Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin and environmental factors. However, PI(3)P function during the Plasmodium stress response was unknown. Here, we used PI3K inhibitors and antimalarial agents to examine the importance of PI(3)P under thermal conditions recapitulating malarial fever. Live cell microscopy using chemical and genetic reporters revealed that PI(3)P stabilizes the digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. A chemoproteomic approach identified PfHsp70-1 as a PI(3)P-binding protein. An Hsp70 inhibitor and knockdown of PfHsp70-1 phenocopy PI(3)P-deficient parasites under heat shock. Furthermore, PfHsp70-1 downregulation hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1 and present a novel PI(3)P function in DV stabilization during heat stress.

Biological tubes must develop and maintain their proper diameter to transport materials efficiently. These tubes are molded and protected in part by apical extracellular matrices (aECMs) that line their lumens. Despite their importance, aECMs are difficult to image in vivo and therefore poorly understood. The Caenorhabditis elegans vulva has been a paradigm for understanding many aspects of organogenesis. Here we describe the vulva luminal matrix, which contains chondroitin proteoglycans, Zona Pellucida (ZP) domain proteins, and other glycoproteins and lipid transporters related to those in mammals. Confocal and transmission electron microscopy revealed, with unprecedented detail, a complex and dynamic aECM. Different matrix factors assemble on the apical surfaces of each vulva cell type, with clear distinctions seen between Ras-dependent (1°) and Notch-dependent (2°) cell types. Genetic perturbations suggest that chondroitin and other aECM factors together generate a structured scaffold that both expands and constricts lumen shape.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e. signaling dynamics) differentially regulate cell behavior. However, the role of signaling dynamics in mediating the effects of cancer driving mutations has not been systematically explored. Here, we show that oncogene expression leads to either pulsatile or sustained ERK activity that correlate with opposing cellular behaviors (i.e. proliferation vs. cell cycle arrest, respectively). Moreover, sustained-but not pulsatile-ERK activity triggers ERK activity waves in unperturbed neighboring cells that depend on the membrane metalloprotease ADAM17 and EGFR activity. Interestingly, the ADAM17-EGFR signaling axis coordinates neighboring cell migration toward oncogenic cells and is required for oncogenic cell extrusion. Overall, our data suggests that the temporal patterns of MAPK activity differentially regulate cell autonomous and non-cell autonomous effects of oncogene expression.

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

JCPyV polyomavirus, a member of the human virome, causes progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease in individuals receiving immunomodulatory therapies. Mutations in the major viral capsid protein, VP1, are common in JCPyV from PML patients (JCPyV-PML) but whether they confer neurovirulence or escape from virus-neutralizing antibody (nAb) in vivo is unknown. A mouse polyomavirus (MuPyV) with a sequence-equivalent JCPyV-PML VP1 mutation replicated poorly in the kidney, a major reservoir for JCPyV persistence, but retained the CNS infectivity, cell tropism, and neuropathology of the parental virus. This mutation rendered MuPyV resistant to a monoclonal Ab (mAb), whose specificity overlapped the endogenous anti-VP1 response. Using cryo-EM and a custom sub-particle refinement approach, we resolved an MuPyV:Fab complex map to 3.2 Å resolution. The structure revealed the mechanism of mAb evasion. Our findings demonstrate convergence between nAb evasion and CNS neurovirulence in vivo by a frequent JCPyV-PML VP1 mutation.

NLRP3 inflammasome activation and complement-mediated inflammation have been implicated in promoting choroidal neovascularization (CNV) in age-related macular degeneration (AMD), but central questions regarding their contributions to AMD pathogenesis remain unanswered. Key open questions are (1) whether NLRP3 inflammasome activation mainly in retinal pigment epithelium (RPE) or rather in non-RPE cells promotes CNV, (2) whether inflammasome activation in CNV occurs via NLRP3 or also through NLRP3-independent mechanisms, and (3) whether complement activation induces inflammasome activation in CNV. Here we show in a neovascular AMD mouse model that NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV. We demonstrate that both NLRP3-dependent and NLRP3-independent inflammasome activation mechanisms induce CNV. Finally, we find that complement and inflammasomes promote CNV through independent mechanisms. Our findings uncover an unexpected role of non-NLRP3 inflammasomes for CNV and suggest that combination therapies targeting inflammasomes and complement may offer synergistic benefits to inhibit CNV.

Single measurements of salivary and plasmatic oxytocin are used as indicators of the physiology of the oxytocin system. However, questions remain about whether they are sufficiently stable to provide valid trait markers of the physiology of the oxytocin system, and whether salivary oxytocin can accurately index its plasmatic concentrations. Using radioimmunoassay, we measured baseline plasmatic and/or salivary oxytocin from two independent datasets. We also administered exogenous oxytocin intravenously and intranasally in a triple dummy, within-subject, placebo-controlled design and compared baseline levels and the effects of routes of administration. Our findings question the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans. Salivary oxytocin is a weak surrogate for plasmatic oxytocin. The increases in salivary oxytocin observed after intranasal oxytocin most likely reflect unabsorbed peptide and should not be used to predict treatment effects.

From cellular activation to drug combinations, immunological responses are shaped by the action of multiple stimuli. Synergistic and antagonistic interactions between stimuli play major roles in shaping immune processes. To understand combinatorial regulation, we present the immune Synergistic/Antagonistic Interaction Learner (iSAIL). iSAIL includes a machine learning classifier to map and interpret interactions, a curated compendium of immunological combination treatment datasets, and their global integration into a landscape of ~30,000 interactions. The landscape is mined to reveal combinatorial control of interleukins, checkpoints, and other immune modulators. The resource helps elucidate the modulation of a stimulus by interactions with other cofactors, showing that TNF has strikingly different effects depending on co-stimulators. We discover new functional synergies between TNF and IFNβ controlling dendritic cell-T cell crosstalk. Analysis of laboratory or public combination treatment studies with this user-friendly web-based resource will help resolve the complex role of interaction effects on immune processes.

Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. Here, we show that a subset of Doublesex+ pC1 neurons in the Drosophila female brain, called pC1d/e, can drive minutes-long changes in female behavior in the presence of males. Using automated reconstruction of a volume electron microscopic (EM) image of the female brain, we map all inputs and outputs to both pC1d and pC1e. This reveals strong recurrent connectivity between, in particular, pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg. We additionally find that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network, including both Doublesex+ and Fruitless+ neurons. Our work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain.

Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.

Molecular examples of evolutionary innovation are scarce and generally involve point mutations. Innovation can occur through larger rearrangements, but here experimental data is extremely limited. Integron integrases innovated from double-strand- toward single-strand-DNA recombination through the acquisition of the I2 α-helix. To investigate how this transition was possible, we have evolved integrase IntI1 to what should correspond to an early innovation state by selecting for its ancestral activity. Using synonymous alleles to enlarge sequence space exploration, we have retrieved 13 mutations affecting both I2 and the multimerization domains of IntI1. We circumvented epistasis constraints among them using a combinatorial library that revealed their individual and collective fitness effects. We obtained up to 104-fold increases in ancestral activity with various asymmetrical trade-offs in single-strand-DNA recombination. We show that high levels of primary and promiscuous functions could have initially coexisted following I2 acquisition, paving the way for a gradual evolution toward innovation.

Despite the importance of programming to modern society, the cognitive and neural bases of code comprehension are largely unknown. Programming languages might 'recycle' neurocognitive mechanisms originally developed for natural languages. Alternatively, comprehension of code could depend on fronto-parietal networks shared with other culturally-invented symbol systems, such as formal logic and symbolic math such as algebra. Expert programmers (average 11 years of programming experience) performed code comprehension and memory control tasks while undergoing fMRI. The same participants also performed formal logic, symbolic math, executive control, and language localizer tasks. A left-lateralized fronto-parietal network was recruited for code comprehension. Patterns of activity within this network distinguish between 'for' loops and 'if' conditional code functions. In terms of the underlying neural basis, code comprehension overlapped extensively with formal logic and to a lesser degree math. Overlap with executive processes and language was low, but laterality of language and code covaried across individuals. Cultural symbol systems, including code, depend on a distinctive fronto-parietal cortical network.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

The assembly of specific neuronal circuits relies on the expression of complementary molecular programs in presynaptic and postsynaptic neurons. In the cerebral cortex, the tyrosine kinase receptor ErbB4 is critical for the wiring of specific populations of GABAergic interneurons, in which it paradoxically regulates both the formation of inhibitory synapses as well as the development of excitatory synapses received by these cells. Here, we found that Nrg1 and Nrg3, two members of the neuregulin family of trophic factors, regulate the inhibitory outputs and excitatory inputs of interneurons in the mouse cerebral cortex, respectively. The differential role of Nrg1 and Nrg3 in this process is not due to their receptor-binding EGF-like domain, but rather to their distinctive subcellular localization within pyramidal cells. Our study reveals a novel strategy for the assembly of cortical circuits that involves the differential subcellular sorting of family-related synaptic proteins.

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

It has been known adipocytes increase p53 expression and activity in obesity, however, only canonical p53 functions (i.e. senescence and apoptosis) are attributed to inflammation-associated metabolic phenotypes. Whether or not p53 is directly involved in mature adipocyte metabolic regulation remains unclear. Here we show p53 protein expression can be up-regulated in adipocytes by nutrient starvation without activating cell senescence, apoptosis, or a death-related p53 canonical pathway. Inducing the loss of p53 in mature adipocytes significantly reprograms energy metabolism and this effect is primarily mediated through a AMP-activated protein kinase (AMPK) pathway and a novel downstream transcriptional target, lysosomal acid lipase (LAL). The pathophysiological relevance is further demonstrated in a conditional and adipocyte-specific p53 knockout mouse model. Overall, these data support a non-canonical p53 function in the regulation of adipocyte energy homeostasis and indicate that the dysregulation of this pathway may be involved in developing metabolic dysfunction in obesity.

With increased life expectancy, age-associated cognitive decline becomes a growing concern, even in the absence of recognizable neurodegenerative disease. The integrated stress response (ISR) is activated during aging and contributes to age-related brain phenotypes. We demonstrate that treatment with the drug-like small-molecule ISR inhibitor ISRIB reverses ISR activation in the brain, as indicated by decreased levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor eIF2. Furthermore, ISRIB treatment reverses spatial memory deficits and ameliorates working memory in old mice. At the cellular level in the hippocampus, ISR inhibition (i) rescues intrinsic neuronal electrophysiological properties, (ii) restores spine density and (iii) reduces immune profiles, specifically interferon and T cell-mediated responses. Thus, pharmacological interference with the ISR emerges as a promising intervention strategy for combating age-related cognitive decline in otherwise healthy individuals.

The infective ability of the opportunistic pathogen Staphylococcus aureus, recognized as the most frequent cause of biofilm-associated infections, is associated with biofilm-mediated resistance to host immune response. Phenol-soluble modulins (PSM) comprise the structural scaffold of S. aureus biofilms through self-assembly into functional amyloids, but the role of individual PSMs during biofilm formation remains poorly understood and the molecular pathways of PSM self-assembly are yet to be identified. Here we demonstrate high degree of cooperation between individual PSMs during functional amyloid formation. PSMα3 initiates the aggregation, forming unstable aggregates capable of seeding other PSMs resulting in stable amyloid structures. Using chemical kinetics we dissect the molecular mechanism of aggregation of individual PSMs showing that PSMα1, PSMα3 and PSMβ1 display secondary nucleation whereas PSMβ2 aggregates through primary nucleation and elongation. Our findings suggest that various PSMs have evolved to ensure fast and efficient biofilm formation through cooperation between individual peptides.