The toad’s warts: Discordance creates bumpy expectations of mitochondrial‐nuclear evolution between species

Discordance between the mitochondrial and nuclear genomes is a prevalent phenomenon in nature, in which the underlying processes responsible are considered to be important in shaping genetic variation in natural populations. Among the evolutionary processes that best explain such genomic mismatches incomplete lineage sorting and introgression are commonly identified, however, many studies are unable to distinguish between these hypotheses, which has become a major challenge in the field. In this issue of Molecular Ecology, Firneno et al. (2020) present an elegant exploration of mitochondrial‐nuclear discordance in Mesoamerican toads. Integrating genome‐scale and spatial data to test between these hypotheses within an empirical model testing framework, they find strong support that incomplete lineage sorting explains the observed discordance. Their work, along with many previous articles in Molecular Ecology, highlights the commonality of mito‐nuclear discordance among species despite the expectations of tightly concerted mitochondrial and nuclear genome evolution. It is increasingly clear that the nuclear genomes of many species are (at least for short periods of evolutionary time) functionally compatible with multiple, divergent mitochondrial haplotypes. As such, we suggest future research not only seeks to understand the processes causing spatial mito‐nuclear discordance (e.g. incomplete lineage sorting, introgression), but also explores those that maintain discordance through time and space (e.g. relaxed selection on mito‐nuclear interactions, heterozygosity, population demographics). We also discuss the vital role that taxonomy plays in interpreting patterns of mito‐nuclear discordance when data‐consistent yet differing taxonomies are used, such as treating allopatrically distributed taxa as multiple isolated populations versus multiple micro‐endemic species.     

The Golgi complex: a hub of the secretory pathway

The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance.     

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin

Oxidative stress and Cu²⁺ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu²⁺, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu²⁺-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu²⁺ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn²⁺, Mg²⁺, and Ca²⁺ does not affect Cu²⁺ binding, indicating selectivity of the Cu²⁺-binding site(s) in α-crystallins. Cu²⁺ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu²⁺ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu²⁺ binding. α-Crystallins rescue amyloid beta peptide, Aβ_1-40, from Cu²⁺-induced aggregation in vitro. α-Crystallins inhibit Cu²⁺-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu²⁺-binding protein, does not inhibit this oxidation process significantly. We find that the Cu²⁺-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu²⁺ binding and redox silencing of Cu²⁺ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu²⁺ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.     

Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway.

The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood. We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor. Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells. The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells. The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs.     

Model‐based approach to test hard polytomies in the Eulaemus clade of the most diverse South American lizard genus Liolaemus (Liolaemini,Squamata)

Lack of resolution in a phylogenetic tree is usually represented as a polytomy, and often adding more data (loci and taxa) resolves the species tree. These are the ‘soft’ polytomies, but in other cases additional data fail to resolve relationships; these are the ‘hard’ polytomies. This latter case is often interpreted as a simultaneous radiation of lineages in the history of a clade. Although hard polytomies are difficult to address, model‐based approaches provide new tools to test these hypotheses. Here, we used a clade of 144 species of the South American lizard clade Eulaemus to estimate phylogenies using a traditional concatenated matrix and three species tree methods: *BEAST, BEST, and minimizing deep coalescences (MDC). The different species tree methods recovered largely discordant results, but all resolved the same polytomy (e.g. very short internodes amongst lineages and low nodal support in Bayesian methods). We simulated data sets under eight explicit evolutionary models (including hard polytomies), tested these against empirical data (a total of 14 loci), and found support for two polytomies as the most plausible hypothesis for diversification of this clade. We discuss the performance of these methods and their limitations under the challenging scenario of hard polytomies. © 2015 The Linnean Society of London     

The importance of metacommunity ecology for environmental assessment research in the freshwater realm

Most bioassessment programs rest on the assumption that species have different niches, and that abiotic environmental conditions and changes therein determine community structure. This assumption is thus equivalent to the species sorting perspective (i.e. that species differ in their responses to environmental variation) in metacommunity ecology. The degree to which basing bioassessment on the species sorting perspective is reasonable is likely to be related to the spatial extent of a study and the characteristics of the organism groups (e.g. dispersal ability) with which the effects of anthropogenic changes are assessed. Recent findings in metacommunity research have stressed that community structure is determined not only by local abiotic environmental conditions but also by biotic interactions and dispersal‐related effects. For example, dispersal limitation may prevent community structure recovery from the effects of a putative stressor, as organisms may not be able to disperse to all sites in a region. Mass effects (i.e. the presence of species in environmentally suboptimal sites due to high dispersal rates from environmentally suitable sites) may, in turn, obscure the effects of a stressor, as dispersal from source sites (e.g. an unaltered site) allows persistence at sink sites (e.g. an anthropogenically altered site). Better bioassessment should thus take both niche‐ and dispersal‐related processes simultaneously into consideration, which can be accomplished by explicitly modelling spatial location as a proxy for dispersal effects. Such an integrated approach should be included in bioassessment programs using general multivariate approaches, predictive modelling, and multimetric indices.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

THE INTERFACE BETWEEN PHYLOGENETICS AND POPULATION GENETICS: INVESTIGATING GENE TREES,SPECIES TREES,AND POPULATION DYNAMICS IN THE PHYLLOPHAGA FRATERNA SPECIES GROUP

This study uses traditional and contemporary phylogenetic and population genetic analyses to assess the causes of discordance (i.e., lineage sorting and introgression) among mitochondrial and nuclear gene trees for a clade of eastern North American scarab beetles (fraterna species group, genus Phyllophaga). I estimated gene trees using individual and combined analysis of one mitochondrial and two nuclear loci in MrBayes , and inferred a species tree using a hierarchical coalescent approach based on all loci in the program Best . Because hybridization violates the assumptions of Best , I tested for introgression by comparing species monophyly between the mitochondrial and nuclear gene trees based on the prediction that cytoplasmic genomes introgress more readily than nuclear genomes. Haplotype exclusivity was identified using Bayesian tests of monophyly and the genealogical sorting index. I used the results of the phylogenetic analyses and monophyly tests to develop an explicit hypothesis of introgression that could be tested in the program IMa. Results from these analyses provided evidence for introgression across clades within the fraterna group. The tiered analytical approach used in this study demonstrated how the use of multiple methods can identify when assumptions are violated and methods are prone to yield misleading results.     

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.