How the other half lives: CRISPR-Cas's influence on bacteriophages

CRISPR-Cas is a genetic adaptive immune system unique to prokaryotic cells used to combat phage and plasmid threats. The host cell adapts by incorporating DNA sequences from invading phages or plasmids into its CRISPR locus as spacers. These spacers are expressed as mobile surveillance RNAs that direct CRISPR-associated (Cas) proteins to protect against subsequent attack by the same phages or plasmids. The threat from mobile genetic elements inevitably shapes the CRISPR loci of archaea and bacteria, and simultaneously the CRISPR-Cas immune system drives evolution of these invaders. Here we highlight our recent work, as well as that of others, that seeks to understand phage mechanisms of CRISPR-Cas evasion and conditions for population coexistence of phages with CRISPR-protected prokaryotes.Comment: 24 pages, 8 figure

Continuous Environmental Tracking: An Engineering Framework to Understand Adaptation and Diversification

We offer a new framework for understanding biological adaptability based on interpreting the findings of 342 journal articles and 67 online reports related to adaptation, bioengineering, and design in view of the assumption that biological functions are most accurately explained by engineering principles. We hypothesize that organisms actively and continuously track environmental variables and respond by self-adjusting to changing environments—utilizing the engineering principles constraining how human-designed objects self-adjust to changes—which results in adaptation. We termed this hypothesis Continuous Environmental Tracking (CET). CET is an engineering-based, organism-focused characterization of adaptation. CET expects to find that organisms adapt via systems with elements analogous to those within human-engineered tracking systems, namely: input sensors, internal logic mechanisms to select suitable responses, and actuators to execute responses. We derived the hypothesis by reinterpreting findings and formalizing biological adaptability within a framework of engineering design, considering: (1) objectives, (2) constraints, (3) variables, and (4) the biological systems related to the previous three. The literature does identify internal mechanisms with elements analogous to engineered systems using sensors coupled to complex logic mechanisms producing highly “targeted” self-adjustments suitable to changes. Adaptive mechanisms were characterized as regulated, rapid, repeatable, and sometimes, reversible. Adaptation happened largely through regulated gene expression and not gene inheritance, per se. These observations, consistent with CET, contrast starkly with the evolutionary framework’s randomness of tiny, accidental “hit-and-miss” phenotypes fractioned out to lucky survivors of deadly challenges. Evolutionists now divide over their framework’s need of modification, and a trend among some seeks to infuse more engineering into biology. This disarray affords a rare, transient opportunity for engineering advocates to frame the issue. CET may fundamentally change how we perceive organisms; from passive modeling clay shaped over time by the vicissitudes of nature, to active, problem-solving creatures that continuously track environmental changes to better fit existing niches or fill new ones

Tracking behavioural and neuronal responses to social pheromones: Insights from a Drosophila model

If eusociality evolved through modification of pre-social mechanisms for regulating personal reproduction, then even insects like Drosophila may be vulnerable to latent effects of \u27queen\u27 pheromone. Here, I test if male fruit flies respond to a eusocial queen bee pheromone. I found that male flies were attracted to queen bee pheromone, and pheromone-treated males raised the intensity of their courting towards conspecific females. These novel observations from Drosophila suggest that male flies have the capacity to respond to queen pheromone in a manner that is comparable to the native response from male (drone) bees. I therefore optimized a nuclear factor of activated T-cell (NFAT) system to label olfactory neurons that are putatively responsive to the pro-reproductive pheromone. The NFAT reporter system implicates three neurons (Or-49b, Or-56a, Or-98a) that, if shown to function similarly in drones, will validate my use of Drosophila to probe otherwise unknown mechanisms of social bee communication

Neural organisation of innate behaviour in zebrafish larvae

Animals’ inner worlds are a hazy imitation of reality, shaped by evolution. Of the infinitude of stimuli that can arise in their natural environment, only a few will bear significance for an animal’s survival and reproductive success. Thus, neural circuits have evolved to extract only these relevant stimuli from the background and connect them to downstream effectors. Sometimes, competing representations of the outside world arise in the brain, and these must be resolved to ensure adaptive behaviour. Through the study of an animal’s behaviour, we can learn about its inner world: which stimuli it cares about; the desires these stimuli engender within it; and how its movements enact and extinguish those desires, allowing new stimuli to emerge that reorchestrate the inner world and refresh the cycle. Here, I present three studies that investigate the emergence of this world in the neural circuits of zebrafish larvae. In the first study, I mapped the behavioural sequences of zebrafish larvae as they pursued and consumed prey. Manipulating their vision with genetic mutants, virtual reality, and lesion studies revealed the dynamic features of stimuli that drive switches in the behaviour. I showed that, by chaining kinematically varied swim types into regular sequences, larvae bring prey to a binocular zone in the near visual field. Here, the fused representation of the stimulus across hemispheres releases stereotyped strike manoeuvres, tuned to the distance to the prey. In the second study, I helped investigate how visual circuits build representations of prey and predator stimuli. Measuring the responses of neurons to visual stimuli revealed how feature selectivity arises from the integration of upstream inputs. Features are unevenly represented across space, matching predicted changes in prey percepts as animals progress through their hunting sequences. When neurons tuned to specific features were ablated, I showed that the detection of prey was altered, no longer eliciting the usual hunting responses from animals. In the third study, I contributed to the discovery of a circuit in the brain that coordinates behavioural responses to competing stimuli. When confronted with multiple threats, animals either ignore one and escape from the other, or average their locations and escape in an intermediate direction. I showed that these two strategies are mediated by distinct swims types. Inhibiting specific neurons in the brain reduced directional escapes, but not intermediate ones, revealing a circuit that contributes to a bottom-up attention mechanism. Together, these three studies reveal the organisation of behaviour within neural circuits of the larval zebrafish brain. Finally, I consider the broader networks in the brain that might implement and modulate responses to salient visual stimuli, and how these circuits could serve as a substrate for behavioural evolution

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

On the adaptive advantage of always being right (even when one is not)

We propose another positive illusion – overconfidence in the generalisability of one’s theory – that fits with McKay & Dennett’s (M&D’s) criteria for adaptive misbeliefs. This illusion is pervasive in adult reasoning but we focus on its prevalence in children’s developing theories. It is a strongly held conviction arising from normal functioning of the doxastic system that confers adaptive advantage on the individual