Browse Articles

Tat-dependent bundling pilus of a halophilic archaeon assembles by a strand donation mechanism and facilitates biofilm formation

Diverse extracellular filaments present on the surface of archaea mediate multiple key processes, such as motility, adhesion, and biofilm formation. Although several archaeal filament types have been characterized in considerable detail, many remain understudied, particularly those utilizing noncanonical secretion systems. Here, we describe the Tafi bundling pilus that facilitates biofilm formation in the haloarchaeon Natrinema sp. J7-2. Unlike previously characterized archaeal pili, Tafi is secreted via the twin-arginine translocation (Tat) pathway, which transports fully folded proteins across the cytoplasmic membrane. Structural analysis reveals that although Tafi pili assemble via a canonical strand-donation mechanism, the pilin subunit (TafE) adopts a distinct structural topology that sets it apart from the previously characterized Sec-dependent pilins that form bundling pili in archaea. Sequence analyses show that TafE homologs are also present in thermophilic archaea from different phyla, but Tat-signal sequences are exclusive to pilins of halophilic archaea. Nevertheless, we find that Tat signal peptides in haloarchaeal TafE-like pili were exchanged back to the Sec signal peptides on multiple independent occasions. These findings expand our understanding of the diversity and evolution of archaeal extracellular filaments and highlight the Tat pathway as a route for pilus assembly in halophilic archaea.

Whole-genome duplication increases genetic diversity and load in outcrossing <i>Arabidopsis arenosa</i>

Genetic variation underpins evolutionary change, but mutation accumulation increases genetic load. Various factors affect the extent of load, such as population size and breeding system, but other important determinants remain unexplored. In particular, whole-genome duplication (WGD)—a pervasive macromutation occurring broadly across Eukaryotes—remains poorly understood in terms of its impact on neutral and selective processes within populations. Using iterative forward simulations and empirical analysis of 632 short- and 16 long-read sequenced individuals of Arabidopsis arenosa (in 23 diploid and 42 natural autotetraploid populations), we measure the effects of WGD on genome-wide diversity and mutation load. Our simulations show how genetic variation gradually rises in autotetraploids due to increased mutational target size. Moreover, mutation load increases due to relaxed purifying selection as ploidies rise, when deleterious mutations are masked by additional chromosome copies. Empirical data confirm these patterns, showing significant increases in nucleotide diversity, ratios of nonsynonymous to synonymous SNPs, and numbers of indels and large structural variants in A. arenosa autotetraploids. However, a rather modest increase in load proxies together with a broad distribution and niche of autotetraploids suggests load accumulation has not yet limited their successful expansion. Overall, we demonstrate a complex interplay between neutral processes and purifying selection in shaping genetic variation following WGD and highlight ploidy as an important determinant of mutation load, genetic diversity, and therefore adaptive potential in natural populations.

PyReconstruct: A fully open-source, collaborative successor to Reconstruct

As the serial section community transitions to volume electron microscopy, tools are needed to balance rapid segmentation efforts with documenting the fine detail of structures that support cell function. New annotation applications should be accessible to users and meet the needs of the neuroscience and connectomics communities while also being useful across other disciplines. Issues not currently addressed by a single, modern annotation application include 1) built-in curation systems with utilities for expert intervention to provide quality assurance, 2) integrated alignment features that allow for image registration on-the-fly as image flaws are found during annotation, 3) simplicity for nonspecialists within and beyond the neuroscience community, 4) a system to store experimental metadata with annotation data in a way that researchers remain masked regarding condition to avoid potential biases, 5) local management of large datasets appropriate for circuit-level analyses, and 6) fully open-source codebase allowing development of new tools, and more. Here, we present PyReconstruct, a modern successor to the Reconstruct annotation tool. PyReconstruct operates in a field-agnostic manner, runs on all major operating systems, breaks through legacy RAM limitations, features an intuitive and collaborative curation system, and employs a flexible and dynamic approach to image registration. It can be used to analyze, display, and publish experimental or connectomics data. PyReconstruct is suited for generating ground truth to implement in automated segmentation, outcomes of which can be returned to PyReconstruct for proofreading and quality control.

Surface delivery quantification reveals distinct trafficking efficiencies among clustered protocadherin isoforms

Proteins that transmit molecules and signals across the plasma membrane are crucial in cell biology because they enable cells to sense and respond to their surroundings. A major challenge for studying cell surface proteins is that often they do not fold or traffic properly to the plasma membrane when produced in heterologous cells. We developed a strategy for quantifying surface localization from fluorescence microscopy images of surface-stained cells. Using clustered protocadherins, a protein family important for cell–cell recognition during neuronal development, we found that surface delivery levels vary among clustered protocadherin isoforms and between wild-type and engineered variants. Quantifying these differences provides evidence that cis dimerization is not tightly coupled to surface delivery for clustered protocadherins. This work establishes a generalizable framework for screening proteins and variants of interest for proper cell surface localization.

Hybridization of molecules via a common photonic mode

Atoms and molecules usually hybridize and form bonds when they come in very close proximity of each other. In this work, we show that molecules can hybridize even through far-field electromagnetic interactions mediated by the shared mode of an optical microcavity. We discuss a collective enhancement of the vacuum Rabi splitting and study super- and subradiant states that arise from the cavity-mediated coupling both in the resonant and dispersive regimes. Moreover, we demonstrate a two-photon transition that emerges between the ground and excited states of the new optical compound. Our experimental data are in excellent agreement with the predictions of the Tavis–Cummings Hamiltonian and open the door to the realization of hybrid light–matter materials.

QnAs with Blake C. Meyers

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Nonsubstrate PI(4,5)P ₂ interacts with the interdomain linker to control electrochemical coupling in voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatase (VSP) comprises a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), achieving a unique electrochemical signal conversion. Previous studies suggest that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), a membrane phospholipid known to be critical for activities of diverse voltage-gated ion channels, associates with a linker connecting the VSD with the CCR of VSP and regulates VSD-CCR coupling. However, the details of PI(4,5)P 2 interaction with the linker of VSP remain elusive. Here, we exploit advantage of sensitivity of a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), to changes in local environment to study interaction between PI(4,5)P 2 and the linker of Ciona intestinalis VSP (Ci-VSP). We found that a conserved tyrosine residue (Y255) as well as neighboring basic residues interacts with PI(4,5)P 2 and this interaction was maintained in G365A Ci-VSP mutant which lacks the substrate PI(4,5)P 2 at the active site and Ci-VSP/human phosphatase and tensin homolog (PTEN) chimera which does not dephosphorylate PI(4,5)P 2 , indicating that the linker interacts with nonsubstrate, regulatory PI(4,5)P 2 outside the active site. Molecular dynamics simulations demonstrated that the linker formed stable interaction with PI(4,5)P 2 in the activated state. These findings indicate that regulation of coupling to an effector region downstream of the VSD through PI(4,5)P 2 binding to the linker is shared among voltage-dependent membrane proteins.

Structural basis for the evolution of a domesticated group II intron–like reverse transcriptase to function in host cell DNA repair

A previous study found that a bacterial group II intron–like reverse transcriptase (G2L4 RT) evolved to function in double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) and that a mobile group II intron-encoded RT has a basal DSBR activity that uses conserved structural features of non-long terminal repeat (non-LTR)-retroelement RTs. Here, we determined G2L4 RT apoenzyme and snap-back DNA synthesis structures revealing unique structural adaptations that optimized its cellular function in DSBR. These included an RT3a structure that stabilizes the apoenzyme in an inactive conformation until encountering a DNA substrate; a longer N-terminal extension/RT0-loop with conserved residues that together with a modified active site favors strand annealing; and a conserved dimer interface that localizes G2L4 RT homodimers to DSBR sites with both monomers positioned for MMEJ. Our findings reveal how an RT can function in DNA repair and suggest ways of optimizing related RTs for genome engineering applications.

Exact linear theory of perturbation response in a space- and feature-dependent cortical circuit model

What are the principles that govern the responses of cortical networks to their inputs and the emergence of these responses from recurrent connectivity? Recent experiments have probed these questions by measuring cortical responses to two-photon optogenetic perturbations of single cells in the mouse primary visual cortex. A robust theoretical framework is needed to determine the implications of these responses for cortical recurrence. Here, we propose a formulation of the dependence of cell-type-specific connectivity on spatial distance that yields an exact analytic solution for the linear perturbation response of a model with multiple cell types and space- and feature-dependent connectivity. Importantly and unlike previous approaches, the solution is valid in regimes of strong as well as weak intracortical coupling. Analysis reveals the structure of connectivity implied by various features of single-cell perturbation responses, such as the surprisingly narrow spatial radius of nearby excitation beyond which inhibition dominates, the number of transitions between mean excitation and inhibition thereafter, and the dependence of these responses on feature preferences. Comparison of these results to existing optogenetic perturbation data yields constraints on cell-type-specific connection strengths and their tuning dependence. Finally, we provide experimental predictions regarding the response of inhibitory neurons to single-cell perturbations and the modulation of perturbation response by neuronal gain.

Mobile gene clusters and coexpressed plant–rhizobium pathways drive partner quality variation in symbiosis

Plant–microbe symbioses such as the legume–rhizobium mutualism are vital in the web of ecological relationships within both natural and managed ecosystems, influencing primary productivity, crop yield, and ecosystem services. The outcome of these interactions for plant hosts varies quantitatively and can range from highly beneficial to even detrimental depending on natural genetic variation in microbial symbionts. Here, we take a systems genetics approach, harnessing the genetic diversity present in wild rhizobial populations to predict genes and molecular pathways crucial in determining partner quality, i.e., the benefits of symbiosis for legume hosts. We combine traits, dual-RNAseq of both partners from active nodules, pangenomics/pantranscriptomics, and Weighted Gene Co-expression Network Analysis (WGCNA) for a panel of 20 Sinorhizobium meliloti strains that vary in symbiotic partner quality. We find that genetic variation in the nodule transcriptome predicts host plant biomass, and WGCNA reveals networks of genes in plants and rhizobia that are coexpressed and associated with high-quality symbiosis. Presence–absence variation of gene clusters on the symbiosis plasmid (pSymA), validated in planta, is associated with high or low-quality symbiosis and is found within important coexpression modules. Functionally our results point to management of oxidative stress, amino acid and carbohydrate transport, and NCR peptide signaling mechanisms in driving symbiotic outcomes. Our integrative approach highlights the complex genetic architecture of microbial partner quality and raises hypotheses about the genetic mechanisms and evolutionary dynamics of symbiosis.

AI–AI bias: Large language models favor communications generated by large language models

Are large language models (LLMs) biased in favor of communications produced by LLMs, leading to possible antihuman discrimination? Using a classical experimental design inspired by employment discrimination studies, we tested widely used LLMs, including GPT-3.5, GPT-4 and a selection of recent open-weight models in binary choice scenarios. These involved LLM-based assistants selecting between goods (the goods we study include consumer products, academic papers, and film-viewings) described either by humans or LLMs. Our results show a consistent tendency for LLM-based AIs to prefer LLM-presented options. This suggests the possibility of future AI systems implicitly discriminating against humans as a class, giving AI agents and AI-assisted humans an unfair advantage.

Coupling of excitation energy to photochemistry in natural marine phytoplankton communities under iron stress

Oxygenic photosynthesis requires excitation energy transfer from light-harvesting complexes (LHCs) to reaction centers (RCs) to drive photochemical redox chemistry. The effective absorption cross section of RCs dynamically responds to the light environment on time scales of seconds to days, allowing rapid acclimations to changes in spectral irradiance and photoprotection under high light, thereby optimizing light absorption for photochemistry. Although energy coupling between LHC–RCs has been studied for decades in laboratory cultures, it remains poorly understood in real-world conditions, where it is potentially influenced by nutrients. In the oceans, one of the most critical micronutrients for photosynthesis is iron (Fe). To investigate the effects of Fe stress on the energetic coupling between LHC–RCs in natural phytoplankton assemblages in the Southern Atlantic Ocean, we assessed photophysiological responses using a pair of custom-built fluorometers measuring chlorophyll-a variable fluorescence and picosecond fluorescence lifetimes. Detailed analysis based on the functional absorption cross section of the oxygen-evolving complex, quantum yield of photochemistry, energetic connectivity of RCs, and the average lifetime of in vivo chlorophyll fluorescence suggested that between 10 and 25% of LHCs remain uncoupled from RCs and do not effectively contribute to photochemical charge separation. Addition of Fe to samples under trace metal-clean on-board incubations indicates relatively rapid recoupling (&lt; 24 h) of antennae to photochemistry, followed by biophysical stabilization of recoupled complexes. Our findings highlight the crucial role of micronutrients in controlling the excitation energy transfer from LHCs to RCs in marine phytoplankton and the overall primary productivity in the real-world oceans.

Synthesis of large single-transcript pathways from oligonucleotide pools: Design of STARBURST, an autobioluminescent reporter

Methods for fast and inexpensive gene synthesis from oligonucleotide pools enable rapid iteration of genetic designs. Here, we describe iggypop (indexed Golden Gate gene assembly from PCR-amplified oligonucleotide pools), a simple computational–experimental pipeline that allows for low-cost design and synthesis of hundreds of genes from oligonucleotide pools using Golden Gate assembly methods. We used iggypop to synthesize a series of single-transcript autonomously bioluminescent reporters (STARBURSTs) that link the five genes of a fungal bioluminescence pathway via ribosomal skipping LP4/2A sequences into a 9.5 kb transcript that function in planta. We also synthesized RUBY reporters (a reporter gene system producing red betalain pigment) recoded to match dicot codon usage, as RUBY was codon optimized for rice codon usage and has a high GC content. Surprisingly, the recoded RUBYs substantially reduced betalain production in transient Nicotiana benthamiana assays. Based on this observation, we synthesized six GC-boosted STARBURSTs, which produced robust luminescence in both transient assays and transgenic Arabidopsis plants. Thus, iggypop enabled the rapid synthesis of multiple genetic designs to deliver a bright single transcript autobioluminescent reporter. Iggypop should enable the facile synthesis and optimization of new genetic parts and complex polycistronic pathways.

<i>Prg4</i> + fibroadipogenic progenitors in muscle are crucial for bone fracture repair

Clinically, compromised fracture healing often occurs at sites with less muscle coverage and muscle flaps can provide the necessary healing environment for appropriate healing in severe bone loss. However, the underlying mechanisms are largely unknown. Here, we established a mouse reporter model for studying muscle cell contribution to bone fracture repair. Analyzing skeletal muscle scRNA-seq datasets revealed that Prg4 marks a fibroadipogenic progenitor (FAP) subpopulation. In mice, Prg4 + cells were specifically located in the skeletal muscle, but not at the periosteum or inside cortical bone. These cells expressed FAP markers, responded to muscle injury, and became periosteal cells under normal and muscle injury conditions. Fracture fragmented muscle fibers, rapidly expanded Prg4 + FAPs at the injury site and promoted their migration into the fracture gap. Later, they gave rise to many chondrocytes, osteoblasts, and osteocytes in the outer periphery of callus next to muscle. In repaired bones, the descendants of Prg4 + FAPs were detected as mesenchymal progenitors in the periosteum and osteocytes at the prior fracture site. A second fracture activated those cells and stimulated them to become osteoblasts in the inner part of callus. Importantly, ablation of Prg4 + FAPs impaired fracture healing and functional repair. In an intramembranous bone injury model (drill-hole), Prg4 + FAPs became periosteal cells, but their contribution to bone defect repair was significantly less than in fractures. Taken together, we demonstrate the critical role of FAPs in endochondral bone repair and uncover a mechanism by which mesenchymal progenitors transform from muscle to cortical bone.

Integrating spatial omics and single-cell mass spectrometry imaging reveals tumor–host metabolic interplay in hepatocellular carcinoma

Metabolic crosstalk among diverse cellular populations contributes to shaping a competitive and symbiotic tumor microenvironment (TME) to influence cancer progression and immune responses, highlighting vulnerabilities that can be exploited for cancer therapy. Using a spatial multiomics platform to study the cell-specific metabolic spectrum in hepatocellular carcinoma (HCC), we map the metabolic interactions between different cells in the HCC TME and identify a unique tumor-immune-cancer-associated fibroblast (CAF) “interface” zone, where cell–cell interactions are enhanced and accompanied by significant upregulation of lactic acid and long-chain polyunsaturated fatty acids. Further combining single-cell mass spectrometry imaging of patient-derived tumor organoids, cocultured CAFs, and macrophages, we demonstrate that CAFs increase glycolysis and secrete lactic acid to the surrounding microenvironment to drive immunosuppressive macrophage M2 polarization. These findings facilitate the understanding of cancer-associated metabolic interactions in complex TME and provide clues for targeted clinical therapies.

How RAG1/2 evolved from ancestral transposases to initiate V(D)J recombination without transposition

The recombination activating genes 1 and 2 (RAG1/2) recombinase, which initiates V(D)J recombination in jawed vertebrates, evolved from RNaseH-like transposases such as Transib and ProtoRAG. However, its postcleavage transposase activity is strictly suppressed. Previous structural studies have focused only on the conserved core domains of RAG1/2, leaving the regulatory mechanisms of the noncore regions unclear. To investigate how RAG1/2 suppresses transposition and regulates DNA cleavage, we determined cryo-electron microscopy (cryo-EM) structures of nearly full-length RAG1/2 complexed with cleaved recombination signal sequences (RSS) in a signal-end complex (SEC) at resolutions up to 2.95 Å. Two key structures, SEC-0 and SEC-Plant Homeodomain (PHD), reveal distinct regulatory roles of RAG2, which is absent in Transib transposase. SEC-0 displays a closed conformation, revealing that the core RAG2 facilitates sequential DNA cleavage by stabilizing the RSS-cleaved states in a “spring-loaded” mechanism. SEC-PHD reveals how RAG2’s noncore PHD and Acidic Hinge (AH), which are absent in ProtoRAG, inhibit target DNA binding in transposition. Histone H3K4me3, which recruits RAG1/2 to RSS sites, does not influence RAG1/2 binding to V, D, or J gene segments bordered by RSS. In contrast, the suppressed transposition can be activated by H3K4me3 peptides that dislodge the inhibitory PHD. To achieve this derepression in vivo, however, would require an unlikely close placement of two nucleosomes flanking a target DNA bent by nearly 180°. Our structural and biochemical results elucidate how RAG1 has acquired RAG2 and utilizes its core and noncore domains to enhance V(D)J recombination and suppress transposition.

Cryo-EM structure and polar assembly of the PS2 S-layer of <i>Corynebacterium glutamicum</i>

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales , this mycomembrane is further supported by a proteinaceous surface layer or “S-layer,” whose function, structure, and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryo-EM reconstruction. PS2 monomers consist of a six-helix bundle “core,” a three-helix bundle “arm,” and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

A granulin-positive macrophage subtype in mycobacterial granulomas alleviates tissue damage by limiting excessive inflammation

Granulomas play a crucial role in the pathology of tuberculosis, but the immune environment governing their formation remains largely unknown. To explore the dynamic changes in the immune microenvironment during the formation of tuberculous granulomas, we infected adult zebrafish with Mycobacterium marinum and then examined uninfected and infected kidneys, as well as large and small granulomas in the kidneys. Using single-cell RNA sequencing technology, we identified two major macrophage subpopulations in the hematopoietic tissue (kidney) of zebrafish under uninfected physiological conditions: monocyte derived and tissue-resident macrophages. Interestingly, the infection induced the emergence of epithelioid cells and a previously undescribed grna.2 + macrophage subpopulation. Depletion of grna.2 + macrophages with the nitroreductase-metronidazole ablation system resulted in shortened zebrafish survival after infection, increased bacterial load, and more granulomas, especially necrotic granulomas. Depletion of grna.2 + macrophages also produced a denser granuloma structure with fewer T cells. RNA-seq and flow cytometry analysis revealed that depletion of grna.2 + macrophages led to upregulated inflammatory signaling pathways, including tnfα and il1β , and increased macrophage lytic cell death. Similarly, in samples from tuberculosis patients, we also identified GRN-positive macrophages, which exhibit similar anti-inflammatory functions. This subset of grna.2 + macrophages present in developing granulomas can suppress excessive inflammatory responses to alleviate macrophage lytic death, reduce tissue damage, promote T cell infiltration and ultimately help control mycobacterial growth in vivo.