rhoA and h-cdc42 bound to RhoGDI, all sharing only 50-55% sequence identity with racE, which suggests that this model is in all probability an accurate prediction of the true tertiary structure of racE.

A user's guide to restriction enzyme-mediated integration in Dictyostelium.

Restriction enzyme-mediated integration (REMI) has been used to study a number of cellular and developmental processes in Dictyostelium discoideum. In this paper we review the basics of this powerful method of introducing random mutations in Dictyostelium. Here we discuss several mutation screens that have been devised and some of the genes that have been discovered through this approach to mutagenesis. Included in this discussion is how one goes about isolating a gene that has been disrupted by REMI, and how one confirms that this disruption is actually responsible for the observed phenotype. Finally, we describe how REMI can be used as an effective teaching tool in undergraduate cell biology laboratory courses.