SignificanceInteractions between the cell nucleus and cytoskeleton regulate cell mechanics and are facilitated by the interplay between the nuclear lamina and linker of nucleoskeleton and cytoskeleton (LINC) complexes. To date, the specific contribution of the four lamin isoforms to nucleocytoskeletal connectivity and whole-cell mechanics remains unknown. We discover that A- and B-type lamins distinctively interact with LINC complexes that bind F-actin and vimentin filaments to differentially modulate cortical stiffness, cytoplasmic stiffness, and contractility of mouse embryonic fibroblasts (MEFs). We propose and experimentally verify an integrated lamin-LINC complex-cytoskeleton model that explains cellular mechanical phenotypes in lamin-deficient MEFs. Our findings uncover potential mechanisms for cellular defects in human laminopathies and many cancers associated with mutations or modifications in lamin isoforms.

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, we knocked down individual NRs through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, we found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of gamma neurons plus de-novo elaboration of both alpha’/beta’ and alpha/beta neurons. Notably, unf is largely dispensable for the initial elaboration of gamma neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity.

The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α'/β' and α/β) and 7 cell subtypes (γd, γm, α'/β'ap, α'/β'm, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker and the α'/β' class marker Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the MB provides a valuable resource for the fly neuroscience community.

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S. cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.

The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates.

Wavefront distortion fundamentally limits the achievable imaging depth and quality in thick tissue. Wavefront correction can help restore the diffraction limited focus albeit with a small field of view (FOV), which limits its imaging applications. In this work, we numerically investigate whether the multi-conjugate configuration, originally developed for astronomical adaptive optics, may increase the correction FOV in random turbid media. The results show that the multi-conjugate configuration can significantly improve the correction area compared to the widely adopted pupil plane correction. Even in the simple case of single-conjugation, it still outperforms the pupil plane correction. This study provides a guideline for designing the optimal wavefront correction system in deep tissue imaging.

Neurodata Without Borders: Neurophysiology (NWB:N) is a data standard for neurophysiology, providing neuroscientists with a common standard to share, archive, use, and build common analysis tools for neurophysiology data. With NWB:N version 2.0 (NWB:N 2.0) we made significant advances towards creating a usable standard, software ecosystem, and vibrant community for standardizing neurophysiology data. In this manuscript we focus in particular on the NWB:N data standard schema and present advances towards creating an accessible data standard for neurophysiology.

In this issue of Neuron, Makino and Malinow and Kleindienst et al. present evidence of a behaviorally induced form of synaptic plasticity that would encourage the development of fine-scale structured input patterns and the binding of features within single neurons.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.