Despite having been identified as the organism that causes tuberculosis in 1882, Mycobacterium tuberculosis has managed to still evade our understanding of the protective immune response against it, defying the development of an effective vaccine. Technology and novel experimental models have revealed much new knowledge, particularly with respect to the heterogeneity of the bacillus and the host response. This review focuses on certain immunological elements that have recently yielded exciting data and highlights the importance of taking a holistic approach to understanding the interaction of M. tuberculosis with the many host cells that contribute to the development of protective immunity.

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid β-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

We report genome-wide data from 33 Ashkenazi Jews (AJ), dated to the 14th century, obtained following a salvage excavation at the medieval Jewish cemetery of Erfurt, Germany. The Erfurt individuals are genetically similar to modern AJ, but they show more variability in Eastern European-related ancestry than modern AJ. A third of the Erfurt individuals carried a mitochondrial lineage common in modern AJ and eight carried pathogenic variants known to affect AJ today. These observations, together with high levels of runs of homozygosity, suggest that the Erfurt community had already experienced the major reduction in size that affected modern AJ. The Erfurt bottleneck was more severe, implying substructure in medieval AJ. Overall, our results suggest that the AJ founder event and the acquisition of the main sources of ancestry pre-dated the 14th century and highlight late medieval genetic heterogeneity no longer present in modern AJ.

Highly potent adult stem cells fuel lifelong tissue homeostasis and regeneration in many aquatic invertebrates, yet their developmental backstories remain obscure. In this issue of Cell, Kimura and colleagues reveal the cellular origin of adult pluripotent stem cells and propose a molecular trajectory for their specification during acoel embryogenesis.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

Chaperones are important for protein folding, but visualizing this process has proven to be exceptionally difficult. In this issue of Cell, Frydman and colleagues have succeeded in watching tubulin being folded by its chaperonin TRiC at near-atomic resolution.

Wound healing in adult mammalian tissues generally involves scarring instead of tissue regeneration. A study in this issue of Cell reveals that after injury, reindeer antler skin regenerates by priming regenerative genes in wound fibroblasts instead of forming a scar through an inflammatory gene program.

Comment in Nature. 2022 Dec;612(7941):595.

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

Comment in Cell Stem Cell. 2023 Jan 5;30(1):5-6.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Cerebrospinal fluid (CSF) contains a tightly regulated immune system. However, knowledge is lacking about how CSF immunity is altered with aging or neurodegenerative disease. Here, we performed single-cell RNA sequencing on CSF from 45 cognitively normal subjects ranging from 54 to 82 years old. We uncovered an upregulation of lipid transport genes in monocytes with age. We then compared this cohort with 14 cognitively impaired subjects. In cognitively impaired subjects, downregulation of lipid transport genes in monocytes occurred concomitantly with altered cytokine signaling to CD8 T cells. Clonal CD8 T effector memory cells upregulated C-X-C motif chemokine receptor 6 (CXCR6) in cognitively impaired subjects. The CXCR6 ligand, C-X-C motif chemokine ligand 16 (CXCL16), was elevated in the CSF of cognitively impaired subjects, suggesting CXCL16-CXCR6 signaling as a mechanism for antigen-specific T cell entry into the brain. Cumulatively, these results reveal cerebrospinal fluid immune dysregulation during healthy brain aging and cognitive impairment.

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of β-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.

The perinatal period represents a critical window for cognitive and immune system development, promoted by maternal and infant gut microbiomes and their metabolites. Here, we tracked the co-development of microbiomes and metabolomes from late pregnancy to 1 year of age using longitudinal multi-omics data from a cohort of 70 mother-infant dyads. We discovered large-scale mother-to-infant interspecies transfer of mobile genetic elements, frequently involving genes associated with diet-related adaptations. Infant gut metabolomes were less diverse than maternal but featured hundreds of unique metabolites and microbe-metabolite associations not detected in mothers. Metabolomes and serum cytokine signatures of infants who received regular-but not extensively hydrolyzed-formula were distinct from those of exclusively breastfed infants. Taken together, our integrative analysis expands the concept of vertical transmission of the gut microbiome and provides original insights into the development of maternal and infant microbiomes and metabolomes during late pregnancy and early life.

Comment on N Engl J Med. 2022 Jan 6;386(1):24-34. Nature. 2022 Nov;611(7934):155-160.

Intraflagellar transport (IFT) is the highly conserved process by which proteins are transported along ciliary microtubules by a train-like polymeric assembly of IFT-A and IFT-B complexes. IFT-A is sandwiched between IFT-B and the ciliary membrane, consistent with its putative role in transporting transmembrane and membrane-associated cargoes. Here, we have used single-particle analysis electron cryomicroscopy (cryo-EM) to determine structures of native IFT-A complexes. We show that subcomplex rearrangements enable IFT-A to polymerize laterally on anterograde IFT trains, revealing a cooperative assembly mechanism. Surprisingly, we discover that binding of IFT-A to IFT-B shields the preferred lipid-binding interface from the ciliary membrane but orients an interconnected network of β-propeller domains with the capacity to accommodate diverse cargoes toward the ciliary membrane. This work provides a mechanistic basis for understanding IFT-train assembly and cargo interactions.

Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.

The assembly and signaling properties of cilia rely on intraflagellar transport (IFT) trains moving proteins into, within, and out of cilia. A flurry of near-atomic models of the multiprotein complexes that make up IFT trains has revealed new conformational changes, which may underlie the switch between anterograde and retrograde intraflagellar transport.

To track and control self-location, animals integrate their movements through space. Representations of self-location are observed in the mammalian hippocampal formation, but it is unknown if positional representations exist in more ancient brain regions, how they arise from integrated self-motion, and by what pathways they control locomotion. Here, in a head-fixed, fictive-swimming, virtual-reality preparation, we exposed larval zebrafish to a variety of involuntary displacements. They tracked these displacements and, many seconds later, moved toward their earlier location through corrective swimming ("positional homeostasis"). Whole-brain functional imaging revealed a network in the medulla that stores a memory of location and induces an error signal in the inferior olive to drive future corrective swimming. Optogenetically manipulating medullary integrator cells evoked displacement-memory behavior. Ablating them, or downstream olivary neurons, abolished displacement corrections. These results reveal a multiregional hindbrain circuit in vertebrates that integrates self-motion and stores self-location to control locomotor behavior.

Comment in Cell Host Microbe. 2023 Jan 11;31(1):135-136.

Comment on Nature. 2022 Oct;610(7931):319-326.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.