Yeast as a model system for studying endocytosis.

Endocytosis is the membrane trafficking process by which plasma membrane components and extracellular material are internalized into cytoplasmic vesicles and delivered to early and late endosomes, eventually either recycling back to the plasma membrane or arriving at the lysosome/vacuole. The budding yeast Saccharomyces cerevisiae has proven to be an invaluable system for identifying proteins involved in endocytosis and elucidating the mechanisms underlying internalization and postinternalization events. Through genetic studies in yeast and biochemical studies in mammalian cells, it has become apparent that multiple cellular processes are linked to endocytosis, including actin cytoskeletal dynamics, ubiquitylation, lipid modification, and signal transduction. In this review, we will highlight the most exciting recent findings in the field of yeast endocytosis. Specifically, we will address the involvement of the actin cytoskeleton in internalization, the role of ubiquitylation as a regulator of multiple steps of endocytosis in yeast, and the sorting of endocytosed proteins into the recycling and vacuolar pathways.     

A novel class of bacterial translation factor RF3 mutations suggests specific structural domains for premature peptidyl-tRNA drop-off

The bacterial translation factor RF3 promotes translation termination by recycling the tRNA-mimicking release factors, RF1 and RF2, after mature polypeptide release. RF3 also enhances the premature peptidyl-tRNA drop-off reaction in the presence of RRF and EF-G. Despite the recently resolved X-ray crystal structure of RF3, the molecular details of the bimodal functionality of RF3 remain obscure. In this report, we demonstrate a novel class of RF3 mutations specifically defective in the tRNA drop-off reaction. These mutations suggest differential molecular pathways closely related to the guanine nucleotide modes of RF3.     

Yeast as a model system for studying lipid homeostasis and function

Lipids are essential eukaryotic cellular constituents. Lipid metabolism has a strong impact on cell physiology, and despite good progress in this area, many important basic questions remain unanswered concerning the functional diversity of lipid species and on the mechanisms that cells employ to sense and adjust their lipid composition. Combining convenient experimental tractability, a large degree of conservation of metabolic pathways with other eukaryotes and the relative simplicity of its genome, proteome and lipidome, yeast represents the most advantageous model organism for studying lipid homeostasis and function. In this review we will focus on the importance of yeast as a model organism and some of the innovative advantages for the lipid research field.     

Intra- and inter-nucleosomal protein-DNA interactions of the core histone tail domains in a model system

The core histone tail domains are key regulators of eukaryotic chromatin structure and function and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here we introduce a method for examining inter-nucleosome interactions of the tail domains in a model dinucleosome and determine the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, we reconstituted a nucleosome containing a single histone site specifically modified with a photoinducible cross-linker within the histone tail domain, and a second nucleosome containing a radiolabeled DNA template. These two nucleosomes were then ligated together and cross-linking induced by brief UV irradiation under various solution conditions. After cross-linking, the two templates were again separated so that cross-linking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal cross-links. Our results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Additionally, we find that binding of a linker histone to the dinucleosome increased the association of the H3 and H4 tails with the linker DNA region.     

ICF syndrome cells as a model system for studying X chromosome inactivation

Mutations in the DNMT3B DNA methyltransferase gene cause the ICF immunodeficiency syndrome. The targets of this DNA methyltransferase are CpG-rich heterochromatic regions, including pericentromeric satellites and the inactive X chromosome. The abnormal hypomethylation in ICF cells provides an important model system for determining the relationships between replication time, CpG island methylation, chromatin structure, and gene silencing in X chromosome inactivation.     

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.KEY WORDS: Caenorhabditis elegans, Cell non-autonomous proteotoxicity, Prion-like spreading     

Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation

Background

Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos.

Methodology/Principal Findings

We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow.

Conclusions/Significance

In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.     

Drosophila as a model system for studying lifespan and neuroprotective activities of plant-derived compounds

The fruit fly, Drosophila melanogaster, has been intensively used as a genetic model system for basic and applied research on human neurological diseases because of advantages over mammalian model systems such as ease of laboratory maintenance and genetic manipulations. Disease-associated gene mutations, whether endogenous or transgenically-inserted, often cause phenotypes in vivo that are similar to the clinical features of the human disorder. The Drosophila genome is simpler than that of mammals, in terms of gene and chromosome number, but nonetheless demonstrates extraordinary phylogenetic conservation of gene structure and function, especially notable among the genes whose mutations cause neurodevelopmental, neuropsychiatric, or neurodegenerative disorders. In addition, its well-established neuroanatomical, developmental, and molecular genetic research techniques allow many laboratories worldwide to study complex biological and genetic processes. Based on these merits of the Drosophila model system, it has been used for screening lifespan expansion and neuroprotective activities of plant extracts or their secondary metabolites to counteract pathological events such as mitochondrial damage by oxidative stress, which may cause sporadic neurodegenerative diseases. In this review, we have summarized that the fruit fly can be used for early-stage drug discovery and development to identify novel plant-derived compounds to protect against neurodegeneration in Alzheimer's disease and Parkinson's disease, and other neurological disorders caused by oxidative stress. Thus, the Drosophila system can directly or indirectly contribute to translational research for new therapeutic strategies to prevent or ameliorate neurodegenerative diseases.     

The PHD finger of human UHRF1 reveals a new subgroup of unmethylated histone H3 tail readers

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.     

Chromatin fiber folding: requirement for the histone H4 N-terminal tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.     

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress

It is well known that sea urchin embryos are able to activate different defense strategies against stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to Cd adopt autophagy as an additional stratagem to safeguard the developmental program. At present, there are no data focusing on the role of this process in embryo development of marine organisms.     

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress

It is well known that sea urchin embryos are able to activate different defense strategies against stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to Cd adopt autophagy as an additional stratagem to safeguard the developmental program. At present, there are no data focusing on the role of this process in embryo development of marine organisms.     

Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9A lysine 9 histone H3 methyltransferase

The activity of recombinant murine G9a toward lysine 9 of histone H3 was investigated. GST fusion proteins containing various lengths of the histone H3 amino-terminal tail were used as substrates in the presence of recombinant G9a enzyme and AdoMet cosubstrate. The minimal substrate methylated by G9a contained seven amino acids (TARKSTG) of the histone H3 tail. Furthermore, mutational analysis of the minimal substrate was performed to identify the amino acids essential for G9a-mediated methylation. All amino acids except Thr-11 were indispensable for the methylation reaction. Steady-state kinetic analysis of the wild-type and histone H3 point mutants, lysine 4 changed to alanine (K4A) or lysine 27 changed to alanine (K27A), with purified G9a revealed similar catalytic efficiency but a reduction in turnover number (k(cat)) from 78 to 58 h(-)(1). G9a methylated synthetic peptide substrates containing the first 13 amino acids of histone H3 efficiently, although methylation, acetylation, or mutation of proximal Lys-4 amino acids reduced Lys-9 methylation. The k(cat) for wild-type peptide substrate vs Lys-4 acetyl- or trimethyl-modified peptide were 88 and 32 h(-)(1), respectively, and the K(m) for the peptides varied from 0.6 to 2.2 muM, resulting in a large difference (15-91) in catalytic efficiency. Ser-10 or Thr-11 phosphorylation resulted in poor methylation by G9a. Immunoprecipitation of unmodified and Ser-10 and Thr-11 phosphorylated histone H3 displayed mostly Lys-4 dimethylation. Dimethylated Lys-9 was reduced in Ser-10 and Thr-11 immunoprecipitated phosphorylated histones as compared to nonphosphorylated H3. In an immunocytochemical assay, GFP fusion SUV39H1 or G9a did not colocalize with phosphorylated histone H3. Thus, Ser-10/Thr-11 phosphorylation impairs Lys-9 methylation. These data suggest that the sequence context of the modified residue affects G9a activity and the modification in the proximal amino acids influences methylation.