eLife

Morphogenetic processes during animal development are remarkably invariant (Duboule, 1994; Hall, 1997; Kalinka et al., 2010; Raff, 1996). This stability is established by the interaction between genetic determination of developmental progression and the constraints imposed by the surrounding embryonic environment (Busby and Steventon, 2021; Gilmour et al., 2017; Gorfinkiel and Martinez Arias, 2021). We discovered that the germ band extension process in Drosophila is rather variable: instead of extending straight towards the head, the germ band tends to twist to the side. Through a combination of experiments and theory, we demonstrated that Scab integrin-mediated attachment to the vitelline envelope stabilizes the germ band and supports its straight extension. Our quantification of germ band extension dynamics also revealed a consistent handedness to the twist of the germ band. We showed that this left-right asymmetry can be altered by manipulating the expression of Myo1D, the molecular determinant of chirality in Drosophila (Lebreton et al., 2018). Our data thus suggest that Myo1D expression causes the early gastrulating blastoderm epithelium to already exhibit inherent chirality and that the resulting destabilization of germ band extension is suppressed by Scab-mediated friction between the blastoderm and the vitelline envelope.

Cholinergic efferent neurons modulate sensory signaling in the peripheral vestibular system, but the cellular mechanisms underlying this modulation remain incompletely understood. In mammalian vestibular organs, type II hair cells (HC-II) receive efferent input and express 910-containing nicotinic acetylcholine receptors (nAChRs) that activate SK potassium channels and produce rapid hyperpolarization. Here, we examined the functional role of mAChRs in mouse vestibular HC-II using whole cell patch clamp recordings in whole tissue preparations of crista ampularis (P13-P17, male and female mice). Activation of mAChRs with oxotremorine-M inhibited voltage dependent outward currents, with the largest effects at depolarized membrane potentials. Further experiments revealed that this effect was mediated by inhibition of large conductance potassium (BK) channels: the BK antagonist iberiotoxin mimicked and occluded the muscarinic effect and muscarinic suppression was absent in mice with BK channel mutations. In contrast, blockade of SK channels with apamin did not prevent the muscarinic effect, indicating that mAChR signaling specifically targets BK mediated currents. In current clamp recordings, mAChR activation enhanced depolarization during strong current injections, consistent with increased hair cell excitability when BK channels were suppressed. These findings identify a previously unrecognized muscarinic efferent pathway in vestibular hair cells and reveal complementary cholinergic mechanisms that suppress responses to weak stimuli while enhancing responses to strong stimulation, providing a cellular basis for dynamic gain control in the vestibular periphery. Significance statementVestibular efferent signaling shapes how head movements are encoded, but its cellular mechanisms are incompletely understood. While nicotinic acetylcholine receptors are known to reduce excitability of type II vestibular hair cells (HC-II) via small conductance (SK) channels, the role of muscarinic receptors has remained unclear. Here we show that muscarinic receptor activation selectively inhibits large conductance (BK) potassium channels in HC-II, enhancing excitability during strong depolarization. This muscarinic pathway is mechanistically distinct from nicotinic signaling and operates at a different voltage range. Together, these findings reveal a dual efferent control strategy that differentially regulates hair cell responses to slow versus fast head movements, providing new insight into how the vestibular system filters sensory input across dynamic ranges.

Neighboring sarcomeres in a cardiomyocyte need not move in perfect synchrony, but it is unclear whether their local timing differences during hyperthermal sarcomeric oscillations (HSOs) are random or organized. Using the same five-sarcomere recordings that previously revealed a constrained 16-state neighboring-pair topology and an amplitude-synchrony relation for the segment-mean rapid signal (Shintani, 2026), we reanalyzed each fast HSO cycle as one local coordination summary. A topology-based circular coordinate showed that the 16 discrete patterns lie on a continuous within-cell order: adjacent positions were enriched for Hamming-1 changes and cycle-to-cycle angular drift was slower than expected by chance. Because each cell has an arbitrary angular zero point and direction, we aligned the cell-wise circles using shared local states as landmarks. This improved same-state concentration across cells from 0.582 to 0.852 (P = 0.0013). The clearest biological translation of the aligned coordinate was mismatch placement along the observed five-sarcomere segment. Aligned angle predicted edge-biased mismatch placement (joint P = 1.15 x 10-6), whereas raw angle did not (P = 0.55). Beat timing was weaker and signed strain less robust. These findings support a mesoscale view in which local HSO nonuniformity is structured: neighboring sarcomeres share a rephasing order, and that order is most readably expressed by where the local mismatch pocket lies along the chain. Significance statementCardiac contraction must convert noisy local events into a stable beat. This study identifies a measurable intermediate-scale variable in living cardiomyocytes. Fast HSO cycles do not wander randomly among local coordination patterns. After cross-cell alignment, they occupy a shared rephasing compass, and the clearest biological readout of that compass is where a local mismatch pocket sits along the observed five-sarcomere segment. This gives a concrete way to describe how local nonuniformity can remain structured rather than merely disruptive. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/714639v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1d0d6deorg.highwire.dtl.DTLVardef@1cab2c8org.highwire.dtl.DTLVardef@9f9cadorg.highwire.dtl.DTLVardef@e75263_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Five consecutive sarcomere-length traces were condensed into one local coordination summary per fast HSO cycle. A topology-based circular coordinate revealed a within-cell order, and cross-cell alignment turned that order into a shared rephasing compass. The clearest biological translation of that compass was where the local mismatch pocket was positioned along the observed five-sarcomere segment. C_FIG

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

Motor adaptation involves the parallel operation of implicit recalibration and explicit re-aiming processes. During naturalistic learning, these systems interact, producing compound behavioral outputs that reflect their combined contributions. It remains unclear whether simultaneously engaged implicit and explicit processes form a single unified representation, or generate parallel memory representations that are merely co-expressed, and how consolidation transforms such representations. We addressed these questions across three visuomotor adaptation experiments (n = 120), in which the implicit process was engaged via gradual cursor rotation and the explicit process via target jump, by systematically manipulating the sequence of learning and the timing of expression. Immediately after learning, behavior reflected an inflexible, integrated memory that could not be decomposed by changing task demands. Following 24-hour consolidation, however, expression became component-selective, with implicit or explicit contributions retrieved in response to task demand. This reorganization had direct consequences on relearning, producing facilitation when the expressed and relearned components matched and interference when they mismatched. Moreover, when implicit adaptation was stabilized prior to compound learning, consolidation preserved the updated state rather than the original implicit representation. Together, these findings demonstrate that consolidation does not merely stabilize compound motor memories. Instead, it actively reorganizes them, transforming the initially integrated representations into independent, context-dependent components.

The subiculum (SUB) is the main output structure of the hippocampus, influencing diverse behaviors through its widespread cortical and subcortical connections. Our previous work creating the mouse Hippocampus Gene Expression Atlas (HGEA) identified four genetically distinct cellular layers across five columnar domains in the SUB, with gene expression boundaries corresponding to distinct connectivity patterns and brain-wide networks involved in spatial navigation, social behavior, and neuroendocrine regulation (Bienkowski et al., 2018). Using the Digital Brain Mouse Projectome Atlas (MPA) tool, we conducted virtual tract-tracing to assess whether connectivity patterns of single-neuron 3D reconstructions aligned with HGEA-defined SUB cell types (Qiu et al., 2024). We classified 689 SUB projection neurons into 12 HGEA cell-type groups based on their laminar and columnar distributions, whose spatial organization recapitulated HGEA-defined 3D boundaries. Using this population sample, we performed a SUB cell-type census, characterized neuronal heterogeneity and projection prevalence, identified common and rare connectivity motifs and axonal collateralization patterns, and defined distinct projection themes for each SUB cell type. Together, this analysis integrates single-neuron and population-level data to advance understanding of SUB cell type organization and its contributions to brain-wide networks regulating diverse behaviors.

Smell allows animals to find food, avoid danger, and communicate through the binding of odorants to chemosensory receptors on olfactory sensory neurons. The vision-priority hypothesis predicts an antagonistic relationship between olfaction and vision, in which olfactory ability increases as visual acuity decreases along evolutionary lineages, a tradeoff that often occurs through expansion and contraction of chemosensory receptor gene families. The Mexican tetra (Astyanax mexicanus), a fish species with both sighted surface-dwelling and blind cave-dwelling populations, presents an ideal model for exploring the mechanisms underlying this tradeoff. Here we show that although cavefish can sense odorants at lower concentrations than surface fish, they do not have an expanded repertoire of chemosensory receptors, increased sensory neuron number or density, or enhanced expression of receptors compared to surface fish. Instead, cavefish have physiological adaptations to the olfactory epithelium, including more motile cilia and decreased flow rate through the olfactory pits. Pharmacological attenuation of flow rate in the olfactory pits in surface fish increased visits to the odorant source, suggesting that the reduced flow rate in cavefish is an adaptation leading to better foraging. This unexpected evolutionary path to enhanced olfaction as a compensation for loss of vision underscores the need for mechanistic understanding of comparative genomics.

Activity regulated transcription modifies the intrinsic excitability of cells, which is proposed to promote neuronal and behavioral plasticity; however, the transcriptional targets involved have not been identified. Here, we identify a single MEF-2 and CRH-1/CREB transcriptional target, gem-4/Copine, whose expression in C. elegans body muscles inhibits gap junction coupling, thereby increasing excitability. We show that gem-4 also promotes a form of experience and CRH-1 dependent neuronal plasticity. Inactivating gem-4 diminishes the ability of AFD thermosensory neurons to adjust their sensory response threshold following shifts in cultivation temperature. These results describe a mechanism linking activity regulated gem-4 expression to the plasticity of intrinsic excitability and sensory responses. Significance StatementActivity induced gene expression (e.g. that produced by activating the transcription factors CREB and MEF-2) is required for behavioral plasticity, including several forms of learning and memory. The effects of activity-induced gene expression on behavior are thought to be mediated by changes in synaptic connectivity and by changes in the intrinsic excitability of neurons. The role of altered excitability on behavioral plasticity has been difficult to assess because specific transcriptional targets regulating excitability have not been identified. Here we identify the gem-4 Copine gene as a MEF-2 and CREB transcriptional target that regulates intrinsic excitability by inhibiting gap junction coupling.

Pupil responses shape the earliest stages of visual perception by regulating the amount of light that enters the eye. How this affects visual processing is still poorly understood. Here we present evidence that pupil constriction causes activity in the human retina and visual system, independently of visual stimulation. Healthy human participants (N=119) viewed brief visual stimuli (light increments or decrements) while pupil size, retinal activity (electroretinogram), and brain activity (electroencephalogram) were recorded. As expected, visual stimulation triggered an initial burst of retinal activity followed by pupil constriction (the pupil light response). Importantly, we used trial-to-trial variability in constriction latency to reveal a previously unknown retinal response that is locked to pupil constriction, rather than to visual stimulation. Presumably, and in line with similar findings in mice, this constriction-locked retinal activity is a response to the sudden decrease in retinal light exposure that accompanies pupil constriction (although contribution of iris muscle activity is not conclusively ruled out). A similar constriction-locked response emerged later over visual cortex. Given these findings, an important open question is how the visual system maintains brightness constancy despite pupil-induced retinal and cortical activity.

The recent movement history shapes motor performance, that is, previous movements can affect current movement characteristics such as trajectory shape. History effects are commonly attributed to carryover of motor-related activity. However, action execution entails sensory feedback; therefore, an alternative is that history effects stem from the sensory information produced by previous movements. To dissociate motor and sensory contributions, we assessed whether history effects emerge from imagined movements, which involve movement planning but not sensory feedback. Overt reaches around an obstacle led to systematic adjustment of the initial reach direction of following reaches - a hallmark of motor history. Imagined reaches around obstacles induced similar biases, albeit with smaller magnitude, presumably due to the need to inhibit overt execution during imagery. By contrast, execution but not imagery induced biases in late, feedback-related measures, suggesting that these history effects depended on sensory rather than motor aspects of the movement history. Thus, motor and sensory signals make distinct and complementary contributions to movement history: recent motor states shape feedforward planning, whereas recent sensory states shape feedback-related movement refinement. Significance StatementRecent movements bias upcoming ones, but these so-called history effects may have their origin either in planning motor commands or in the executed movements sensory consequences. By leveraging motor imagery to retain movement planning but remove movement-related sensory feedback, we show that feedforward, planning-related biases persist without sensory consequences, whereas feedback-dependent biases only emerge from prior sensory feedback. Thus, motor and sensory processes induce specific histories that affect distinct aspects of the current movement.

Mammalian two-pore channels (TPCs) are endolysosomal cation channels that regulate membrane trafficking and ionic homeostasis and have been strongly implicated in nicotinic acid adenine dinucleotide phosphate (NAADP) signaling. We previously identified Lsm12 as an NAADP receptor and TPC-interacting protein required for NAADP-evoked Ca2+ mobilization from acidic organelles; however, how NAADP-Lsm12 coupling regulates TPC gating has remained unclear. Here, we show that Lsm12 acts as a potent antagonist of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-dependent TPC activation. Purified Lsm12 strongly inhibited PI(3,5)P2-evoked currents of TPC1 and TPC2, and endogenous Lsm12 similarly suppressed TPC2 activity in cells. Mechanistically, Lsm12 reduces the apparent sensitivity of TPC2 to PI(3,5)P2 through a competitive mechanism that depends on the concentrations of both Lsm12 and PI(3,5)P2, as well as on Lsm12-TPC interaction. Importantly, NAADP specifically and dose-dependently reverses Lsm12-mediated inhibition, restoring TPC currents only in the presence of PI(3,5)P2 or an intact PI(3,5)P2-binding site on TPCs. Consistently, acute sequestration of endogenous PI(3,5)P2 reduces NAADP-evoked cytosolic Ca2+ signals. These findings support a model in which Lsm12 tonically restrains PI(3,5)P2-dependent TPC gating, whereas NAADP binding to Lsm12 relieves this inhibition to permit channel activation. Our study therefore establishes a mechanistic link between NAADP signaling and phosphoinositide-dependent TPC gating and provides a working model for understanding NAADP-evoked Ca2+ release from acidic stores.

Pennaceous feathers are fundamental to avian flight, yet their early function in non-volant dinosaurs remains unknown. Early-diverging pennaraptorans had simple pennaceous feathers on proto-wings and tails, which were unsuitable for flight but may have enhanced visual signals. However, the visual display hypothesis has not been empirically tested. To address this, we used computer animations of early pennaraptoran displays to measure responses in a well-established animal model of a visually sensitive neural pathway. We show that pennaceous proto-wings and tails enhance the efficiency of motion-based displays across a range of anatomically plausible movements. Integrating these results with comparative and paleontological evidence, we suggest that early pennaceous feathers functioned in diverse signaling contexts and were subsequently exapted for aerodynamic use.

Recent advancements in tomography produce imaging data of geological materials (rocks and fossils) at trillion-voxel scales with multi-channels. Such high-resolution datasets are potentially keys to unveil evolutionary biological information with various shapes and sizes that have not been ever discovered. Volume rendering is an ideal visualization approach for them because it treats all voxels without relying on user-defined surface boundaries. However, these large-scale real-world tomographic data have rarely been volume-rendered at their native resolution, limiting the examination of rich morphological information. Here, we demonstrate a de facto standard volume-rendering pipeline running on a graphical processing unit (GPU)-equipped supercomputing system toward multi-channel, trillion-voxel tomographic data. Our workflow preserves original resolution, capturing detailed morphological information spanning microscopic to macroscopic scales. Systematic comparison of node types shows that GPU memory, rather than host memory, is the primary bottleneck. Our results establish a baseline for large-scale, multi-channel volume rendering of real tomographic data and demonstrate its applicability to geological samples. This work is presented as a practical demonstration of large-scale volume visualization.

The brain stores information by changing the strength of its synapses, a process that has at least two phases: Late long-term potentiation (L-LTP) is thought to result from the consolidation of early LTP (E-LTP), just as long-term memory requires the prior establishment of short-term memory. Recently, inhibitory avoidance experiments under CaMKII inhibition have challenged this notion, demonstrating long-term fear memory without measurable short-term memory. Here we use optogenetic activation and inhibition of CaMKII during induction of spike-timing-dependent potentiation (tLTP) to dissect the signaling pathways. While CaMKII activation in CA1 neurons was sufficient to induce E-LTP, growth of the postsynaptic density and spine neck expansion, we found that CaMKII-induced LTP does not give rise to L-LTP. Conversely, inhibition of CaMKII during tLTP induction prevented E-LTP, but FOS and L-LTP were still expressed, driven by CaMKK and PKM{zeta}. Thus, both long-term memory and L-LTP form in the absence of CaMKII activation.

HighlightsBinocular rotational stimulation enhances OKR in wild-type mice along both axes. Chrnb2tm mice show spontaneous horizontal eye oscillations regardless of input. Frmd7tm mice lack binocular enhancement of vertical OKR. The optokinetic reflex (OKR) stabilizes retinal images during global motion and depends on retinal direction-selective (DS) circuits. Although multiple mutant mouse strains exhibit impaired DS circuits via distinct mechanisms, differences in OKR phenotypes remain unexplained. Here, we developed a behavioral system to quantify mouse eye movements under controlled rotational and translational visual stimuli. Using this platform, we examined OKR in wild-type (WT) mice and two DS circuit mutants: Frmd7tm mice, which lack horizontal DS tuning and horizontal OKR, and Chrnb2tm mice, which have disrupted {beta}2-nAChR-dependent cholinergic spontaneous activity during development. Consistent with previous research, both mutants lacked horizontal OKR across conditions, while vertical OKR was preserved. We found that Chrnb2tm mice exhibited spontaneous horizontal eye oscillations regardless of visual input. This phenotype was absent in Frmd7tm mice, suggesting that defective retinal waves in Chrnb2tm mice may induce circuit-level instability distinct from the loss of horizontal DS tuning alone. In addition, WT mice showed enhanced vertical OKR under binocular rotational stimulation, which was absent in Frmd7tm mice. Together, these findings provide a functional comparison of Frmd7tm and Chrnb2tm mice and establish a quantitative framework for dissecting how specific genetic perturbations alter the retinal computations underlying horizontal and vertical OKR.

Persistent detection of high-risk human papillomavirus (HPV) is required for cervical carcinogenesis, yet the metabolic phenotype associated with distinct HPV transition states remains incompletely defined. We analyzed vaginal metabolomics data from 71 HIV-negative, non-smoking, premenopausal women without other sexually transmitted infections, grouped by three-visit HPV trajectories: persistent negative (NNN, n=20), late incident positivity (NNP, n=9), conversion with persistence (NPP, n=13), clearance after prior positivity (PPN, n=16), and persistent positive (PPP, n=13). After detection-based filtering, 186 putative and 64 quantitatively estimated metabolites were retained for integrated univariate, multivariate, network, pathway, and machine learning analyses. Global class separation was weak by PERMANOVA and by five-class classification, indicating that the vaginal metabolome does not reorganize broadly across all HPV states. In contrast, trajectory-specific signals were reproducible. The strongest pairwise contrast was NNP versus PPP (best cross-validated ROC AUC 0.778; permutation p=0.039). Glycolic acid was the dominant single metabolite, particularly for NNP versus PPP (Mann-Whitney p=6.96x10^-4, FDR=0.0446, AUROC=0.902; detection 88.9% versus 15.4%; combined abundance+detection FDR=0.0010). Persistent positivity was characterized by a focused uracil-high, methyl-donor/redox-low signature, including lower glycolic acid, S-adenosylmethionine, NAD+, and betaine, together with higher uracil. Ratio mining further sharpened discrimination, with uracil/S-adenosylmethionine and uracil/creatinine among the best PPP classifiers, and glucose 1-phosphate/isovaleric acid-valeric acid strongly separating NNP from NPP. These data support a model in which HPV trajectory is encoded by targeted metabolic states rather than a diffuse HPV-positive versus HPV-negative metabolomic shift.

Witchweeds (Striga spp.) are parasitic plants that severely constrain cereal production across sub-Saharan Africa, threatening food security for millions of people (Runo and Kuria, 2018). Striga infection begins when dormant seeds germinate in response to host-derived biomolecules, primarily strigolactones which plants emit to regulate shoot branching and to communicate with beneficial microbes. This obligate dependence on host signals can be exploited for Striga control through suicidal germination, whereby strigolactone-like compounds induce parasite germination in the absence of a host. Although this strategy proved highly effective during Striga eradication efforts in the United States using ethylene gas as a Striga germination inducer (Eplee, 1975; Iverson et al., 2011), its deployment in Africa has been limited by capacity to synthesize cost-effective strigolactone-like Striga germination inducers. Here, we show that structure-guided in silico screening of chemical libraries using AlphaFold2-modeled receptor-ligand interactions improve the efficiency and likelihood of identifying previously unknown strigolactone analogs. Using this approach, we identify a structurally simple synthetic lactone scaffold that induces Striga germination at nanomolar concentrations. These results present new avenues for the development of strigolactone analogs and support revisiting suicidal germination as a practical Striga control strategy in Africa.

Carbohydrates are classically catabolized by fermentation or oxidation, a choice that impacts many cellular functions including proliferation. Proliferating cells including somatic stem and progenitor cells are thought to favor fermentation over oxidation, and most proliferating cells in vitro depend on lactate production. However, it has not been tested if fermentation and oxidation are the universal obligatory terminal fates for carbohydrates in vivo because the key enzymes, lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), have not been simultaneously deleted in any cell type. Here we show that both fermentation and oxidation are dispensable for the survival and function of hematopoietic stem cells (HSC). Combined LDHA and LDHB deletion to ablate LDH did not impair HSC function, suggesting that HSCs and rapidly proliferating hematopoietic progenitors surprisingly do not require fermentation. Combined LDHA, LDHB, and PDH deletion abolished both glucose oxidation and fermentation, but did not impair HSC function. Glycolysis was preserved, suggesting the operation of an alternative endpoint. LDH/PDH-deficient HSCs terminated glycolysis through pyruvate export. Pyruvate export by HSCs and progenitors was a physiological response to changing nutrient levels. Quadruple deletion of LDHA/B, PDH, and the pyruvate transporter MCT1 impaired HSC function. This suggested that an essential role of glycolysis termination is not to produce acetyl-CoA or lactate but to remove pyruvate. Therefore, in contrast to classical theories and to in vitro metabolism, carbohydrate metabolism in vivo does not require oxidation or fermentation but can terminate directly in pyruvate export, and this alternative pathway is sufficient to support stem cell function.

The nsp16 2-O-Methyltransferase is an essential non-structural protein of SARS-CoV-2, which methylates the viral mRNA cap structure, enabling it to evade the host immune response for higher translation efficiency. However, nsp16 is only active when it is bound to its cofactor, namely the non-structural protein-10 (nsp10). Understanding how nsp10 binds to and activates nsp16 function can help to develop targeted inhibitors; however, given the varying degree of disorder in both nsp10 and nsp16, characterizing this interaction has been challenging. Using long-timescale molecular dynamics simulations and AI/ML methods, we posit that the nsp16/nsp10 binding process is mediated by a hydrophobic latch formed with Leu4298 from nsp10 and a hydrophobic concave on the nsp16 protein surface. Our study highlights how the nsp16 S-adenosyl-L-methionine (SAM) pocket closes in its monomer state, which in turn deactivates the MTase function. We also observe that the nsp16/nsp10 complex allows for the RNA binding site to open with the empty SAM pocket. The results reveal how the SAM pocket loops facilitate SAM binding while allowing for the by-product S-adenosyl-L-homocysteine (SAH) to exit. Our study thus provides valuable atomistic-level mechanistic insights into understanding the activation of nsp16 MTase function while highlighting the challenges of studying protein-protein interactions mediated by largely flexible/disordered regions. SIGNIFICANCENsp16 carries out the methylation of the viral mRNA to gain immune evasion and translation efficiency. Understanding its complex molecular machinery can help us develop better therapeutic treatments. Here, we explore the key activation conditions for the SARS-CoV-2 nsp16 function via molecular dynamics simulation and AI/ML methods. The results demonstrate the role of nsp16 loops in different stages preparing for the methylation reaction from nsp16/nsp10 binding, (de)activation of nsp16 function and how the nsp16 SAM binding pocket can affect the RNA binding loops. This research explains the role of the nsp16 loops, which orchestrate its molecular function, and provides valuable insight to develop more targeted therapeutic approaches to disrupt viral immune evasion activity.

The network of interactions comprising CD28, CTLA4 and PD-1 and their ligands CD80, CD86, and PD-L1 have profound implications for T cell activation. Despite their critical role, factors that determine their receptor occupancy when all three ligands are present remains incompletely understood. Using a supported lipid bilayer to replicate the antigen-presenting cell membrane facilitated a quantitative analysis of CD80, CD86, and PD-L1 interactions. Our observations reveal that PD-1 blockade operates through a previously unrecognized competitive mechanism beyond conventional checkpoint inhibition: liberated PD-L1 molecules redistribute to form CD80-PD-L1 heterodimers that strengthen CD28-CD80 interactions and outcompete CD86 for CD28 binding. This preferential binding enhances T cell activation through superior CD80 co-stimulation. Furthermore, the redistribution by PD-1 blockade reduces CD80 homodimer availability, limiting CTLA4 binding to CD80 and enhancing CD28 signaling. These findings provide vital insights into the competitive principles that allow B7-CD28 family receptors to regulate T cell activation through network-level effects.