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19 pages, 4728 KiB

Open AccessFeature PaperArticle

Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L Subcellular Localization and Function

by Teresa Fernández-Acero, Eleonora Bertalmio, Sandra Luna, Janire Mingo, Ignacio Bravo-Plaza, Isabel Rodríguez-Escudero, María Molina, Rafael Pulido and Víctor J. Cid

Cells 2019, 8(12), 1512; https://doi.org/10.3390/cells8121512 - 26 Nov 2019

Cited by 9 | Viewed by 4194

Abstract

The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP₃ counteracts [...] Read more.

The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP₃ counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP₂-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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19 pages, 4521 KiB

Open AccessArticle

Dyskerin Mutations Present in Dyskeratosis Congenita Patients Increase Oxidative Stress and DNA Damage Signalling in Dictyostelium Discoideum

by Javier Rodriguez-Centeno, Rosario Perona and Leandro Sastre

Cells 2019, 8(11), 1406; https://doi.org/10.3390/cells8111406 - 08 Nov 2019

Cited by 6 | Viewed by 3080

Abstract

Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, [...] Read more.

Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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22 pages, 4042 KiB

Open AccessFeature PaperArticle

Modelling of Neuronal Ceroid Lipofuscinosis Type 2 in Dictyostelium discoideum Suggests That Cytopathological Outcomes Result from Altered TOR Signalling

by Paige K. Smith, Melodi G. Sen, Paul R. Fisher and Sarah J. Annesley

Cells 2019, 8(5), 469; https://doi.org/10.3390/cells8050469 - 16 May 2019

Cited by 14 | Viewed by 3513

Abstract

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 [...] Read more.

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 variant of the disease, Tripeptidyl peptidase (Tpp1), in the cellular slime mould Dictyostelium discoideum. Knocking down Tpp1 in Dictyostelium resulted in the accumulation of autofluorescent material, a characteristic trait of Batten disease. Phenotypic characterisation of the mutants revealed phenotypic deficiencies in growth and development, whilst endocytic uptake of nutrients was enhanced. Furthermore, the severity of the phenotypes correlated with the expression levels of Tpp1. We propose that the phenotypic defects are due to altered Target of Rapamycin (TOR) signalling. We show that treatment of wild type Dictyostelium cells with rapamycin (a specific TOR complex inhibitor) or antisense inhibition of expression of Rheb (Ras homologue enriched in the brain) (an upstream TOR complex activator) phenocopied the Tpp1 mutants. We also show that overexpression of Rheb rescued the defects caused by antisense inhibition of Tpp1. These results suggest that the TOR signalling pathway is responsible for the cytopathological outcomes in the Dictyostelium Tpp1 model of Batten disease. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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17 pages, 3968 KiB

Open AccessArticle

Supramolecular Structures of the Dictyostelium Lamin NE81

by Marianne Grafe, Petros Batsios, Irene Meyer, Daria Lisin, Otto Baumann, Martin W. Goldberg and Ralph Gräf

Cells 2019, 8(2), 162; https://doi.org/10.3390/cells8020162 - 16 Feb 2019

Cited by 5 | Viewed by 4253

Abstract

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and [...] Read more.

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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Review

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17 pages, 501 KiB

Open AccessFeature PaperReview

Dictyostelium: A Model for Studying the Extracellular Vesicle Messengers Involved in Human Health and Disease

by Irène Tatischeff

Cells 2019, 8(3), 225; https://doi.org/10.3390/cells8030225 - 08 Mar 2019

Cited by 16 | Viewed by 4424

Abstract

Cell-derived extracellular vesicles (EVs) are newly uncovered messengers for intercellular communication. They are released by almost all cell types in the three kingdoms, Archeabacteria, Bacteria and Eukaryotes. They are known to mediate important biological functions and to be increasingly involved in cell physiology [...] Read more.

Cell-derived extracellular vesicles (EVs) are newly uncovered messengers for intercellular communication. They are released by almost all cell types in the three kingdoms, Archeabacteria, Bacteria and Eukaryotes. They are known to mediate important biological functions and to be increasingly involved in cell physiology and in many human diseases, especially in oncology. The aim of this review is to recapitulate the current knowledge about EVs and to summarize our pioneering work about Dictyostelium discoideum EVs. However, many challenges remain unsolved in the EV research field, before any EV application for theranostics (diagnosis, prognosis, and therapy) of human cancers, can be efficiently implemented in the clinics. Dictyostelium might be an outstanding eukaryotic cell model for deciphering the utmost challenging problem of EV heterogeneity, and for unraveling the still mostly unknown mechanisms of their specific functions as mediators of intercellular communication. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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16 pages, 1768 KiB

Open AccessReview

Recent Insights into NCL Protein Function Using the Model Organism Dictyostelium discoideum

by Meagan D. McLaren, Sabateeshan Mathavarajah and Robert J. Huber

Cells 2019, 8(2), 115; https://doi.org/10.3390/cells8020115 - 02 Feb 2019

Cited by 22 | Viewed by 5459

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating neurological disorders that have a global distribution and affect people of all ages. Commonly known as Batten disease, this form of neurodegeneration is linked to mutations in 13 genetically distinct genes. The precise [...] Read more.

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating neurological disorders that have a global distribution and affect people of all ages. Commonly known as Batten disease, this form of neurodegeneration is linked to mutations in 13 genetically distinct genes. The precise mechanisms underlying the disease are unknown, in large part due to our poor understanding of the functions of NCL proteins. The social amoeba Dictyostelium discoideum has proven to be an exceptional model organism for studying a wide range of neurological disorders, including the NCLs. The Dictyostelium genome contains homologs of 11 of the 13 NCL genes. Its life cycle, comprised of both single-cell and multicellular phases, provides an excellent system for studying the effects of NCL gene deficiency on conserved cellular and developmental processes. In this review, we highlight recent advances in NCL research using Dictyostelium as a biomedical model. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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15 pages, 891 KiB

Open AccessReview

Yeast to Study Human Purine Metabolism Diseases

by Bertrand Daignan-Fornier and Benoît Pinson

Cells 2019, 8(1), 67; https://doi.org/10.3390/cells8010067 - 17 Jan 2019

Cited by 25 | Viewed by 6735

Abstract

Purine nucleotides are involved in a multitude of cellular processes, and the dysfunction of purine metabolism has drastic physiological and pathological consequences. Accordingly, several genetic disorders associated with defective purine metabolism have been reported. The etiology of these diseases is poorly understood and [...] Read more.

Purine nucleotides are involved in a multitude of cellular processes, and the dysfunction of purine metabolism has drastic physiological and pathological consequences. Accordingly, several genetic disorders associated with defective purine metabolism have been reported. The etiology of these diseases is poorly understood and simple model organisms, such as yeast, have proved valuable to provide a more comprehensive view of the metabolic consequences caused by the identified mutations. In this review, we present results obtained with the yeast Saccharomyces cerevisiae to exemplify how a eukaryotic unicellular organism can offer highly relevant information for identifying the molecular basis of complex human diseases. Overall, purine metabolism illustrates a remarkable conservation of genes, functions and phenotypes between humans and yeast. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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13 pages, 1771 KiB

Open AccessReview

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Dictyostelium

by Tetsuya Muramoto, Hoshie Iriki, Jun Watanabe and Takefumi Kawata

Cells 2019, 8(1), 46; https://doi.org/10.3390/cells8010046 - 12 Jan 2019

Cited by 11 | Viewed by 11405

Abstract

In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, [...] Read more.

In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2–3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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21 pages, 4347 KiB

Open AccessReview

Dictyostelium: An Important Source of Structural and Functional Diversity in Drug Discovery

by Yuzuru Kubohara and Haruhisa Kikuchi

Cells 2019, 8(1), 6; https://doi.org/10.3390/cells8010006 - 21 Dec 2018

Cited by 21 | Viewed by 7160

Abstract

The cellular slime mold Dictyostelium discoideum is an excellent model organism for the study of cell and developmental biology because of its simple life cycle and ease of use. Recent findings suggest that Dictyostelium and possibly other genera of cellular slime molds, are [...] Read more.

The cellular slime mold Dictyostelium discoideum is an excellent model organism for the study of cell and developmental biology because of its simple life cycle and ease of use. Recent findings suggest that Dictyostelium and possibly other genera of cellular slime molds, are potential sources of novel lead compounds for pharmacological and medical research. In this review, we present supporting evidence that cellular slime molds are an untapped source of lead compounds by examining the discovery and functions of polyketide differentiation-inducing factor-1, a compound that was originally isolated as an inducer of stalk-cell differentiation in D. discoideum and, together with its derivatives, is now a promising lead compound for drug discovery in several areas. We also review other novel compounds, including secondary metabolites, that have been isolated from cellular slime molds. Full article

(This article belongs to the Special Issue Simple Organisms for Complex Problems: Modeling Human Disease in Yeast and Dictyostelium)

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