Daniel S. Fisher

High-Throughput Sequencing of the Zebrafish Antibody Repertoire

Antibody Repertoire Revealed Antibodies produced by B cells protect us against infection by a wide array of pathogens. Such wide-ranging responses are possible because the specific domain of the antibody that binds to the invader is highly variable owing to the somatic recombination of an inherited set of gene segments that encode the antibody molecule. Although antibody diversity within an individual organism is well-established, the specific antibody repertoire of an individual organism has not been characterized. Using high-throughput sequencing technology, Weinstein et al. (p. 807) characterized the diversity of the antigen-binding domain of the antibody heavy chain in zebrafish. The antibody repertoire of individual fish covered at least 50% of the possible gene combinations. Although the specific gene combinations differed between fish, a similar frequency distribution of the repertoire was observed. Unexpected examples of evolutionary conversion were also seen, with the same antibody observed in different animals. Sequencing of immunoglobulin messenger RNA characterizes the diversity of the antibody repertoire in individual zebrafish. Despite tremendous progress in understanding the nature of the immune system, the full diversity of an organism’s antibody repertoire is unknown. We used high-throughput sequencing of the variable domain of the antibody heavy chain from 14 zebrafish to analyze VDJ usage and antibody sequence. Zebrafish were found to use between 50 and 86% of all possible VDJ combinations and shared a similar frequency distribution, with some correlation of VDJ patterns between individuals. Zebrafish antibodies retained a few thousand unique heavy chains that also exhibited a shared frequency distribution. We found evidence of convergence, in which different individuals made the same antibody. This approach provides insight into the breadth of the expressed antibody repertoire and immunological diversity at the level of an individual organism.

Beneficial Mutation–Selection Balance and the Effect of Linkage on Positive Selection

When beneficial mutations are rare, they accumulate by a series of selective sweeps. But when they are common, many beneficial mutations will occur before any can fix, so there will be many different mutant lineages in the population concurrently. In an asexual population, these different mutant lineages interfere and not all can fix simultaneously. In addition, further beneficial mutations can accumulate in mutant lineages while these are still a minority of the population. In this article, we analyze the dynamics of such multiple mutations and the interplay between multiple mutations and interference between clones. These result in substantial variation in fitness accumulating within a single asexual population. The amount of variation is determined by a balance between selection, which destroys variation, and beneficial mutations, which create more. The behavior depends in a subtle way on the population parameters: the population size, the beneficial mutation rate, and the distribution of the fitness increments of the potential beneficial mutations. The mutation–selection balance leads to a continually evolving population with a steady-state fitness variation. This variation increases logarithmically with both population size and mutation rate and sets the rate at which the population accumulates beneficial mutations, which thus also grows only logarithmically with population size and mutation rate. These results imply that mutator phenotypes are less effective in larger asexual populations. They also have consequences for the advantages (or disadvantages) of sex via the Fisher–Muller effect; these are discussed briefly.

The Speed of Evolution and Maintenance of Variation in Asexual Populations

BACKGROUND The rate at which beneficial mutations accumulate determines how fast asexual populations evolve, but this is only partially understood. Some recent clonal-interference models suggest that evolution in large asexual populations is limited because smaller beneficial mutations are outcompeted by larger beneficial mutations that occur in different lineages within the same population. This analysis assumes that the important mutations fix one at a time; it ignores multiple beneficial mutations that occur in the lineage of an earlier beneficial mutation, before the first mutation in the series can fix. We focus on the effects of such multiple mutations. RESULTS Our analysis predicts that the variation in fitness maintained by a continuously evolving population increases as the logarithm of the population size and logarithm of the mutation rate and thus yields a similar logarithmic increase in the speed of evolution. To test these predictions, we evolved asexual budding yeast in glucose-limited media at a range of population sizes and mutation rates. CONCLUSIONS We find that their evolution is dominated by the accumulation of multiple mutations of moderate effect. Our results agree with our theoretical predictions and are inconsistent with the one-by-one fixation of mutants assumed by recent clonal-interference analysis.

Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination

High-throughput sequencing suggests age-related differences in the changes in the human antibody repertoire after influenza vaccination. Antibodies Act Their Age One of the main advantages of the immune system in fighting infection is its ability to diversify—antibody and T cell receptor genes physically rearrange, creating a repertoire of potential responses that can be called upon and expanded if needed. However, the very diversity of this repertoire is what makes immune responses hard to study. Although we can know how any individual B cell or antibody responds to stimulation, getting the big picture is much more difficult. Adding in another variable, such as time, further complicates things. Now, Jiang et al. use high-throughput long read sequencing to characterize the human antibody response after influenza vaccination. People of age are thought to have altered immune systems compared to younger individuals. However, why exactly how the antibody repertoire changes with age remains unclear. By analyzing more than 5 million antibody heavy chain sequences, the authors were able to compare isotype diversity, lineage structure, and mutational activity in differently aged populations. They found that the elderly have fewer lineages, with reduced diversity, compared with younger subjects; however, the antibodies present before vaccination had higher levels of mutation. These same techniques can be used to study individual-specific immune responses and may aid in optimizing vaccination in the future. The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell’s genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

The rate at which asexual populations cross fitness valleys.

Complex traits often involve interactions between different genetic loci. This can lead to sign epistasis, whereby mutations that are individually deleterious or neutral combine to confer a fitness benefit. In order to acquire the beneficial genotype, an asexual population must cross a fitness valley or plateau by first acquiring the deleterious or neutral intermediates. Here, we present a complete, intuitive theoretical description of the valley-crossing process across the full spectrum of possible parameter regimes. We calculate the rate at which a population crosses a fitness valley or plateau of arbitrary width, as a function of the mutation rates, the population size, and the fitnesses of the intermediates. We find that when intermediates are close to neutral, a large population can cross even wide fitness valleys remarkably quickly, so that valley-crossing dynamics may be common even when mutations that directly increase fitness are also possible. Thus the evolutionary dynamics of large populations can be sensitive to the structure of an extended region of the fitness landscape - the population may not take directly uphill paths in favor of paths across valleys and plateaus that lead eventually to fitter genotypes. In smaller populations, we find that below a threshold size, which depends on the width of the fitness valley and the strength of selection against intermediate genotypes, valley-crossing is much less likely and hence the evolutionary dynamics are less influenced by distant regions of the fitness landscape.

Statistics of Earthquakes in Simple Models of Heterogeneous Faults

Simple models for ruptures along a heterogeneous earthquake fault zone are studied, focussing on the interplay between the roles of disorder and dynamical effects. A class of models are found to operate naturally at a critical point whose properties yield power law scaling of earthquake statistics. Various dynamical effects can change the behavior to a distribution of small events combined with characteristic system size events. The studies employ various analytic methods as well as simulations.

Collective transport in random media: from superconductors to earthquakes

Abstract In these lectures, a variety of non-equilibrium transport phenomena are introduced that all involve, in some way, elastic manifolds being driven through random media. A simple class of models is studied focussing on the behavior near to the critical “depinning” force above which persistent motion occurs in these systems. A simple mean field theory and a “toy” model of “avalanche” processes are analyzed and used to motivate the general scaling picture found in recent renormalization group studies. The general ideas and results are then applied to various systems: sliding charge density waves, critical current behavior of vortices in superconductors, dynamics of cracks, and simple models of a geological fault. The roles of thermal fluctuations, defects, inertia, and elastic wave propagation are all discussed briefly.

Slide-and-cluster models for spindle assembly

BACKGROUND Mitotic and meiotic spindles are assemblies of microtubules (MTs) that form during cell division to physically separate sister chromosomes. How the various components of spindles act together to establish and maintain the dynamic bipolar structure of spindles is not understood. Interactions between MTs and motors have been studied both experimentally and theoretically in many contexts, including the self-organization of arrays of MTs by motors and the competition between different classes of motors to move a single load. This work demonstrates how the interplay between two types of motors together with continual nucleation of MTs by chromosomes could organize the MTs into spindles. RESULTS We propose a slide-and-cluster model based on four known molecular activities: MT nucleation near chromosomes, the sliding of MTs by a plus-end-directed motor, the clustering of their minus ends by a minus-end-directed motor, and the loss of MTs by dynamic instability. Our model applies to overlapping, nonkinetochore MTs in anastral spindles, and perhaps also to interpolar MTs in astral spindles. We show mathematically that the slide-and-cluster mechanism robustly forms bipolar spindles with sharp poles and a stable steady-state length. This model accounts for several experimental observations that were difficult to explain with existing models. Three new predictions of the model were tested and verified in Xenopus egg extracts. CONCLUSIONS We show that a simple two-motor model could create stable, bipolar spindles under a wide range of physical parameters. Our model is the first self-contained model for anastral spindle assembly and MT sliding (known as poleward flux). Our experimental results support the slide-and-cluster scenario; most significantly, we find that MT sliding slows near spindle poles, confirming the models primary prediction.

Quantitative evolutionary dynamics using high-resolution lineage tracking

Evolution of large asexual cell populations underlies ∼30% of deaths worldwide, including those caused by bacteria, fungi, parasites, and cancer. However, the dynamics underlying these evolutionary processes remain poorly understood because they involve many competing beneficial lineages, most of which never rise above extremely low frequencies in the population. To observe these normally hidden evolutionary dynamics, we constructed a sequencing-based ultra high-resolution lineage tracking system in Saccharomyces cerevisiae that allowed us to monitor the relative frequencies of ∼500,000 lineages simultaneously. In contrast to some expectations, we found that the spectrum of fitness effects of beneficial mutations is neither exponential nor monotonic. Early adaptation is a predictable consequence of this spectrum and is strikingly reproducible, but the initial small-effect mutations are soon outcompeted by rarer large-effect mutations that result in variability between replicates. These results suggest that early evolutionary dynamics may be deterministic for a period of time before stochastic effects become important.

DYNAMICS AND INSTABILITIES OF PLANAR TENSILE CRACKS IN HETEROGENEOUS MEDIA

The dynamics of tensile crack fronts restricted to advance in a plane are studied. In an ideal linear elastic medium, a propagating mode along the crack front with a velocity slightly less than the Rayleigh wave velocity, is found to exist. But the dependence of the effective fracture toughness