The N6-methyladenosine (mA) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous mA-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

Innate behavioral biases and preferences can vary significantly among individuals of the same genotype. Though individuality is a fundamental property of behavior, it is not currently understood how individual differences in brain structure and physiology produce idiosyncratic behaviors. Here we present evidence for idiosyncrasy in olfactory behavior and neural responses in We show that individual female from a highly inbred laboratory strain exhibit idiosyncratic odor preferences that persist for days. We used in vivo calcium imaging of neural responses to compare projection neuron (second-order neurons that convey odor information from the sensory periphery to the central brain) responses to the same odors across animals. We found that, while odor responses appear grossly stereotyped, upon closer inspection, many individual differences are apparent across antennal lobe (AL) glomeruli (compact microcircuits corresponding to different odor channels). Moreover, we show that neuromodulation, environmental stress in the form of altered nutrition, and activity of certain AL local interneurons affect the magnitude of interfly behavioral variability. Taken together, this work demonstrates that individual exhibit idiosyncratic olfactory preferences and idiosyncratic neural responses to odors, and that behavioral idiosyncrasies are subject to neuromodulation and regulation by neurons in the AL.

The influence of peripheral physiology on goal-directed behavior involves specialized interoceptive sensory neurons that signal internal state to the brain. Here, we review recent progress to examine the impact of these specialized cell types on neurons and circuits throughout the central nervous system. These new approaches are important for understanding how the needs of the body interact and guide goal-directed behaviors.

Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLC neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLC neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLC neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLC neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLC neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.

We present a method for microtubule tracking in electron microscopy volumes. Our method first identifies a sparse set of voxels that likely belong to microtubules. Similar to prior work, we then enumerate potential edges between these voxels, which we represent in a candidate graph. Tracks of microtubules are found by selecting nodes and edges in the candidate graph by solving a constrained optimization problem incorporating biological priors on microtubule structure. For this, we present a novel integer linear programming formulation, which results in speed-ups of three orders of magnitude and an increase of 53% in accuracy compared to prior art (evaluated on three 1 . 2 × 4 × 4µm volumes of Drosophila neural tissue). We also propose a scheme to solve the optimization problem in a block-wise fashion, which allows distributed tracking and is necessary to process very large electron microscopy volumes. Finally, we release a benchmark dataset for microtubule tracking, here used for training, testing and validation, consisting of eight 30 x 1000 x 1000 voxel blocks (1 . 2 × 4 × 4µm) of densely annotated microtubules in the CREMI data set (https://github.com/nilsec/micron).

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

Calcium imaging with fluorescent protein sensors is widely used to record activity in neuronal populations. The transform between neural activity and calcium-related fluorescence involves nonlinearities and low-pass filtering, but the effects of the transformation on analyses of neural populations are not well understood. We compared neuronal spikes and fluorescence in matched neural populations in behaving mice. We report multiple discrepancies between analyses performed on the two types of data, including changes in single-neuron selectivity and population decoding. These were only partially resolved by spike inference algorithms applied to fluorescence. To model the relation between spiking and fluorescence we simultaneously recorded spikes and fluorescence from individual neurons. Using these recordings we developed a model transforming spike trains to synthetic-imaging data. The model recapitulated the differences in analyses. Our analysis highlights challenges in relating electrophysiology and imaging data, and suggests forward modeling as an effective way to understand differences between these data.