Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Mitochondria as Cellular and Organismal Signaling Hubs.

Mitochondria are traditionally known as the powerhouse of the cell, but their functions extend far beyond energy production. They are vital in cellular and organismal pathways that direct metabolism, stress responses, immunity, and cellular fate. To accomplish these tasks, mitochondria have established networks of both intra- and extracellular communication. Intracellularly, these communication routes comprise direct contacts between mitochondria and other subcellular components as well as indirect vesicle transport of ions, metabolites, and other intracellular messengers. Extracellularly, mitochondria can induce stress responses or other cellular changes that secrete mitochondrial cytokine (mitokine) factors that can travel between tissues as well as respond to immune challenges from extracellular sources. Here we provide a current perspective on the major routes of communication for mitochondrial signaling, including their mechanisms and physiological impact. We also review the major diseases and age-related disorders associated with defects in these signaling pathways. An understanding of how mitochondrial signaling controls cellular homeostasis will bring greater insight into how dysfunctional mitochondria affect health in disease and aging.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes.

The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1ß and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring guanylate-binding proteins. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structural integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. Overall, our results reveal IRGB10 as part of a conserved signaling hub at the interface between cell-autonomous immunity and innate immune sensing pathways.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

Intercellular protein movement: deciphering the language of development.

Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.

Defected twisted ring topology for autonomous periodic flip-spin-orbit soft robot.

Periodic spin-orbit motion is ubiquitous in nature, observed from electrons orbiting nuclei to spinning planets orbiting the Sun. Achieving autonomous periodic orbiting motions, along circular and noncircular paths, in soft mobile robotics is crucial for adaptive and intelligent exploration of unknown environments-a grand challenge yet to be accomplished. Here, we report leveraging a closed-loop twisted ring topology with a defect for an autonomous soft robot capable of achieving periodic spin-orbiting motions with programmed circular and re-programmed irregular-shaped trajectories. Constructed by bonding a twisted liquid crystal elastomer ribbon into a closed-loop ring topology, the robot exhibits three coupled periodic self-motions in response to constant temperature or constant light sources: inside-out flipping, self-spinning around the ring center, and self-orbiting around a point outside the ring. The coupled spinning and orbiting motions share the same direction and period. The spinning or orbiting direction depends on the twisting chirality, while the orbital radius and period are determined by the twisted ring geometry and thermal actuation. The flip-spin and orbiting motions arise from the twisted ring topology and a bonding site defect that breaks the force symmetry, respectively. By utilizing the twisting-encoded autonomous flip-spin-orbit motions, we showcase the robot's potential for intelligently mapping the geometric boundaries of unknown confined spaces, including convex shapes like circles, squares, triangles, and pentagons and concaves shapes with multi-robots, as well as health monitoring of unknown confined spaces with boundary damages.

miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity.

Homeostatic synaptic plasticity is a process by which neurons adjust their synaptic strength to compensate for perturbations in neuronal activity. Whether the highly diverse synapses on a neuron respond uniformly to the same perturbation remains unclear. Moreover, the molecular determinants that underlie synapse-specific homeostatic synaptic plasticity are unknown. Here, we report a synaptic tagging mechanism in which the ability of individual synapses to increase their strength in response to activity deprivation depends on the local expression of the spine-apparatus protein synaptopodin under the regulation of miR-124. Using genetic manipulations to alter synaptopodin expression or regulation by miR-124, we show that synaptopodin behaves as a "postsynaptic tag" whose translation is derepressed in a subpopulation of synapses and allows for nonuniform homeostatic strengthening and synaptic AMPA receptor stabilization. By genetically silencing individual connections in pairs of neurons, we demonstrate that this process operates in an input-specific manner. Overall, our study shifts the current view that homeostatic synaptic plasticity affects all synapses uniformly to a more complex paradigm where the ability of individual synapses to undergo homeostatic changes depends on their own functional and biochemical state.

LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

Autonomous Artificial Intelligence vs Artificial Intelligence-Assisted Human Optical Diagnosis of Colorectal Polyps: A Randomized Controlled Trial.

BACKGROUND & AIMS: Artificial intelligence (AI)-based optical diagnosis systems (CADx) have been developed to allow pathology prediction of colorectal polyps during colonoscopies. However, CADx systems have not yet been validated for autonomous performance. Therefore, we conducted a trial comparing autonomous AI to AI-assisted human (AI-H) optical diagnosis. METHODS: We performed a randomized noninferiority trial of patients undergoing elective colonoscopies at 1 academic institution. Patients were randomized into (1) autonomous AI-based CADx optical diagnosis of diminutive polyps without human input or (2) diagnosis by endoscopists who performed optical diagnosis of diminutive polyps after seeing the real-time CADx diagnosis. The primary outcome was accuracy in optical diagnosis in both arms using pathology as the gold standard. Secondary outcomes included agreement with pathology for surveillance intervals. RESULTS: A total of 467 patients were randomized (238 patients/158 polyps in the autonomous AI group and 229 patients/179 polyps in the AI-H group). Accuracy for optical diagnosis was 77.2% (95% confidence interval [CI], 69.7-84.7) in the autonomous AI group and 72.1% (95% CI, 65.5-78.6) in the AI-H group (P = .86). For high-confidence diagnoses, accuracy for optical diagnosis was 77.2% (95% CI, 69.7-84.7) in the autonomous AI group and 75.5% (95% CI, 67.9-82.0) in the AI-H group. Autonomous AI had statistically significantly higher agreement with pathology-based surveillance intervals compared to AI-H (91.5% [95% CI, 86.9-96.1] vs 82.1% [95% CI, 76.5-87.7]; P = .016). CONCLUSIONS: Autonomous AI-based optical diagnosis exhibits noninferior accuracy to endoscopist-based diagnosis. Both autonomous AI and AI-H exhibited relatively low accuracy for optical diagnosis; however, autonomous AI achieved higher agreement with pathology-based surveillance intervals. (ClinicalTrials.gov, Number NCT05236790).

The role of IFN-γ-mediated host immune responses in monitoring and the elimination of Toxoplasma gondii infection.

Toxoplasma gondii is a pathogenic protozoan parasite of the Apicomplexa family that affects approximately 30% of the world's population. Symptoms are usually mild in immunocompetent hosts, but it can pose significant health risks to immunosuppressed patients and pregnant women. Current treatment options are limited, and new therapies and vaccines are needed. The innate immune system is the first to recognize T. gondii infection and activates pro-inflammatory cytokines and chemokines to promote acquired immunity. The IL-12/IFN-γ axis is particularly important, and when this pathway is inhibited, infection becomes uncontrolled and lethal. In mice, receptors such as Toll-like receptor 11 (TLR11), TLR12, and chemokine receptors are involved in T. gondii recognition and the modulation of immune responses. In humans, where TLR11 and TLR12 are absent, other mechanisms have been reported as the innate immune sensing system in T. gondii infection. Immune cells activated in response to infection produce interleukin (IL)-12, which stimulates the proliferation of natural killer cells and T cells and promotes the production of interferon (IFN)-γ. Several IFN-γ-induced anti-T. gondii defense mechanisms inhibit parasite growth. These include nitric oxide (NO) production, indoleamine 2,3-dioxygenase, and the destruction of parasitophorous vacuoles by IFN-γ-inducible immunity related GTPase groups (IRGs and GBPs). Recent studies focusing on the diversity of IRGs in rodents and effector molecules in T. gondii suggest that host immune mechanisms and pathogen immune evasion mechanisms have co-evolved. Furthermore, it has been suggested that cysts are not simply dormant during chronic infection. This review summarizes recent findings on anti-T. gondii innate, adaptive, and cell-autonomous immune responses.

De-stressing plaques attenuates atherosclerosis progression.

Atherogenesis is accompanied by formation of immune cell aggregates, so-called artery tertiary lymphoid organs (ATLOs), in the outermost layer of diseased arteries. In a recent study, Mohanta, Habenicht, and colleagues revealed that there are distinct interactions between ATLOs and the autonomous nervous system, which are critically implicated in atherosclerosis progression.

Caresses, whispers and affective faces: A theoretical framework for a multimodal interoceptive mechanism underlying ASMR and affective touch: An evolutionary and developmental perspective for understanding ASMR and affective touch as complementary processes within affiliative interactions.

Autonomous sensory meridian response (ASMR) and affective touch (AT) are two phenomena that have been independently investigated from separate lines of research. In this article, I provide a unified theoretical framework for understanding and studying them as complementary processes. I highlight their shared biological basis and positive effects on emotional and psychophysiological regulation. Drawing from evolutionary and developmental theories, I propose that ASMR results from the development of biological mechanisms associated with early affiliative behaviour and self-regulation, similar to AT. I also propose a multimodal interoceptive mechanism underlying both phenomena, suggesting that different sensory systems could specifically respond to affective stimulation (caresses, whispers and affective faces), where the integration of those inputs occurs in the brain's interoceptive hubs, allowing physiological regulation. The implications of this proposal are discussed with a view to future research that jointly examines ASMR and AT, and their potential impact on improving emotional well-being and mental health.

Twisting for soft intelligent autonomous robot in unstructured environments.

Soft robots that can harvest energy from environmental resources for autonomous locomotion is highly desired; however, few are capable of adaptive navigation without human interventions. Here, we report twisting soft robots with embodied physical intelligence for adaptive, intelligent autonomous locomotion in various unstructured environments, without on-board or external controls and human interventions. The soft robots are constructed of twisted thermal-responsive liquid crystal elastomer ribbons with a straight centerline. They can harvest thermal energy from environments to roll on outdoor hard surfaces and challenging granular substrates without slip, including ascending loose sandy slopes, crossing sand ripples, escaping from burying sand, and crossing rocks with additional camouflaging features. The twisting body provides anchoring functionality by burrowing into loose sand. When encountering obstacles, they can either self-turn or self-snap for obstacle negotiation and avoidance. Theoretical models and finite element simulation reveal that such physical intelligence is achieved by spontaneously snapping-through its soft body upon active and adaptive soft body-obstacle interactions. Utilizing this strategy, they can intelligently escape from confined spaces and maze-like obstacle courses without any human intervention. This work presents a de novo design of embodied physical intelligence by harnessing the twisting geometry and snap-through instability for adaptive soft robot-environment interactions.

Spontaneous Activity of the Local GABAergic Synaptic Network Causes Irregular Neuronal Firing in the External Globus Pallidus.

Autonomously firing GABAergic neurons in the external globus pallidus (GPe) form a local synaptic network. In slices, most GPe neurons receive a continuous inhibitory synaptic barrage from 1 or 2 presynaptic GPe neurons. We measured the barrage's effect on the firing rate and regularity of GPe neurons in male and female mice using perforated patch recordings. Silencing the firing of parvalbumin-positive (PV+) GPe neurons by activating genetically expressed Archaerhodopsin current increased the firing rate and regularity of PV- neurons. In contrast, silencing Npas1+ GPe neurons with Archaerhodopsin had insignificant effects on Npas1- neuron firing. Blocking spontaneous GABAergic synaptic input with gabazine reproduced the effects of silencing PV+ neuron firing on the firing rate and regularity of Npas1+ neurons and had similar effects on PV+ neuron firing. To simulate the barrage, we constructed conductance waveforms for dynamic clamp based on experimentally measured inhibitory postsynaptic conductance trains from 1 or 2 unitary local connections. The resulting inhibition replicated the effect on firing seen in the intact active network in the slice. We then increased the number of unitary inputs to match estimates of local network connectivity in vivo As few as 5 unitary inputs produced large increases in firing irregularity. The firing rate was also reduced initially, but PV+ neurons exhibited a slow spike-frequency adaptation that partially restored the rate despite sustained inhibition. We conclude that the irregular firing pattern of GPe neurons in vivo is largely due to the ongoing local inhibitory synaptic barrage produced by the spontaneous firing of other GPe neurons.SIGNIFICANCE STATEMENT Functional roles of local axon collaterals in the external globus pallidus (GPe) have remained elusive because of difficulty in isolating local inhibition from other GABAergic inputs in vivo, and in preserving the autonomous firing of GPe neurons and detecting their spontaneous local inputs in slices. We used perforated patch recordings to detect spontaneous local inputs during rhythmic firing. We found that the autonomous firing of single presynaptic GPe neurons produces inhibitory synaptic barrages that significantly alter the firing regularity of other GPe neurons. Our findings suggest that, although GPe neurons receive input from only a few other GPe neurons, each local connection has a large impact on their firing.

MED8 regulates floral transition in Arabidopsis by interacting with FPA.

Success in plant reproduction is highly dependent on the correct timing of the floral transition, which is tightly regulated by the flowering pathways. In the model plant Arabidopsis thaliana, the central flowering repressor FLOWERING LOCUS C (FLC) is precisely regulated by multiple flowering time regulators in the vernalization pathway and autonomous pathway, including FPA. Here we report that Arabidopsis MEDIATOR SUBUNIT 8 (MED8) promotes floral transition in Arabidopsis by recruiting FPA to the FLC locus to repress FLC expression. Loss of MED8 function leads to a significant late-flowering phenotype due to increased FLC expression. We further show that MED8 directly interacts with FPA in the nucleus and recruits FPA to the FLC locus. Moreover, MED8 is indispensable for FPA's function in controlling flowering time and regulating FLC expression. Our study thus reveals a flowering mechanism by which the Mediator subunit MED8 represses FLC expression by facilitating the binding of FPA to the FLC locus to ensure appropriate timing of flowering for reproductive success.

Hybrid sterility genes in mice (Mus musculus): a peculiar case of PRDM9 incompatibility.

Hybrid sterility is a critical step in the evolution of reproductive barriers between diverging taxa during the process of speciation. Recent studies of young subspecies of the house mouse revealed a multigenic nature and frequent polymorphism of hybrid sterility genes as well as the recurrent engagement of the meiosis-specific gene PR domain-containing 9 (Prdm9) and X-linked loci. Prdm9-controlled hybrid sterility is essentially chromosomal in nature, conditioned by the sequence divergence between subspecies. Depending on the Prdm9 interallelic interactions and the X-linked Hstx2 locus, the same homologs either regularly recombine and synapse, or show impaired DNA DSB repair, asynapsis, and early meiotic arrest. Thus, Prdm9-dependent hybrid sterility points to incompatibilities affecting meiotic recombination as a possible mechanism of reproductive isolation between (sub)species.