Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy.

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

Human Ageing Genomic Resources: new and updated databases.

In spite of a growing body of research and data, human ageing remains a poorly understood process. Over 10 years ago we developed the Human Ageing Genomic Resources (HAGR), a collection of databases and tools for studying the biology and genetics of ageing. Here, we present HAGR's main functionalities, highlighting new additions and improvements. HAGR consists of six core databases: (i) the GenAge database of ageing-related genes, in turn composed of a dataset of >300 human ageing-related genes and a dataset with >2000 genes associated with ageing or longevity in model organisms; (ii) the AnAge database of animal ageing and longevity, featuring >4000 species; (iii) the GenDR database with >200 genes associated with the life-extending effects of dietary restriction; (iv) the LongevityMap database of human genetic association studies of longevity with >500 entries; (v) the DrugAge database with >400 ageing or longevity-associated drugs or compounds; (vi) the CellAge database with >200 genes associated with cell senescence. All our databases are manually curated by experts and regularly updated to ensure a high quality data. Cross-links across our databases and to external resources help researchers locate and integrate relevant information. HAGR is freely available online (http://genomics.senescence.info/).

Identification of UBact, a ubiquitin-like protein, along with other homologous components of a conjugation system and the proteasome in different gram-negative bacteria.

Systems analogous to the eukaryotic ubiquitin-proteasome system have been previously identified in Archaea, and Actinobacteria (gram-positive), but not in gram-negative bacteria. Here, we report the bioinformatic identification of a novel prokaryotic ubiquitin-like protein, which we name UBact. The phyletic distribution of UBact covers at least five gram-negative bacterial phyla, including Nitrospirae, Armatimonadetes, Verrucomicroba, Nitrospinae, and Planctomycetes. Additionally, it was identified in seven candidate (uncultured) phyla and one Archaeon. UBact might have been overlooked because only few species in the phyla where it is found have been sequenced. In most of the species where we identified UBact, its neighbors in the genome code for proteins homologous to those involved in conjugation and/or degradation of Pup and Pup-tagged substrates. Among them are PafA-, Dop-, Mpa- and proteasome-homologous proteins. This gene association as well as UBact's size and conserved C-terminal G[E/Q] motif, strongly suggest that UBact is used as a conjugatable tag for degradation. With regard to its C-terminus, UBact differs from ubiquitin and most ubiquitin-like proteins (including the mycobacterial Pup) in that it lacks the characteristic C-terminal di-glycine motif, and it usually ends with the sequence R[T/S]G[E/Q]. The phyla that contain UBact are thought to have diverged over 3000 million years ago, indicating that either this ubiquitin-like conjugation system evolved early in evolution or that its occurrence in distant gram-negative phyla is due to multiple instances of horizontal gene transfer.

Ubiquitination of specific mitochondrial matrix proteins.

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins.

On the linkage between the ubiquitin-proteasome system and the mitochondria.

Several metabolic pathways critical for cellular homeostasis occur in the mitochondria. Because of the evolution of mitochondria and their physical separation, these pathways have traditionally been thought to be free from regulation by the ubiquitin-proteasome system. This perception has recently been challenged by evidence for the presence of ubiquitin system components in the mitochondria. Furthermore, it has been shown that certain mitochondrial proteins are conjugated by ubiquitin, and some of them are degraded by the proteasome. Of particular interest is the finding that some of these proteins are localized to the inner membrane and matrix, which rules out that their targeting is mediated by the cytosolic ubiquitin system. However, the extent of the involvement of the ubiquitin system in mitochondrial regulation is not known. The present study addresses this surprising finding, employing several independent approaches. First, we identified reported ubiquitin conjugates in human and yeast mitochondria and found that a large fraction of the mitochondrial proteome (62% in human) is ubiquitinated, with most proteins localized to the inner membrane and matrix. Next, we searched the literature and found that numerous ubiquitin system components localize to the mitochondria and/or contain mitochondrial targeting sequences. Finally, we identified reported protein-protein interactions between ubiquitin system components and mitochondrial proteins. These unexpected findings suggest that mitochondrial regulation by the ubiquitin system is fundamental and may have broad biomedical implications.

MitoAge: a database for comparative analysis of mitochondrial DNA, with a special focus on animal longevity.

Mitochondria are the only organelles in the animal cells that have their own genome. Due to a key role in energy production, generation of damaging factors (ROS, heat), and apoptosis, mitochondria and mtDNA in particular have long been considered one of the major players in the mechanisms of aging, longevity and age-related diseases. The rapidly increasing number of species with fully sequenced mtDNA, together with accumulated data on longevity records, provides a new fascinating basis for comparative analysis of the links between mtDNA features and animal longevity. To facilitate such analyses and to support the scientific community in carrying these out, we developed the MitoAge database containing calculated mtDNA compositional features of the entire mitochondrial genome, mtDNA coding (tRNA, rRNA, protein-coding genes) and non-coding (D-loop) regions, and codon usage/amino acids frequency for each protein-coding gene. MitoAge includes 922 species with fully sequenced mtDNA and maximum lifespan records. The database is available through the MitoAge website (www.mitoage.org or www.mitoage.info), which provides the necessary tools for searching, browsing, comparing and downloading the data sets of interest for selected taxonomic groups across the Kingdom Animalia. The MitoAge website assists in statistical analysis of different features of the mtDNA and their correlative links to longevity.

Human Ageing Genomic Resources: integrated databases and tools for the biology and genetics of ageing.

The Human Ageing Genomic Resources (HAGR, http://genomics.senescence.info) is a freely available online collection of research databases and tools for the biology and genetics of ageing. HAGR features now several databases with high-quality manually curated data: (i) GenAge, a database of genes associated with ageing in humans and model organisms; (ii) AnAge, an extensive collection of longevity records and complementary traits for >4000 vertebrate species; and (iii) GenDR, a newly incorporated database, containing both gene mutations that interfere with dietary restriction-mediated lifespan extension and consistent gene expression changes induced by dietary restriction. Since its creation about 10 years ago, major efforts have been undertaken to maintain the quality of data in HAGR, while further continuing to develop, improve and extend it. This article briefly describes the content of HAGR and details the major updates since its previous publications, in terms of both structure and content. The completely redesigned interface, more intuitive and more integrative of HAGR resources, is also presented. Altogether, we hope that through its improvements, the current version of HAGR will continue to provide users with the most comprehensive and accessible resources available today in the field of biogerontology.

NUMT ("new mighty") hypothesis of longevity.

Maximum life span (MLS) and abundance of mitochondrial DNA (mtDNA) insertions in the nuclear DNA (NUMTs) were analyzed in 17 animal species with completely sequenced mitochondrial and nuclear genomes. Highly significant positive correlations were found between MLS and NUMT number, total size, or density (both in mammals and all animal species). In mammals, NUMT abundance correlated positively with the mtDNA guanine content and negatively with adenine and thymine contents, but did not correlate with such longevity-associated variables as the body mass and resting metabolic rate.

The Human Ageing Genomic Resources: online databases and tools for biogerontologists.

Aging is a complex, challenging phenomenon that requires multiple, interdisciplinary approaches to unravel its puzzles. To assist basic research on aging, we developed the Human Ageing Genomic Resources (HAGR). This work provides an overview of the databases and tools in HAGR and describes how the gerontology research community can employ them. Several recent changes and improvements to HAGR are also presented. The two centrepieces in HAGR are GenAge and AnAge. GenAge is a gene database featuring genes associated with aging and longevity in model organisms, a curated database of genes potentially associated with human aging, and a list of genes tested for their association with human longevity. A myriad of biological data and information is included for hundreds of genes, making GenAge a reference for research that reflects our current understanding of the genetic basis of aging. GenAge can also serve as a platform for the systems biology of aging, and tools for the visualization of protein-protein interactions are also included. AnAge is a database of aging in animals, featuring over 4000 species, primarily assembled as a resource for comparative and evolutionary studies of aging. Longevity records, developmental and reproductive traits, taxonomic information, basic metabolic characteristics, and key observations related to aging are included in AnAge. Software is also available to aid researchers in the form of Perl modules to automate numerous tasks and as an SPSS script to analyse demographic mortality data. The HAGR are available online at http://genomics.senescence.info.

Do mitochondrial DNA and metabolic rate complement each other in determination of the mammalian maximum longevity?

In animal cells, mitochondria are semiautonomous organelles of virtually "hostile" (bacterial) origin, with their own code and genome (mtDNA). The semiautonomy and restricted resources could result in occasional "conflicts of interests" with other cellular components, in which mitochondria have greater chances to be "the weakest link," thus limiting longevity. Two principal questions are addressed: (1) to what extent the mammalian maximum life span (MLS) is associated with mtDNA base composition? (2) Does mtDNA base composition correlate with another important mitochondria-associated variable-resting metabolic rate (RMR)-and whether they complement each other in determination of MLS? Analysis of 140 mammalian species revealed significant correlations between MLS and the content of the four mtDNA nucleotides, especially noted for GC pairs (r(2) = 0.42; p < 10(-17)). The most remarkable finding of this study is that multivariate stepwise analysis selected only the GC content and RMR, which together explained 77% of variation in MLS (p < 10(-25)). To the authors' knowledge, it is the highest coefficient of MLS determination that has ever been reported for a comparable sample size. Taking into account substantial errors in estimation of MLS and RMR, it could mean that the GC and RMR explain most of the MLS biological variation. Other putative players in MLS determination should have relatively small contribution or their effects should be realized via the same channels. Although further research is clearly warranted, the extraordinary high correlation of mtDNA GC and RMR with MLS suggests a "direct hitting" of the core longevity targets, inferring mitochondria as a primary object for longevity-promoting interventions.

Mitochondrial genome anatomy and species-specific lifespan.

Several lines of evidence implicate mtDNA in the mechanisms of aging and longevity. The authors examined possible links between mtDNA composition and maximum lifespan of multicellular eukaryotes, including 102 mammals. MLS correlates positively with cytosine and negatively with adenine or thymine content, whereas guanine has no apparent effect. This is especially noted for primates. It appears that an increase in MLS of mammals is associated with thymine-to-cytosine substitution. The results suggest that the MLS may be associated with stability and/or mutability of mtDNA and call for further investigation of the mitochondrial genome as a potential target for lifespan-extending interventions.