Germinal Centers.

Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

Ligand binding initiates single-molecule integrin conformational activation.

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels.

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.

Time-resolved single-cell transcriptomics defines immune trajectories in glioblastoma.

Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.

Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

Anteromedial thalamus gates the selection and stabilization of long-term memories.

Memories initially formed in hippocampus gradually stabilize to cortex over weeks-to-months for long-term storage. The mechanistic details of this brain re-organization remain poorly understood. We recorded bulk neural activity in circuits that link hippocampus and cortex as mice performed a memory-guided virtual-reality task over weeks. We identified a prominent and sustained neural correlate of memory in anterior thalamus, whose inhibition substantially disrupted memory consolidation. More strikingly, gain amplification enhanced consolidation of otherwise unconsolidated memories. To gain mechanistic insights, we developed a technology for simultaneous cellular-resolution imaging of hippocampus, thalamus, and cortex throughout consolidation. We found that whereas hippocampus equally encodes multiple memories, the anteromedial thalamus preferentially encodes salient memories, and gradually increases correlations with cortex to facilitate tuning and synchronization of cortical ensembles. We thus identify a thalamo-cortical circuit that gates memory consolidation and propose a mechanism suitable for the selection and stabilization of hippocampal memories into longer-term cortical storage.

A genetic history of the Balkans from Roman frontier to Slavic migrations.

The rise and fall of the Roman Empire was a socio-political process with enormous ramifications for human history. The Middle Danube was a crucial frontier and a crossroads for population and cultural movement. Here, we present genome-wide data from 136 Balkan individuals dated to the 1st millennium CE. Despite extensive militarization and cultural influence, we find little ancestry contribution from peoples of Italic descent. However, we trace a large-scale influx of people of Anatolian ancestry during the Imperial period. Between ∼250 and 550 CE, we detect migrants with ancestry from Central/Northern Europe and the Steppe, confirming that "barbarian" migrations were propelled by ethnically diverse confederations. Following the end of Roman control, we detect the large-scale arrival of individuals who were genetically similar to modern Eastern European Slavic-speaking populations, who contributed 30%-60% of the ancestry of Balkan people, representing one of the largest permanent demographic changes anywhere in Europe during the Migration Period.

Tracking cell-type-specific temporal dynamics in human and mouse brains.

Progenitor cells are critical in preserving organismal homeostasis, yet their diversity and dynamics in the aged brain remain underexplored. We introduced TrackerSci, a single-cell genomic method that combines newborn cell labeling and combinatorial indexing to characterize the transcriptome and chromatin landscape of proliferating progenitor cells in vivo. Using TrackerSci, we investigated the dynamics of newborn cells in mouse brains across various ages and in a mouse model of Alzheimer's disease. Our dataset revealed diverse progenitor cell types in the brain and their epigenetic signatures. We further quantified aging-associated shifts in cell-type-specific proliferation and differentiation and deciphered the associated molecular programs. Extending our study to the progenitor cells in the aged human brain, we identified conserved genetic signatures across species and pinpointed region-specific cellular dynamics, such as the reduced oligodendrogenesis in the cerebellum. We anticipate that TrackerSci will be broadly applicable to unveil cell-type-specific temporal dynamics in diverse systems.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

The Structural Dynamics of Translation.

Accurate protein synthesis (translation) relies on translation factors that rectify ribosome fluctuations into a unidirectional process. Understanding this process requires structural characterization of the ribosome and translation-factor dynamics. In the 2000s, crystallographic studies determined high-resolution structures of ribosomes stalled with translation factors, providing a starting point for visualizing translation. Recent progress in single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution of numerous structures sampled in heterogeneous complexes (ensembles). Ensemble and time-resolved cryo-EM have now revealed unprecedented views of ribosome transitions in the three principal stages of translation: initiation, elongation, and termination. This review focuses on how translation factors help achieve high accuracy and efficiency of translation by monitoring distinct ribosome conformations and by differentially shifting the equilibria of ribosome rearrangements for cognate and near-cognate substrates.

Membrane phosphoinositides regulate GPCR-ß-arrestin complex assembly and dynamics.

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Enhanced DNA repair through droplet formation and p53 oscillations.

Living organisms are constantly exposed to DNA damage, and optimal repair is therefore crucial. A characteristic hallmark of the response is the formation of sub-compartments around the site of damage, known as foci. Following multiple DNA breaks, the transcription factor p53 exhibits oscillations in its nuclear concentration, but how this dynamics can affect the repair remains unknown. Here, we formulate a theory for foci formation through droplet condensation and discover how oscillations in p53, with its specific periodicity and amplitude, optimize the repair process by preventing Ostwald ripening and distributing protein material in space and time. Based on the theory predictions, we reveal experimentally that the oscillatory dynamics of p53 does enhance the repair efficiency. These results connect the dynamical signaling of p53 with the microscopic repair process and create a new paradigm for the interplay of complex dynamics and phase transitions in biology.

Cell-type-specific population dynamics of diverse reward computations.

Computational analysis of cellular activity has developed largely independently of modern transcriptomic cell typology, but integrating these approaches may be essential for full insight into cellular-level mechanisms underlying brain function and dysfunction. Applying this approach to the habenula (a structure with diverse, intermingled molecular, anatomical, and computational features), we identified encoding of reward-predictive cues and reward outcomes in distinct genetically defined neural populations, including TH+ cells and Tac1+ cells. Data from genetically targeted recordings were used to train an optimized nonlinear dynamical systems model and revealed activity dynamics consistent with a line attractor. High-density, cell-type-specific electrophysiological recordings and optogenetic perturbation provided supporting evidence for this model. Reverse-engineering predicted how Tac1+ cells might integrate reward history, which was complemented by in vivo experimentation. This integrated approach describes a process by which data-driven computational models of population activity can generate and frame actionable hypotheses for cell-type-specific investigation in biological systems.

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.