Pairwise sequence covariations are a signal of conserved RNA secondary structure. We describe a method for distinguishing when lack of covariation signal can be taken as evidence against a conserved RNA structure, as opposed to when a sequence alignment merely has insufficient variation to detect covariations. We find that alignments for several long noncoding RNAs previously shown to lack covariation support do have adequate covariation detection power, providing additional evidence against their proposed conserved structures.

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

Many biological applications require the segmentation of cell bodies, membranes and nuclei from microscopy images. Deep learning has enabled great progress on this problem, but current methods are specialized for images that have large training datasets. Here we introduce a generalist, deep learning-based segmentation algorithm called Cellpose, which can very precisely segment a wide range of image types out-of-the-box and does not require model retraining or parameter adjustments. We trained Cellpose on a new dataset of highly-varied images of cells, containing over 70,000 segmented objects. To support community contributions to the training data, we developed software for manual labelling and for curation of the automated results, with optional direct upload to our data repository. Periodically retraining the model on the community-contributed data will ensure that Cellpose improves constantly.

Many animals rely on acoustic cues to decide what action to take next. Unraveling the wiring patterns of the auditory neural pathways is prerequisite for understanding such information processing. Here we reconstructed the first step of the auditory neural pathway in the fruit fly brain, from primary to secondary auditory neurons, at the resolution of transmission electron microscopy. By tracing axons of two major subgroups of auditory sensory neurons in fruit flies, low-frequency tuned Johnston's organ (JO)-B neurons and high-frequency tuned JO-A neurons, we observed extensive connections from JO-B neurons to the main second-order neurons in both the song-relay and escape pathways. In contrast, JO-A neurons connected strongly to a neuron in the escape pathway. Our findings suggest that heterogeneous JO neuronal populations could be recruited to modify escape behavior whereas only specific JO neurons contribute to courtship behavior. We also found that all JO neurons have postsynaptic sites at their axons. Presynaptic modulation at the output sites of JO neurons could affect information processing of the auditory neural pathway in flies. This article is protected by copyright. All rights reserved.

While cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant embryonic divisions and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, cytokinesis is implemented in a specialized way during epithelial polarization and Aurora B has a new role in the formation of the apical surface.

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.

The Mushroom Body (MB) is the primary location of stored associative memories in the Drosophila brain. We discuss recent advances in understanding the MB's neuronal circuits made using advanced light microscopic methods and cell-type-specific genetic tools. We also review how the compartmentalized nature of the MB's organization allows this brain area to form and store memories with widely different dynamics.

This chapter describes many of the technologies, which have the potential to provide new insights into fundamental aspects of liver biology. Imaging live liver tissue in an animal with multiphoton microscopy coupled with photoactivatable fluorescent proteins and/or additional fluorescent proteins could be used to follow the lineage and fates of individual transplanted stem cells or developing transgenic cells in liver. Proteins or other molecules are labeled with a dye that can be excited with light source. Cells and proteins are generally too small to detect with the naked eye, relatively transparent when imaged by light microscopy, and are highly dynamic. With the increased signal to noise, isotropic and volumetric imaging and high speeds lattice light sheet allows for 3D super‐resolution microscopy, as well. Photomultiplier tubes, while capable of detecting and counting single photons, are less useful for high‐speed imaging because they normally only detect a single pixel at a time.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

The motor cortex controls skilled arm movement by sending temporal patterns of activity to lower motor centres. Local cortical dynamics are thought to shape these patterns throughout movement execution. External inputs have been implicated in setting the initial state of the motor cortex, but they may also have a pattern-generating role. Here we dissect the contribution of local dynamics and inputs to cortical pattern generation during a prehension task in mice. Perturbing cortex to an aberrant state prevented movement initiation, but after the perturbation was released, cortex either bypassed the normal initial state and immediately generated the pattern that controls reaching or failed to generate this pattern. The difference in these two outcomes was probably a result of external inputs. We directly investigated the role of inputs by inactivating the thalamus; this perturbed cortical activity and disrupted limb kinematics at any stage of the movement. Activation of thalamocortical axon terminals at different frequencies disrupted cortical activity and arm movement in a graded manner. Simultaneous recordings revealed that both thalamic activity and the current state of cortex predicted changes in cortical activity. Thus, the pattern generator for dexterous arm movement is distributed across multiple, strongly interacting brain regions.