How closely related neuronal subtypes integrate into different circuits remains poorly understood. Deep and superficial hippocampal CA3 pyramidal neurons (PNs) differ in intrinsic firing and mossy fiber (MF) input at specialized postsynaptic structures termed thorny excrescences (TEs). Combining developmental single-nucleus multiomics with genetic targeting, morphological analysis and electrophysiology, we identify the transcription factor ST18 as a regulator of deep CA3 subtype identity. ST18 marks sparsely thorny and burst-firing neurons, and gene regulatory analyses predict that it represses a superficial-associated program involved in dendritic spine development, synaptic organization, and electrophysiological properties. Postnatal ST18 loss shifts the transcriptional and chromatin landscape of deep CA3 PNs toward a superficial-like state. These molecular changes are accompanied by increased TE elaboration approaching superficial levels, enhanced excitatory synaptic activity, and reduced burst-firing. Together, these findings identify ST18-dependent restriction of an alternative subtype program as a mechanism linking molecular identity to distinct input connectivity patterns and intrinsic firing properties during postnatal circuit maturation.

Vimentin intermediate filaments (VIFs) undergo active microtubule-dependent transport throughout the cell, with trajectories resembling those of motor-driven organelles, yet our recent work showed that only a small subset of VIFs lies close to microtubules. How VIFs engage the microtubule-based transport machinery, and whether additional transport routes contribute, remain unresolved. We hypothesized that VIFs move indirectly, by hitchhiking on motile organelles, and tested this using mitochondria. Live imaging showed mitochondria and adjacent VIFs moving together, and volume electron microscopy revealed VIFs closely following mitochondrial surfaces at sites distinct from microtubule contacts. Depleting the mitochondrial motor adaptor TRAK1 to inhibit anterograde mitochondrial transport shifted both mitochondria and VIFs toward the nucleus. Driving mitochondria toward the cell periphery with a tailless kinesin-1 in cells lacking endogenous kinesin-1, or toward the cell center with a minus-end-directed motor, redistributed a subset of VIFs in the same direction. Thus, VIF transport to a large extend is accomplished by hitchhiking on motile mitochondria.

Power-law scaling in coarse-grained data suggests critical dynamics, but the true source of this scaling often remains unclear. Here, we analyze neural activity recorded during spatial navigation, reproducing power-law scaling under a phenomenological renormalization group (PRG) procedure that clusters units by activity similarity. Such scaling was previously linked to criticality. Here, we show that the iterative nature of the procedure itself leads to the emergence of power laws when applied to heterogeneous, non-interacting units obeying spatially structured activity without requiring critical interactions. Furthermore, the scaling exponents produced by heteregeneous non-interacting units match the observed exponents in recorded neural data. A simplified version of the PRG further reveals how heterogeneity smooths transitions across scales, mimicking critical behavior. The resulting exponents depend systematically on system and population size, predictions confirmed by subsampling the data.

Mitochondria are integral to the metabolism and cell biology of a neuron. Electron microscopy images of fly brain volumes, taken for connectomics, can be analyzed for mitochondria as well as the cells and synapses already reported. Here, from the Drosophila Hemibrain connectome dataset, we extract, classify, and measure approximately 6 million mitochondria, with the majority located among more than 20 thousand neurons and over 5500 cell types. Each mitochondrion is annotated with its location, orientation, voxel size, and appearance (dark and dense, light and sparse, or medium), and each synapse is linked to its closest mitochondrion. Using these data, we show how the most basic characteristics of mitochondria—volume, distance from synapses, and appearance—vary considerably between cell types and between brain regions. Mitochondria are larger and closer at presynapses than at postsynapses, and presynapses typically have a mitochondrion within one micron. However, cells important for learning and memory, Kenyon cells, have unusually small and sparse mitochondria that are placed with unusual precision near only half of presynapses. We find that mitochondria occupy a greater fraction of cell volume in inhibitory neurons than excitatory cells, dopaminergic neurons have a distinct synaptic positioning of mitochondria, and that glutamatergic neurons have a high fraction of mitochondria with a light appearance. We also find that synapses with more postsynaptic partners have larger presynaptic mitochondria and more distant postsynaptic mitochondria. Finally, we have extended the data model of our public web interface, neuPrint, to record our mitochondria data as searchable subcellular components of neurons in a connectomic database. These results indicate wide-ranging principles for how mitochondrial organization varies by cell type in a dataset of unprecedented size and coverage.

Many regulatory mechanisms function by changing a molecule’s conformation such that it is switched between active and inactive states. Engineering macromolecules such as proteins to show robust conformational changes in response to arbitrary, well-defined molecular stimuli is of great interest in fields such as synthetic biology and nanoscale engineering, because it allows the development of “smart” molecular tools. We present a strategy that can convert “simple” affinity binders such as nanobodies into protein switches that undergo clear structural rearrangements in response to their innate targets, by introducing a peptide that competes for the binding site. We demonstrate proof-of-concept implementations of this “NanoBlock” design by developing genetically encoded fluorescent biosensors based on two entirely different binder frameworks, the ALFA nanobody and the PDZ domain of Erbin. We show that our design allows the construction of well-performing protein switches that can reliably detect their targets in vitro, in mammalian cells, as part of fluorescence-activated cell sorting (FACS) experiments, and that also generalizes to the creation of stimulus-responsive enzymes via the integration of a bioluminescent readout. We furthermore develop a theoretical model that rationalizes the observations and provides insight into the underlying molecular mechanisms. Overall, our strategy expands the possibilities to create “smart” protein-based molecules and effectors.

This protocol describes a method for endogenous protein tagging in postmitotic primary neurons using homology-independent targeted integration (HITI), a CRISPR/Cas9-based approach that exploits non-homologous end joining (NHEJ). Unlike homology-directed repair (HDR), HITI does not require cell division, making it uniquely suited for postmitotic neurons. SpCas9 is delivered by a replication-incompetent simian immunodeficiency lentiviral vector (pLenti SIV), while the sgRNA, donor tag cassette, and a fluorescent plasma membrane marker are co-packaged in a single AAV vector. The two vectors are co-delivered at DIV5, and endogenous tagging is assessed at DIV15-23. The workflow is illustrated using Junctophilin-3 (JPH3) tagged with mEmerald at its endogenous locus. Airyscan microscopy imaging of endogenously tagged JPH3 at native expression levels revealed its localization at endoplasmic reticulum-plasma membrane (ER-PM) contact sites and uncovered a periodic distribution of ER-PM junctions along the dendritic arbor, enabling identification of a previously unrecognized ladder-like organization of the ER in neuronal dendrites. Genomic integration of the tag is validated at both 5' and 3' junctions by next-generation sequencing. The protocol can be adapted to other neuronal proteins by redesigning the guide RNA and donor sequence, and supports fluorescent proteins as well as epitope and self-labeling tags. The plasma membrane marker can also be replaced with other fluorescent reporters or markers for intracellular organelles to assess infection efficiency and verify subcellular localization of the protein of interest. The full workflow takes 5-6 weeks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Protocols included in this article: Basic Protocol 1: sgRNA design and HITI donor vector cloning Basic Protocol 2: Primary rat neuron culture and viral co-transduction Basic Protocol 3: Live-cell imaging of endogenously tagged proteins Support Protocol 1: Production of lentiviral and AAV particles Support Protocol 2: Validation of knock-in by next-generation sequencing (NGS).

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remain unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases nuclear membrane rupture during confinement. Together, these data argue that septins act as a dynamic mechanosensitive protective network to buffer mechanical forces on the nucleus.

Expansion Microscopy (ExM) enables nanoscale imaging of biological samples while preserving structural and molecular details. The Robust Non-rigid Registration Challenge for ExM (RnR-ExM) was launched to benchmark 3D registration algorithms on real-world ExM datasets. The challenge released 24 pairs of high-resolution 3D volumes from zebrafish, mouse, and C. elegans, deliberately selected for their difficulty with standard alignment methods. To evaluate performance, we provided curated segmentation masks of nuclei and blood vessels as landmarks for half of the dataset. To encourage learning-based approaches, we released code for generating synthetic training data reflecting the deformation and appearance of the unlabeled pairs. By submission, 168 participants registered, with six teams submitting results by the deadline; the top three were invited to present at ISBI 2023. Accurate registration of ExM volumes is essential for downstream biological analyses such as multiplexed molecular mapping and cell-type-specific structural profiling. In this paper, we detail the methods and results of these top teams, highlighting current capabilities and future directions for 3D registration in ExM. All datasets and code are available at https://rnr-exm.grand-challenge.org/

Metabolic state is tightly linked to cell fate, and expression of the mitochondrial pyruvate carrier (MPC), which imports pyruvate into mitochondria, is a robust marker of it. The transcriptional regulators that set metabolic state, however, remain poorly defined. In the liver, β-catenin is one of the determinants of metabolic zonation, and activating CTNNB1 mutations are frequent in hepatocellular carcinoma. Here we show that β-catenin regulates metabolic state in liver cancer cells. An unbiased CRISPR screen that uses endogenous MPC1transcript abundance as the marker identifies CTNNB1. Activating β-catenin pharmacologically or genetically reduces MPC1 and MPC2 transcript and protein levels, and MPC expression is also low in β-catenin-driven liver tumors in vivo. Beyond the MPC, β-catenin activation impairs assembly of respiratory Complexes III and IV, reduces mitochondrial DNA content and respiratory capacity, elongates mitochondrial networks and remodels the lipidome. Stable isotope tracing shows reduced entry of glucose- and lactate-derived carbon into the TCA cycle alongside increased oxidation of palmitate and oleate. These findings identify β-catenin as a regulator of metabolic state that drives a fuel switch from carbohydrate to fatty acid oxidation in liver cancer cells.

The coagulation cascade generates a three-dimensional fibrin gel that seals wounds and prevents the loss of blood. Extensive research has defined the structural and mechanical characteristics of fibrin gels and the various biochemical, physiological, and pathological factors that alter those properties. However, there has been no direct visualization of fibrin polymerization with the spatial and temporal resolution to characterize the fibrin branching process. Thus, a mechanistic explanation for the origins of fibrin structures remains unresolved, with several hypothesized models for how fibrin branching occurs. Using SimView light-sheet microscopy, we captured videos of fibrin fibers forming junctions and gelling. Results: We observed four distinct pathways for fibrin fibers to interact and bind, all predicated on the observation that tumbling and diffusing fibers collide and stick together. Branching occurred due to the binding of mature fibers, not through the splitting of fibers or through the lateral association of small fibrin oligomers (protofibrils), as had been previously hypothesized. Diffusion simulations of rod-like fibers confirmed the branching mechanisms, the relative frequency of the various binding pathways, and the distribution of fiber angles after binding. Scanning Electron Microscopy (SEM) imaging also corroborated the relative frequency of various types of fibrin junctions. Moreover, SEM enabled the visualization of junctions at different timepoints, demonstrating that additional fibrin strands adhere to the initial binding site, strengthening the interaction. These results establish a mechanistic framework for fibrin branching and gel formation and define an innovative experimental approach for future interrogations of fibrin polymerization.