Experimental Virus Evolution Reveals a Role of Plant Microtubule Dynamics and TORTIFOLIA1/SPIRAL2 in RNA Trafficking

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms.     

Matrix and Mineral in the Sea Urchin Larval Skeleton

The endoskeletal spicules of sea urchin larvae are composed of calcite, a surrounding extracellular matrix, and small amounts of occluded matrix proteins. The spicules are formed by primary mesenchyme cells (PMCs) in the blastocoel of the embryo, where they adopt stereotypical locations, thereby specifying where spicules will form. PMCs also fuse to form cytoplasmic cords connecting the cell bodies, and it is within the cords that spicules arise. The mineral phase contains 5% Mg as well as Ca, and about 0.1% of the mass is protein. The matrix and mineral form concentric plies, and the composite has different physical properties than those of pure calcite. The calcite diffracts as a single crystal and is composed of well-ordered, but not perfectly ordered, microdomains. There is evidence for adsorption of matrix proteins to specific crystal faces at domain boundaries, which may help regulate crystal growth and texture. Immature spicules contain considerable precipitated amorphous CaCO3, and PMCs also have vesicles that contain amorphous CaCO3. This suggests the hypothesis that the cellular precursor to the spicules is actually amorphous CaCO3 stabilized in the cell by protein. The spicule s enveloped by the PMC cord, but is topologically exterior to the cell. The PMC plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tip. The characteristics, localization, and possible function of the four identified matrix proteins are discussed. SM50, SM37, and PM27 all primarily enclose the mineral, though small amounts are occluded. SM30 is found in cellular vesicles and is probably the principal occluded protein of the spicule.     

Lineage-Specific Evolution of Echinoderm Mitochondrial ATP Synthase Subunit 8

Peculiar evolutionary properties of the subunit 8 of mitochondrial ATP synthase (ATPase8) are revealed by comparative analyses carried out between both closely and distantly related species of echinoderms. The analysis of nucleotide substitution in the three echinoids demonstrated a relaxation of amino acid functional constraints. The deduced protein sequences display a well conserved domain at the N-terminus, while the central part is very variable. At the C-terminus, the broad distribution of positively charged amino acids, which is typical of other organisms, is not conserved in the two different echinoderm classes of the sea urchins and of the sea stars. Instead, a motif of three amino acids, so far not described elsewhere, is conserved in sea urchins and is found to be very similar to the motif present in the sea stars. Our results indicate that the N-terminal region seems to follow the same evolutionary pattern in different organisms, while the maintenance of the C-terminal part in a phylum-specific manner may reflect the co-evolution of mitochondrial and nuclear genes.     

Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release

Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.     

Experimental Evolution of Adenylate Kinase Reveals Contrasting Strategies toward Protein Thermostability

Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK_BSUB Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK_BSUB G213E decreases protein stability by >25°C, the same mutation in the background of AK_BSUB Q199R increases stability by 3.4°C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK_BSUB double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.     

Experimental Evolution Reveals Interplay between Sch9 and Polyploid Stability in Yeast

Polyploidization has crucial impacts on the evolution of different eukaryotic lineages including fungi, plants and animals. Recent genome data suggest that, for many polyploidization events, all duplicated chromosomes are maintained and genome reorganizations occur much later during evolution. However, newly-formed polyploid genomes are intrinsically unstable and often quickly degenerate into aneuploidy or diploidy. The transition between these two states remains enigmatic. In this study, laboratory evolution experiments were conducted to investigate this phenomenon. We show that robust tetraploidy is achieved in evolved yeast cells by increasing the abundance of Sch9—a protein kinase activated by the TORC1 (Target of Rapamycin Complex 1) and other signaling pathways. Overexpressing SCH9, but not TOR1, allows newly-formed tetraploids to exhibit evolved phenotypes and knocking out SCH9 diminishes the evolved phenotypes. Furthermore, when cells were challenged with conditions causing ancestral cells to evolve aneuploidy, tetraploidy was maintained in the evolved lines. Our results reveal a determinant role for Sch9 during the early stage of polyploid evolution.     

Experimental Approach to the Role of Protozoa in Aquatic Ecosystems

In enrichment batch experiments, samples from three water bodieswere alternatively supplemented by various amounts of organicmaterial and incubated at 20 C. Colpidium campylum reached itshighest total cell volumes in cultures with the highest initialtotal cell volumes in cultures with the highest initial concentrationsof organics; Cyclidium glaucoma preferred lower concentrations;and Glaucoma chattoni occupied the intermediate position. Noneof the species preferred any special type of organic material.In two-stage continuous-flow units, a mixed culture of bacteriawas kept in stage I and the clones of ciliates were maintainedin stage II. The interrelations between the total cell volumesof ciliates at various concentrations of bactopeptone were inaccordance with the results from the enrichment experiments.Since the growth of bacteria continued in the presence of ciliates,a four-stage apparatus was constructed in which a bacterialculture was raised and diluted in three stages before enteringthe culture ofColpidium. The bacterial growth in the presenceof Colpidium was not eliminated even by this arrangement, asdemonstrated by dosing antibiotics along with bacteria. An effectof ciliate metabolites on bacterial growth rate is suggested,completing a metabolic cycle in the bacteria-protozoa system.     

Levels of Homoplasy in the Evolution of the Mammalian Skeleton

It is commonly believed that there are differences in the evolutionary lability of the crania, dentition, and postcrania of mammals, the latter two being more prone to homoplasy because of strong selective pressures for feeding and locomotion, respectively. Further, because of the fragmentary nature of fossils, phylogenetic analyses of extinct taxa often must utilize characters based on only one of these systems. In this paper the levels of homoplasy (as measured by the consistency index; CI) were compared in characters based on these three anatomical systems in therian mammals. No statistically significant differences were found in the overall CIs of 41 data sets based on dental, cranial, or postcranial characters. Differences in homoplasy within data sets with two or three kinds of data were not statistically significant. These findings suggest that dental, cranial, and postcranial characters can be equally prone to homoplasy and none should be automatically dismissed, disregarded, or systematically weighted in phylogenetic analyses. The level of homoplasy in characters derived from a given region of the skeleton may differ depending on the taxonomic level of the taxa considered. Dental, cranial, and postcranial characters may not constitute natural classes, yet examination of the phylogenetic signal of these subsets of data previous to a simultaneous analysis can shed light on significant aspects of the evolutionary process.     

Experimental Evolution and Its Role in Evolutionary Physiology

Four general approaches to the study of evolutionary physiology—phylogenetically-basedcomparisons, genetic analyses and manipulations, phenotypicplasticity and manipulation, and selection studies—areoutlined and discussed. We provide an example of the latter,the application of laboratory selection experiments to the studyof a general issue in environmental adaptation, differencesin adaptive patterns of generalists and specialists. A cloneof the bacterium Escherichia coli that had evolved in a constantenvironment of 37°C was replicated into 6 populations andallowed to reproduce for 2,000 generations in a variable thermalenvironment alternating between 32 and 42°C. As predictedby theory, fitness and efficiency of resource use increasedin this new environment, as did stress resistance. Contraryto predictions, however, fitness and efficiency in the constantancestral environment of 37°C did not decrease, nor didthermal niche breadth or phenotypic plasticity increase. Selectionexperiments can thus provide a valuable approach to testinghypotheses and assumptions about the evolution of functionalcharacters.     

Activity Physiology and Evolution of the Vertebrate Skeleton

SYNOPSIS. Vertebrates frequently rely on intramuscular glycolysisas the major source of ATP utilized during bouts of intenseexercise. This is often followed by extended periods of markedsystemic pH fluctuation. Such a pattern of activity physiologyis unique among the Metazoa and probably dates back to the veryearliest vertebrates. The origin of bone may have been necessitated by requirementfor a skeletal matrix with chemical stability over the broadrange of tissue pH associated with vertebrate exercise physiology.     

Functional Constraints on the Evolution of Larval Forms of Marine Invertebrates: Experimental and Comparative Evidence

Marine invertebrate larvae are well known for their distinctivebody shapes and elaborate patterns of ciliation. In this studyI take a physically based approach to investigate the functionalconsequences of variations in body shape and patterns of ciliation.With experimental models I demonstrate that shape as well assurface area contributes to drag of larval forms. Based on flowfields around larvae tethered in still water and flowing waterI argue that drag, which acts as a partial tether, may influencehow water is processed and food is captured by cilia. With mechanicalmodels of cilia I show that placement of cilia on the surfacescan influence the effectiveness with which water is moved andthe steepness of the velocity gradient through the ciliary layer.These models indicate that placement of cilia on ridges, atextreme anterior ends, and at extreme posterior ends of larvalbodies increases the volume of water moved per ciliary strokerelative to placement of cilia on a flat surface. A comparativesurvey of46 larval forms indicates that distributions of bodyshape and patterns of ciliation reflect functional requirementsof swimming and feeding by larvae. The experimental and comparativeapproaches together suggest functional constraints on the evolutionof larval forms which may lead to convergence in patterns ofciliation and conservation of larval forms within taxa.     

Genetic Perturbation of TIA1 Reveals a Physiological Role in Fear Memory

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The Developmental Origins of Mosaic Evolution in the Primate Limb Skeleton

The central hypothesis of this paper is that basic properties of vertebrate limb development bias the generation of phenotypic variation in certain directions, and that these biases establish focal units, or regions, of evolutionary change within the primate hand and foot. These focal units include (1) a preaxial domain (digit I, hallux or pollex, metapodial and proximal phalanx), (2) a postaxial domain (metapodials and phalanges of digits II?CV), and (3) a digit tip domain (terminal phalanges and nails/claws of rays I?CV). The existence of these focal units therefore provides a mechanistic basis for mosaic evolution within the hand and foot, and can be applied to make specific predictions about which features of the limb skeleton are most likely to be altered in primate adaptive radiations over time. Examination of the early primate fossil record provides support for this model, and suggests that the existence of variational tendencies in limb development has played a major role in guiding the origin and evolution of primate skeletal form.     

Experimental Relocation of the Mitochondrial ATP9 Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.     

Experimental Evolution of a Bacteriophage Virus Reveals the Trajectory of Adaptation across a Fecundity/Longevity Trade-Off

Life history theory attempts to account for how organisms lead their lives, balancing the conflicting demands of reproduction and survival. Here, we track the genomic and phenotypic evolution of the bacteriophage virus T7 across a postulated fecundity/longevity constraint. We adapted T7 to a challenging survival environment (6M urea). Our evolved strain displayed a significant improvement in propagule survival, coupled with a significant loss of fecundity (reduced growth rate on host cells). However, the increased resistance to urea did not generalise to increased resistance against temperature stress, highlighting that propagule durability is environment dependent. Previous comparative studies predicted that changes in propagule resistance would be mediated by changes in capsid proteins or gene deletions. In contrast, we found that point mutations in internal core protein genes (6.7 and 16) were responsible for the increased urea resistance of our evolved strain. Prior to the emergence of the 6.7 and 16 mutations, a distinct set of 5-point mutations peaked at over 20% prevalence before attenuating, suggestive of negative epistatic interactions during adaptation. Our results illustrate that parasites can adapt to specific transmission environments, and that this adaptation can impose costs on the subsequent ability to exploit host cells, potentially constraining durable parasites to lower virulence.     

Role of Calcitonin Gene-Related Peptide in Functional Adaptation of the Skeleton

Peptidergic sensory nerve fibers innervating bone and periosteum are rich in calcitonin gene-related peptide (CGRP), an osteoanabolic neurotransmitter. There are two CGRP isoforms, CGRPα and CGRPβ. Sensory fibers are a potential means by which the nervous system may detect and respond to loading events within the skeleton. However, the functional role of the nervous system in the response of bone to mechanical loading is unclear. We used the ulna end-loading model to induce an adaptive modeling response in CGRPα and CGRPβ knockout mouse lines and their respective wildtype controls. For each knockout mouse line, groups of mice were treated with cyclic loading or sham-loading of the right ulna. A third group of mice received brachial plexus anesthesia (BPA) of the loaded limb before mechanical loading. Fluorochrome labels were administered at the time of loading and 7 days later. Ten days after loading, bone responses were quantified morphometrically. We hypothesized that CGRP signaling is required for normal mechanosensing and associated load-induced bone formation. We found that mechanically-induced activation of periosteal mineralizing surface in mice and associated blocking with BPA were eliminated by knockout of CGRPα signaling. This effect was not evident in CGRPβ knockout mice. We also found that mineral apposition responses to mechanical loading and associated BPA blocking were retained with CGRPα deletion. We conclude that activation of periosteal mineralizing surfaces in response to mechanical loading of bone is CGRPα-dependent in vivo. This suggests that release of CGRP from sensory peptidergic fibers in periosteum and bone has a functional role in load-induced bone formation.