In a recent publication, Ma et al [1] claim that a transformer-based cellular segmentation method called Mediar [2] — which won a Neurips challenge — outperforms Cellpose [3] (0.897 vs 0.543 median F1 score). Here we show that this result was obtained by artificially impairing Cellpose in multiple ways. When we removed these impairments, Cellpose outperformed Mediar (0.861 vs 0.826 median F1 score on the updated test set). To further investigate the performance of transformers for cellular segmentation, we replaced the Cellpose backbone with a transformer. The transformer-Cellpose model also did not outperform the standard Cellpose (0.848 median F1 test score). Our results suggest that transformers do not advance the state-of-the-art in cellular segmentation.

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

In most animals, a relatively small number of descending neurons (DNs) connect higher brain centers in the animal’s head to motor neurons (MNs) in the nerve cord of the animal’s body that effect movement of the limbs. To understand how brain signals generate behavior, it is critical to understand how these descending pathways are organized onto the body MNs. In the fly, Drosophila melanogaster, MNs controlling muscles in the leg, wing, and other motor systems reside in a ventral nerve cord (VNC), analogous to the mammalian spinal cord. In companion papers, we introduced a densely-reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC, Takemura et al., 2023), including cell type and developmental lineage annotation (Marin et al., 2023), which provides complete VNC connectivity at synaptic resolution. Here, we present a first look at the organization of the VNC networks connecting DNs to MNs based on this new connectome information. We proofread and curated all DNs and MNs to ensure accuracy and reliability, then systematically matched DN axon terminals and MN dendrites with light microscopy data to link their VNC morphology with their brain inputs or muscle targets. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify communities of intrinsic neurons linked to control of different motor systems, including putative ventral circuits for walking, dorsal circuits for flight steering and power generation, and intermediate circuits in the lower tectulum for coordinated action of wings and legs. Our analysis generates hypotheses for future functional experiments and, together with the MANC connectome, empowers others to investigate these and other circuits of the Drosophila ventral nerve cord in richer mechanistic detail.

In most animals, a relatively small number of descending neurons (DNs) connect higher brain centers in the animal’s head to circuits and motor neurons (MNs) in the nerve cord of the animal’s body that effect movement of the limbs. To understand how brain signals generate behavior, it is critical to understand how these descending pathways are organized onto the body MNs. In the fly, Drosophila melanogaster, MNs controlling muscles in the leg, wing, and other motor systems reside in a ventral nerve cord (VNC), analogous to the mammalian spinal cord. In companion papers, we introduced a densely-reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC, (Takemura et al., 2024)), including cell type and developmental lineage annotation (Marin et al., 2024), which provides complete VNC connectivity at synaptic resolution. Here, we present a first look at the organization of the VNC networks connecting DNs to MNs based on this new connectome information. We proofread and curated all DNs and MNs to ensure accuracy and reliability, then systematically matched DN axon terminals and MN dendrites with light microscopy data to link their VNC morphology with their brain inputs or muscle targets. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify communities of intrinsic neurons linked to control of different motor systems, including putative ventral circuits for walking, dorsal circuits for flight steering and power generation, and intermediate circuits in the lower tectulum for coordinated action of wings and legs. Our analysis generates hypotheses for future functional experiments and, together with the MANC connectome, empowers others to investigate these and other circuits of the Drosophila ventral nerve cord in richer mechanistic detail.

The generation of post-gastrulation stem cell-derived mouse embryo models (SEMs) exclusively from naive embryonic stem cells (nESCs) has underscored their ability to give rise to embryonic and extra-embryonic lineages. However, existing protocols for mouse SEMs rely on the separate induction of extra-embryonic lineages and on ectopic expression of transcription factors to induce nESC differentiation into trophectoderm (TE) or primitive endoderm (PrE). Here, we demonstrate that mouse nESCs and naive induced pluripotent stem cells (niPSCs) can be simultaneously co-induced, via signaling pathway modulation, to generate PrE and TE extra-embryonic cells that self-organize into embryonic day (E) 8.5-E8.75 transgene-free (TF) SEMs. We also devised an alternative condition (AC) naive media that in vitro stabilizes TF-SEM-competent OCT4+/NANOG+ nESC colonies that co-express antagonistic CDX2 and/or GATA6 extra-embryonic fate master regulators and self-renew while remaining poised for TE and PrE differentiation, respectively. These findings improve mouse SEM strategies and shed light on amplifying an inherent and dormant extra-embryonic plasticity of mouse naive pluripotent cells in vitro.

Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply-inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly-inverted chromosome 3R in D. simulans that serves as an effective balancer chromosome.

Translation is the fundamental biological process converting mRNA information into proteins. Single molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here we report Single molecule Imaging of NAscent PeptideS (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 AA/s). In primary neurons mRNAs are translated in proximal dendrites but repressed in distal dendrites and display “bursting” translation. This technology provides a tool to address the spatiotemporal translation mechanism of single mRNAs in living cells.

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport.