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Summary ASAP1 is a multi-domain ArfGAP that controls cell migration, spreading, and focal adhesion dynamics. Although its GAP activity contributes to remodeling of the actin cytoskeleton, it does not fully explain all cellular functions of ASAP1. Here we find that ASAP1 regulates actin filament assembly directly through its N-BAR domain and controls stress fiber maintenance. ASAP1 depletion caused defects in stress fiber organization. Conversely, overexpression of ASAP1 enhanced actin remodeling. The BAR-PH fragment was sufficient to affect actin. ASAP1 with the BAR domain replaced with the BAR domain of the related ACAP1 did not affect actin. The BAR-PH tandem of ASAP1 bound and bundled actin filaments directly, whereas the presence of the ArfGAP and the C-terminal linker/SH3 domain reduced binding and bundling of filaments by BAR-PH. Together these data provide evidence that ASAP1 may regulate the actin cytoskeleton through direct interaction of the BAR-PH domain with actin filaments., Graphical Abstract, Highlights • Downregulation of ArfGAP ASAP1 leads to loss of stress fibers • ASAP1 N-BAR domain organizes actin filaments into bundles • Actin bundling by the BAR domain is intramolecularly inhibited by C-terminal domains • Actin remodeling property is not shared with a related ArfGAP ACAP1, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary Transforming growth factor beta (TGF-β) signaling contributes to tissue fibrosis. Here we demonstrate that TGF-β enhances CatS and CatK expression but reduces CatB and CatL expression in mouse kidney tubular epithelial cells (TECs). CatS- and CatK deficiency reduces TEC nuclear membrane importer importin-β expression, Smad-2/3 activation, and extracellular matrix (ECM) production. Yet CatB- and CatL-deficiency displays the opposite observations with reduced nuclear membrane exporter RanBP3 expression. CatS and CatK form immunocomplexes with the importin-β and RanBP3 more effectively than do CatB and CatL. On the plasma membrane, CatS and CatK preferentially form immunocomplexes with and activate TGF-β receptor-2, whereas CatB and CatL form immunocomplexes with and inactivate TGF-β receptor-1. Unilateral ureteral obstruction-induced renal injury tests differential cathepsin activities in TGF-β signaling and tissue fibrosis. CatB- or CatL-deficiency exacerbates fibrosis, whereas CatS- or CatK-deficiency protects kidneys from fibrosis. These cathepsins exert different effects in the TGF-β signaling cascade independent of their proteolytic properties., Graphical Abstract, Highlights • Cathepsins exert different activities in kidney TEC TGF-β signaling and fibrosis • Cathepsins regulate nuclear membrane transporter expression and form immunocomplexes • Cathepsins regulate plasma membrane TGF-β receptor expression and form immunocomplexes • Cathepsins play different roles in acute injury-induced kidney fibrosis, Fibrosis; Molecular Mechanism of Behavior; Functional Aspects of Cell Biology

Summary Both caloric restriction (CR) and mitochondrial proteostasis are linked to longevity, but how CR maintains mitochondrial proteostasis in mammals remains elusive. MicroRNAs (miRNAs) are well known for gene silencing in cytoplasm and have recently been identified in mitochondria, but knowledge regarding their influence on mitochondrial function is limited. Here, we report that CR increases miRNAs, which are required for the CR-induced activation of mitochondrial translation, in mouse liver. The ablation of miR-122, the most abundant miRNA induced by CR, or the retardation of miRNA biogenesis via Drosha knockdown significantly reduces the CR-induced activation of mitochondrial translation. Importantly, CR-induced miRNAs cause the overproduction of mtDNA-encoded proteins, which induces the mitochondrial unfolded protein response (UPRmt), and consequently improves mitochondrial proteostasis and function. These findings establish a physiological role of miRNA-enhanced mitochondrial function during CR and reveal miRNAs as critical mediators of CR in inducing UPRmt to improve mitochondrial proteostasis., Graphical Abstract, Highlights • CR increases miRNA biogenesis and the global expression of miRNAs in mitochondria • miRNAs are critical for CR-induced activation of mitochondrial translation • CR-induced miRNAs cause overproduction of mtDNA-encoded proteins and induce UPRmt, Biological Sciences; Molecular Biology; Cell Biology; Functional Aspects of Cell Biology

Summary The erythropoietin receptor (EPOR) plays an essential role in erythropoiesis and other cellular processes by forming distinct signaling complexes composed of EPOR homodimers or hetero-oligomers between the EPOR and another receptor, but the mechanism of heteroreceptor assembly and signaling is poorly understood. We report here a 46-residue, artificial transmembrane protein aptamer, designated ELI-3, that binds and activates the EPOR and induces growth factor independence in murine BaF3 cells expressing the EPOR. ELI-3 requires the transmembrane domain and JAK2-binding sites of the EPOR for activity, but not the cytoplasmic tyrosines that mediate canonical EPOR signaling. Instead, ELI-3-induced proliferation and activation of JAK/STAT signaling requires the transmembrane and cytoplasmic domains of the cytokine receptor β-common subunit (βcR) in addition to the EPOR. Moreover, ELI-3 fails to induce erythroid differentiation of primary human hematopoietic progenitor cells but inhibits nonhematopoietic cell death induced by serum withdrawal., Graphical Abstract, Highlights • 46-residue artificial transmembrane protein can activate the erythropoietin receptor • Activation of the EPOR also requires the cytokine receptor β-common chain • Activation does not require the EPOR cytoplasmic tyrosines • Active heteroreceptor supports cell proliferation but not erythroid differentiation, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary How actin-bundling factors cooperatively regulate shank-localized actin bundles remains largely unexplored. Here we demonstrate that FIM5 and PLIM2a/PLIM2b decorate shank-localized actin bundles and that loss of function of PLIM2a and/or PLIM2b suppresses phenotypes associated with fim5 mutants. Specifically, knockout of PLIM2a and/or PLIM2b partially suppresses the disorganized actin bundle and intracellular trafficking phenotype in fim5 pollen tubes. PLIM2a/PLIM2b generates thick but loosely packed actin bundles, whereas FIM5 generates thin but tight actin bundles that tend to be cross-linked into networks in vitro. Furthermore, PLIM2a/PLIM2b and FIM5 compete for binding to actin filaments in vitro, and PLIM2a/PLIM2b decorate disorganized actin bundles in fim5 pollen tubes. These data together suggest that the disorganized actin bundles in fim5 mutants are at least partially due to gain of function of PLIM2a/PLIM2b. Our data suggest that the balance between FIM5 and PLIM2a/PLIM2b is crucial for the normal bundling and organization of shank-localized actin bundles in pollen tubes., Graphical Abstract, Highlights • The transcription of PLIM2a and PLIM2b is upregulated in fim5 pollen tubes • Downregulation of PLIM2a and/or PLIM2b suppresses the defects in fim5 pollen tubes • Both FIM5 and PLIM2a/PLIM2b decorate shank-localized actin filaments • FIM5 can inhibit the binding of PLIM2a and PLIM2b to actin filaments, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Plant Biology

Summary A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner., Graphical Abstract, Highlights • CFLARs Tg mice develop severe ileitis in utero • Intestinal epithelial cells die by apoptosis and necroptosis in CFLARs Tg mice • Blockade of necroptosis rescues lethality of CFLARs Tg mice • Necroptosis activates type 3 innate lymphoid cells, resulting in severe ileitis, Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Summary To address the question of why more than one myosin-II isoform is expressed in a single cell to drive cytokinesis, we analyzed the roles of the myosin-II isoforms, Myo2 and Myp2, of the fission yeast Schizosaccharomyces pombe, in cytokinesis under normal and stressed conditions. We found that Myp2 controls the disassembly, stability, and constriction initiation of the Myo2 ring in response to high-salt stress. A C-terminal coiled-coil domain of Myp2 is required for its immobility and contractility during cytokinesis, and when fused to the tail of the dynamic Myo2, renders the chimera the low-turnover property. We also found, by following distinct processes in real time at the single-cell level, that Myo2 and Myp2 are differentially required but collectively essential for guiding extracellular matrix remodeling during cytokinesis. These results suggest that the dynamic and immobile myosin-II isoforms are evolved to carry out cytokinesis with robustness under different growth conditions., Graphical Abstract, Highlights • The myosin-II isoforms Myo2 and Myp2 display distinct responses to cellular stress • Myp2 controls the constriction initiation of Myo2 during stress response • A C-terminal region of Myp2 is required for its immobility during cytokinesis • Myo2 and Myp2 are differentially required for guiding ECM remodeling during cytokinesis, Molecular Mechanism of Behavior; Cell Biology; Functional Aspects of Cell Biology

Summary The sensory organelle cilium is involved in sensing and transducing important signaling cascades in almost all cells of our body. These ciliary-mediated pathways affect cellular homeostasis and metabolisms profoundly. However, it is almost completely unknown whether the cellular metabolic state affects the assembly of cilia. This study is to investigate how O-linked β-N-acetylglucosamine (O-GlcNAc), a sensor of cellular nutrients, regulates the cilia length. Pharmacologic or genetic inhibition of O-GlcNAcylation led to longer cilia, and vice versa. Further biochemical assays revealed that both α-tubulin and HDAC6 (histone deacetylase 6) were O-GlcNAcylated in vivo. In vitro enzymatic assays showed that O-GlcNAcylation of either tubulin or HDAC6 promoted microtubule disassembly, which likely in turn caused ciliary shortening. Taken together, these results uncovered a negative regulatory role of O-GlcNAc in modulating the ciliary microtubule assembly. The cross talk between O-GlcNAc and cilium is likely critical for fine-tuning the cellular response to nutrients., Graphical Abstract, Highlights • Cellular O-GlcNAc level inversely regulates ciliary length • Attenuated O-GlcNAc level increases the percentage of ciliated cells in hTERT-RPE1 • O-GlcNAcylation of α-tubulin promotes ciliary axoneme disassembly • O-GlcNAcylation of HDAC6 promotes its deacetylase activity, Biological Sciences; Molecular Biology; Cell Biology; Functional Aspects of Cell Biology

Summary Mitochondria are dynamic organelles undergoing fission, fusion, and translocation. These processes have been studied in cultured cells; however, little is known about their regulation in cells within tissues in vivo. We applied four-dimensional intravital microscopy to address this in secretory cells of the salivary gland. We found that mitochondria are organized in two populations: one juxtaposed to the basolateral plasma membrane and the other dispersed in the cytosol. Under basal conditions, central mitochondria exhibit microtubule-dependent motility and low fusion rate, whereas basolateral mitochondria are static and display high fusion rate. Increasing cellular energy demand by β-adrenergic stimulation of regulated exocytosis selectively enhanced motility and fusion of central mitochondria. Inhibition of microtubule polymerization led to inhibition of central mitochondrial motility and fusion and a marked reduction in exocytosis. This study reveals a conserved heterogeneity in mitochondrial positioning and dynamics in exocrine tissues that may have fundamental implications in organ pathophysiology., Graphical Abstract, Highlights • In the salivary glands, mitochondria exist in two populations: basolateral and central • Basolateral mitochondria are static and frequently fuse • Central mitochondria are highly motile and rarely fuse • Exocytosis elicits selective, microtubule-dependent response in central mitochondria, Optical Imaging; Cell Biology; Functional Aspects of Cell Biology

Summary Glycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes. In addition, we found two proteins, LAPTM4A and TM9SF2, for which physiological roles remain elusive. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity but instead disrupted localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family. These results provide mechanistic insight into the post-translational regulation of the activity and localization of Gb3 synthase., Graphical Abstract, Highlights • Genome-wide CRISPR knockout screening using Shiga toxin-induced cell death • Both LAPTM4A and TM9SF2 are required for Gb3 biosynthesis • Loss of LAPTM4A reduces Gb3 synthase activity post-transcriptionally • TM9SF2 regulates the subcellular localization of Gb3 synthase, Molecular Biology; Molecular Mechanism of Behavior; Cell Biology; Functional Aspects of Cell Biology

Summary Timely centrosome separation is critical for accurate chromosome separation. It is initiated by Nek2A at the onset of mitosis, but the mechanism for the strict requirement of phosphorylated Nek2A for its own activation remains unclear. In this study, we have found that Plk1 interacts with Cep85 and forms a ternary complex with Cep85-Nek2A. Nek2A binding, but not its kinase activity, is pre-required for Cep85 to be phosphorylated by Plk1. Nek2A-dependent Cep85 phosphorylation, in turn, leads to the dissociation of phosphorylated Cep85 exclusively from phospho-Nek2A, thereby increasing the freed phospho-Nek2A activity. Both kinases are also required for phosphorylating endogenous Cep85 in cells, and timely phosphorylation of Cep85 and Nek2A is crucial for initiating centrosome disjunction at G2/M. Overall, our study has uncovered a previously unrecognized role of Plk1 and Nek2A and identified Cep85 as a missing piece directly relaying Plk1 activity to Nek2A for its activation in centrosome disjunction., Graphical Abstract, Highlights • Cep85 prevents centrosome separation by binding to and inhibiting Nek2A in interphase • Plk1 binds to Cep85 and forms a ternary Plk1-Cep85-Nek2A complex in late G2 • Nek2A-assisting Cep85 phosphorylation by Plk1 releases phospho-Nek2A from Cep85 • Freed phospho-Nek2A initiates centrosome separation in G2/M, Molecular Biology; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology

Summary Dynamic processes such as fusion, fission, and trafficking are important in the regulation of cellular organelles, with an abundant literature focused on mitochondria. Mitochondrial dynamics not only help shape its network within cells but also are involved in the modulation of respiration and integrity. Disruptions of mitochondrial dynamics are associated with neurodegenerative disorders. Although proteins that directly bind mitochondria to promote membrane fusion/fission have been studied intensively, machineries that regulate dynamic mitochondrial processes remain to be explored. We have identified an interaction between the mitochondrial fission GTPase Dnm1/DRP1 and the actin-regulatory protein Srv2/CAP at mitochondria. Deletion of Srv2 causes elongated-hyperfused mitochondria and reduces the reserved respiration capacity in yeast cells. Our results further demonstrate that the irregular network morphology in Δsrv2 cells derives from disrupted actin assembly at mitochondria. We suggest that Srv2 functions as a pro-fission factor in shaping mitochondrial dynamics and regulating activity through its actin-regulatory effects., Graphical Abstract, Highlights • Srv2 interacts with fission protein Dnm1 on mitochondria in yeast cells • Srv2 deletion causes an irregular, hyperfused-elongated mitochondrial network • The irregular network derives from loss of Srv2-mediated actin assembly at mitochondria • Srv2 modulates both mitochondrial dynamics and activity, Molecular Biology; Cell Biology; Functional Aspects of Cell Biology

Summary Angiogenesis, the development of new blood vessels, is a key process in disease. We reported that insulin promotes translocation of transforming growth factor β (TGF-β) receptors to the plasma membrane of epithelial and fibroblast cells, thus enhancing TGF-β responsiveness. Since insulin promotes angiogenesis, we addressed whether increased autocrine TGF-β signaling participates in endothelial cell responses to insulin. We show that insulin enhances TGF-β responsiveness and autocrine TGF-β signaling in primary human endothelial cells, by inducing a rapid increase in cell surface TGF-β receptor levels. Autocrine TGF-β/Smad signaling contributed substantially to insulin-induced gene expression associated with angiogenesis, including TGF-β target genes encoding angiogenic mediators; was essential for endothelial cell migration; and participated in endothelial cell invasion and network formation. Blocking TGF-β signaling impaired insulin-induced microvessel outgrowth from neonatal aortic rings and modified insulin-stimulated blood vessel formation in zebrafish. We conclude that enhanced autocrine TGF-β signaling is integral to endothelial cell and angiogenic responses to insulin., Graphical Abstract, Highlights • Insulin promotes enhanced autocrine TGF-β responsiveness in endothelial cells • Autocrine TGF-β signaling contributes to insulin-induced angiogenesis gene expression • Insulin-induced endothelial migration and sprouting require autocrine TGF-β signaling • Enhanced autocrine TGF-β signaling is integral to angiogenic responses to insulin, Molecular Biology; Molecular Mechanism of Behavior; Cell Biology; Functional Aspects of Cell Biology

Summary DNA double-strand break (DSB) repair by homologous recombination (HR) is essential for ensuring genome stability. Abnormal spindle-like microcephaly-associated (ASPM) gene encodes a spindle protein that is commonly implicated in primary microcephaly. We found that ASPM is recruited to sites of DNA damage in a PARP2-dependent manner. ASPM interacts with BRCA1 and its E3 ligase HERC2, preventing HERC2 from accessing to BRCA1 and ensuring BRCA1 stability. Inhibition of ASPM expression promotes HERC2-mediated BRCA1 degradation, compromises HR repair efficiency and chromosome stability, and sensitizes cancer cells to ionizing radiation. Moreover, we observed a synergistic effect between ASPM and PARP inhibition in killing cancer cells. This research has uncovered a novel function for ASPM in facilitating HR-mediated repair of DSBs by ensuring BRCA1 stability. ASPM might constitute a promising target for synthetic lethality-based cancer therapy., Graphical abstract, Highlights • ASPM is recruited to sites of DNA damage in a PARP2-dependent manner. • ASPM promotes DSB-end resection to facilitate HR repair. • ASPM prevents HERC2 from accessing to BRCA1 and ensuring BRCA1 stability. • Inhibition of ASPM sensitizes cancer cells to ionizing radiation and PARP inhibitor., Molecular biology; Cell biology; Functional aspects of cell biology

Summary Defects in endothelial cells cause deterioration in kidney function and structure. Here, we found that endothelial SIRT3 regulates metabolic reprogramming and fibrogenesis in the kidneys of diabetic mice. By analyzing, gain of function of the SIRT3 gene by overexpression in a fibrotic mouse strain conferred disease resistance against diabetic kidney fibrosis, whereas its loss of function in endothelial cells exacerbated the levels of diabetic kidney fibrosis. Regulation of endothelial cell SIRT3 on fibrogenic processes was due to tight control over the defective central metabolism and linked activation of endothelial-to-mesenchymal transition (EndMT). SIRT3 deficiency in endothelial cells stimulated the TGFβ/Smad3-dependent mesenchymal transformations in renal tubular epithelial cells. These data demonstrate that SIRT3 regulates defective metabolism and EndMT-mediated activation of the fibrogenic pathways in the diabetic kidneys. Together, our findings show that endothelial SIRT3 is a fundamental regulator of defective metabolism regulating health and disease processes in the kidney., Graphical abstract, Highlights • Endothelial SIRT3 protects against renal fibrosis in diabetes • Endothelial SIRT3 regulates mesenchymal metabolic shifts • Amelioration of endothelial SIRT3 could be a potential therapeutic approach • Targeting defective metabolism offers new therapeutics against kidney diseases, Biological Sciences ; Cell Biology ; Functional Aspects of Cell Biology

Summary The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut., Graphical Abstract, Highlights • Propionate activates Gαi/o but no Gαq/11 signaling in STC-1 cells or colonic crypts • Acute Gαi/o signaling via FFA2 requires endocytosis and mediates GLP-1 release • FFA2 traffics to very early endosomes to control rapid recycling and Gαi/o signals • Propionate-induced internalization of FFA2 drives p38 signaling and GLP-1 release, Cell Biology; Functional Aspects of Cell Biology

Summary Homeostatic generation of T cells, which occurs in the thymus, is controlled at least in part by endogenous cytokines and ligands. In addition, nutritional factors are other key regulators for the homeostasis of host immunity, but whether and how nutrition affects the homeostatic generation of thymocytes remains to be established. Here, we showed that vitamin B1 deficiency resulted in a bias toward the maturation of γδ thymocytes accompanied by decreased differentiation into double-positive thymocytes during thymic involution. These events were mediated through the increased production of TGF-β superfamily members due to the accumulation of branched-chain α-keto acids in thymic stromal cells. These findings revealed essential roles of vitamin B1 in the appropriate differentiation of T cells through the metabolism of thymic stromal cells., Graphical Abstract, Highlights • Vitamin B1 deficiency induces excessive BCKA production in the thymic stromal cells • BCKAs increase the production of TGF-β superfamily from thymic stromal cells • TGF-β superfamily induces unbalanced differentiation of thymocytes • Vitamin B1 deficiency induces thymic involution, Cellular Physiology; Immunology; Cell Biology; Functional Aspects of Cell Biology

Summary Mitochondrial DNA (mtDNA) encodes thirteen core components of OXPHOS complexes, and its steady expression is crucial for cellular energy homeostasis. However, the regulation of mtDNA expression machinery, along with its sensing mechanism to energetic stresses, is not fully understood. Here, we identified SQSTM1/p62 as an important regulator of mtDNA expression machinery, which could effectively induce mtDNA expression and the effects were mediated by p38-dependent upregulation of mitochondrial ribosomal protein L12 (MRPL12) in renal tubular epithelial cells (TECs), a highly energy-demanding cell type related to OXPHOS. We further identified a direct binding site within the MRPL12 promoter to ATF2, the downstream effector of p38. Besides, SQSTM1/p62-induced mtDNA expression is involved in both serum deprivation and hypoxia-induced mitochondrial response, which was further highlighted by kidney injury phenotype of TECs-specific SQSTM1/p62 knockout mice. Collectively, these data suggest that SQSTM1/p62 is a key regulator and energetic sensor of mtDNA expression machinery., Graphical Abstract, Highlights • SQSTM1/p62 is an important regulator of mtDNA expression machinery • SQSTM1/p62 induces MRPL12 expression via activating p38/ATF2 signaling pathway • SQSTM1/p62 maintains TECs mitochondrial homeostasis and kidney function, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Molecular Biology

Summary The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed HBV replication and prevented HBV surface Ag expression. We further demonstrate that the adoptive transfer significantly increased T cell accumulation and production of antiviral cytokines. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the liver and suppress HBV replication through producing antiviral cytokines., Graphical Abstract, Highlights • Generation of functional viral Ag-specific CTLs from iPSCs, i.e., iPSC-CTLs • Viral Ag-specific iPSC-CTLs suppress HBV replication in a murine model • Adoptive transfer of viral Ag-specific iPSC-CTLs generates persistent α-HBV T cells • Adoptive transfer of viral Ag-specific iPSC-CTLs produces antiviral IFN-γ and TNF-⍺, Immunology; Functional Aspects of Cell Biology

Summary During collective cell migration, front cells tend to extend a predominant leading protrusion, which is rarely present in cells at the side or rear positions. Using Drosophila border cells (BCs) as a model system of collective migration, we revealed the presence of a supracellular actomyosin network at the peripheral surface of BC clusters. We demonstrated that the Myosin II-mediated mechanical tension as exerted by this peripheral supracellular network not only mediated cell-cell communication between leading BC and non-leading BCs but also restrained formation of prominent protrusions at non-leading BCs. Further analysis revealed that a cytoplasmic dendritic actin network that depends on the function of Arp2/3 complex interacted with the actomyosin network. Together, our data suggest that the outward pushing or protrusive force as generated from Arp2/3-dependent actin polymerization and the inward restraining force as produced from the supracellular actomyosin network together determine the collective and polarized morphology of migratory BCs., Graphical Abstract, Highlights • A supracellular actomyosin network encompasses the surface of migratory BC cluster • The network mediates cell-cell communication and restrains protrusion formation • Dynamics of myosin patches correlate with protrusion behaviors • A cytoplasmic dendritic actin population interacts with the actomyosin network, Biological Sciences; Cell Biology; Organizational Aspects of Cell Biology; Functional Aspects of Cell Biology

Summary The proteasome is a therapeutic target in cancer, but resistance to proteasome inhibitors often develops owing to the induction of compensatory pathways. Through a genome-wide siRNA screen combined with RNA sequencing analysis, we identified hexokinase and downstream O-GlcNAcylation as cell survival factors under proteasome impairment. The inhibition of O-GlcNAcylation synergistically induced massive cell death in combination with proteasome inhibition. We further demonstrated that O-GlcNAcylation was indispensable for maintaining proteasome activity by enhancing biogenesis as well as proteasome degradation in a manner independent of Nrf1, a well-known compensatory transcription factor that upregulates proteasome gene expression. Our results identify a pathway that maintains proteasome function under proteasome impairment, providing potential targets for cancer therapy., Graphical Abstract, Highlights • O-GlcNAcylation suppresses cell death under proteasome impairment • Combined inhibition of O-GlcNAcylation and proteasome induces massive tumor cell death • O-GlcNAcylation maintains proteasome activity independently of Nrf1 • O-GlcNAcylation enhances proteasome turnover under the proteasome impairment, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Cancer

Summary Despite common consensus about the importance of planar cell polarity (PCP) proteins in tissue orientation, little is known about the mechanisms used by PCP proteins to promote planar polarization of cytoskeletons within individual cells. One PCP protein Fzd6 asymmetrically localizes to the apical cell membrane of multi-ciliated ependymal cells lining the lateral ventricular (LV) wall on the side that contacts cerebrospinal fluid flow. Individual ependymal cells have planar polarized microtubules that connect ciliary basal bodies (BBs) with the cell cortex of the Fzd side to coordinate cilia orientation. Here, we report that cytoplasmic dynein is anchored to the cell cortex of the Fzd side via an adapter protein Daple that regulates microtubule dynamics. Asymmetric localization of cortical dynein generates a pulling force on dynamic microtubules connected to BBs, which in turn orients BBs toward the Fzd side. This is required for coordinated cilia orientation on the LV wall., Graphical Abstract, Highlights • Daple anchors cytoplasmic dynein to the cell cortex of ependymal cells on LV wall • Cytoplasmic dynein is anchored to the Fzd6/Dvl1/Daple side of the cell cortex • Cytoplasmic dynein functions include BB positioning and orientation • Cortex-anchored dynein generates a pulling force on microtubules connected to BBs, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Specialized Functions of Cells

Summary Increased flux of glucose through glycolysis is a hallmark of inflammatory macrophages and is essential for optimal effector functions. Solute carrier (SLC) 37A2 is an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter that is highly expressed in macrophages and neutrophils. We demonstrate that SLC37A2 plays a pivotal role in murine macrophage inflammatory activation and cellular metabolic rewiring. Toll-like receptor (TLR) 4 stimulation by lipopolysaccharide (LPS) rapidly increases macrophage SLC37A2 protein expression. SLC37A2 deletion reprograms macrophages to a hyper-glycolytic process and accelerates LPS-induced inflammatory cytokine production, which partially depends on nicotinamide adenine dinucleotide (NAD+) biosynthesis. Blockade of glycolysis normalizes the differential expression of pro-inflammatory cytokines between control and SLC37A2 deficient macrophages. Conversely, overexpression of SLC37A2 lowers macrophage glycolysis and significantly reduces LPS-induced pro-inflammatory cytokine expression. In conclusion, our study suggests that SLC37A2 dampens murine macrophage inflammation by down-regulating glycolytic reprogramming as a part of macrophage negative feedback system to curtail acute innate activation., Graphical Abstract, Highlights • LPS treatment rapidly elevates macrophage SLC37A2 protein expression • SLC37A2 dampens early glycolytic reprogramming in acute macrophage inflammation • SLC37A2 suppresses macrophage cell-surface and endosomal TLR activation • SLC37A2 attenuates macrophage cellular ROS production, Cell Biology; Functional Aspects of Cell Biology; Immunology; Metabolism

Summary Many metabolic diseases disrupt endoplasmic reticulum (ER) homeostasis, but little is known about how metabolic activity is communicated to the ER. Here, we show in hepatocytes and other metabolically active cells that decreasing the availability of substrate for the tricarboxylic acid (TCA) cycle diminished NADPH production, elevated glutathione oxidation, led to altered oxidative maturation of ER client proteins, and attenuated ER stress. This attenuation was prevented when glutathione oxidation was disfavored. ER stress was also alleviated by inhibiting either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity increased NADPH production, glutathione reduction, and ER stress. Validating these findings, deletion of the Mitochondrial Pyruvate Carrier—which is known to decrease TCA cycle activity and protect the liver from steatohepatitis—also diminished NADPH, elevated glutathione oxidation, and alleviated ER stress. Together, our results demonstrate a novel pathway by which mitochondrial metabolic activity is communicated to the ER through the relay of redox metabolites., Graphical Abstract, Highlights • Inhibiting nutrient catabolism alleviates ER stress in metabolically active cells • NADPH production and glutathione redox link TCA activity to ER homeostasis • ER client protein oxidation, maturation, and ERAD respond to metabolic activity • Cells lacking the Mitochondrial Pyruvate Carrier are resistant to ER stress, biological sciences; cell biology; functional aspects of cell biology

Summary Signaling pathways that promote oligodendrocyte development improve oligodendrocyte regeneration and myelin recovery from demyelinating pathologies. Sox factors critically control myelin gene expression and oligodendroglial fate, but little is known about signaling events underlying Sox-mediated oligodendroglial regeneration. In this study of the SoxF member Sox17, we demonstrate that Sox17-induced oligodendrocyte regeneration in adult myelin lesions occurs by suppressing lesion-induced Wnt/beta-catenin signaling which is inhibitory to oligodendrocyte regeneration and by increasing Sonic Hedgehog/Smoothened/Gli2 activity. Hedgehog signaling through Smoothened critically supports adult oligodendroglial viability and is an upstream regulator of beta-catenin. Gli2 ablation in adult oligodendrocyte progenitor cells indicates that Gli2 regulates beta-catenin differentially in wild-type and Sox17-overexpressing white matter. Myelin lesions in Sox17-deficient mice show beta-catenin hyperactivation, regenerative failure, and loss of oligodendrogenesis, despite exogenous Hedgehog stimulation. These studies indicate the benefit of Sox17 signaling targets to enhance oligodendrocyte regeneration after demyelination injury by modulating both Hedgehog and Wnt/beta-catenin signaling., Graphical Abstract, Highlights • Sox17 suppresses demyelination-induced Wnt/beta-catenin increase • Sox17 enhances Hedgehog signaling in adult white matter lesions • Adult Hedgehog-Smoothened activity promotes oligodendrocyte maintenance • Oligodendrocyte regeneration by Smoothened activation is Sox17 dependent, Neuroscience; Molecular Neuroscience; Functional Aspects of Cell Biology

Summary Stress-driven ribosome dysfunction triggers an eIF2α-mediated integrated stress response to maintain cellular homeostasis. Among four key eIF2α kinases, protein kinase R (PKR) expression positively associates with poor prognoses for colorectal cancer (CRC) patients. We identified PKR-linked Wnt signaling networks that facilitate early inflammatory niche and epithelial-mesenchymal transitions of tumor tissues in response to ribosomal insults. However, the downstream Wnt signaling target fibrogenic connective tissue growth factor (CTGF/CCN2) regulates the nuclear translocation of β-catenin in a negative feedback manner. Moreover, dwindling expression of the Wnt/β-catenin pathway-regulator CTGF triggers noncanonical Wnt pathway-mediated exacerbation of intestinal cancer progression such as an increase in cancer stemness and acquisition of chemoresistance in the presence of ribosomal insults. The Wnt-CTGF-circuit-associated landscape of oncogenic signaling events was verified with clinical genomic profiling. This ribosome-associated wave of crosstalk between stress and oncogenes provides valuable insight into potential molecular interventions against intestinal malignancies., Graphical Abstract, Highlights • PKR expression positively associates with poor prognoses for CRC patients • CTGF/CCN2 mediates tumor niche remodeling under PKR-activating ribosomal stress • CTGF/CCN2 antagonism of Wnt regulates cancer stemness and chemoresistance, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Cancer

Summary Actin stress fibers guide cell migration and morphogenesis. During centripetal flow, actin transverse arcs fuse accompanied by the formation of myosin II stacks to generate mechanosensitive actomyosin bundles. However, whether myosin II stack formation plays a role in cell mechano-sensing has remained elusive. Myosin-18B is a “glue” molecule for assembling myosin II stacks. By examining actin networks and traction forces, we find that cells abolishing myosin-18B resemble Ca2+∕calmodulin-dependent kinase kinase 2 (CaMKK2)-defective cells. Inhibition of CaMKK2 activity reverses the strong actin network to thin filaments in myosin-18B-overexpressing cells. Moreover, AMP-activated protein kinase (AMPK) activation is able to relieve the thin stress fibers by myosin-18B knockout. Importantly, lack of myosin-18B compromises AMPK-vasodilator-stimulated phosphoprotein and RhoA-myosin signaling, thereby leading to defective persistent migration, which can be rescued only by full-length and C-extension-less myosin-18B. Together, these results reveal a critical role of myosin-18B in the mechanosensitive regulation of migrating cells., Graphical Abstract, Highlights • Myosin-18B knockout cells resemble cells dampening mechano-sensing signaling pathway • Myosin-18B depletion decreases the phosphorylation level of AMPK-VASP and MLC • Myosin-18B knockout cells show compromised persistent migration • The N-extension and coiled-coil domain of myosin-18B is indispensable in cell migration, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary Obesity commonly co-exists with fatty liver disease with increasing health burden worldwide. Family with Sequence Similarity 13, Member A (FAM13A) has been associated with lipid levels and fat mass by genome-wide association studies (GWAS). However, the function of FAM13A in maintaining metabolic homeostasis in vivo remains unclear. Here, we demonstrated that rs2276936 in this locus has allelic-enhancer activity in massively parallel reporter assays (MPRA) and reporter assay. The DNA region containing rs2276936 regulates expression of endogenous FAM13A in HepG2 cells. In vivo, Fam13a−/− mice are protected from high-fat diet (HFD)-induced fatty liver accompanied by increased insulin sensitivity and reduced glucose production in liver. Mechanistically, loss of Fam13a led to the activation of AMP-activated protein kinase (AMPK) and increased mitochondrial respiration in primary hepatocytes. These findings demonstrate that FAM13A mediates obesity-related dysregulation of lipid and glucose homeostasis. Targeting FAM13A might be a promising treatment of obesity and fatty liver disease., Graphical Abstract, Highlights • SNP rs2276936 regulates expression of endogenous FAM13A • Fam13a−/− mice are protected from high-fat-diet-induced obesity and fatty liver. • Fam13a−/− hepatocytes show increased mitochondrial respiration and AMPK activity, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary Activation of the hexosamine pathway (HP) through gain-of-function mutations in its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT-1) ameliorates proteotoxicity and increases lifespan in Caenorhabditis elegans. Here, we investigate the role of the HP in mammalian protein quality control. In mouse neuronal cells, elevation of HP activity led to phosphorylation of both PERK and eIF2α as well as downstream ATF4 activation, identifying the HP as a modulator of the integrated stress response (ISR). Increasing uridine 5′-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) levels through GFAT1 gain-of-function mutations or supplementation with the precursor GlcNAc reduces aggregation of the polyglutamine (polyQ) protein Ataxin-3. Blocking PERK signaling or autophagy suppresses this effect. In C. elegans, overexpression of gfat-1 likewise activates the ISR. Consistently, co-overexpression of gfat-1 and proteotoxic polyQ peptides in muscles reveals a strong protective cell-autonomous role of the HP. Thus, the HP has a conserved role in improving protein quality control through modulation of the ISR., Graphical Abstract, Highlights • Hexosamine pathway (HP) activation induces the integrated stress response (ISR) • HP activation ameliorates poly-glutamine aggregation via the ISR and autophagy • In C. elegans, the HP/ISR axis improves cell autonomous protein homeostasis • The proteoprotective role of longevity-associated HP is evolutionarily conserved, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary It has been well documented that the ER responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tm), and dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, indicating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that TG induces Golgi fragmentation through elevating intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including phorbol 12-myristate 13-acetate and histamine, modulates Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function., Graphical Abstract, Highlights • Thapsigargin (TG) treatment leads to Golgi fragmentation independent of ER stress • TG induces Golgi fragmentation through elevated cytosolic Ca2+ • TG-induced cytosolic Ca2+ spikes activate PKCα that phosphorylates GRASP55 • Histamine modulates the Golgi structure and function by a similar mechanism, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets., Graphical Abstract, Highlights • RBC and RBC-EVs contain unequal amount of RNA • Efficient detection of miRNA in vitro by MBs using nano-flow cytometry • CPPs effectively deliver cargo to cells and extracellular vesicles • MB-CPP can be used to detect cell-specific EV miRNAs, Biochemistry Methods; Biomolecules; Cell Biology; Functional Aspects of Cell Biology

Summary To quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins., Graphical Abstract, Highlights • MITNCAT combines BONCAT, pSILAC, and TMT to quantify protein synthesis rates • MITNCAT quantified up-regulation of protein folding chaperones during the UPR • MITNCAT revealed EGF-driven protein synthesis in four distinct temporal waves • MITNCAT identified delayed synthesis proteins with enriched rare codons, Functional Aspects of Cell Biology; Methodology in Biological Sciences; Proteomics

Summary Rapid cell migration requires efficient rear de-adhesion. It remains undetermined whether cells mechanically detach or biochemically disassemble integrin-mediated rear adhesion sites in highly motile cells such as keratocytes. Using molecular tension sensor, we calibrated and mapped integrin tension in migrating keratocytes. Our experiments revealed that high-level integrin tension abbreviated as HIT, in the range of 50–100 pN (piconewton) and capable of rupturing integrin-ligand bonds, is exclusively and narrowly generated at cell rear margin during cell migration. Co-imaging of HIT and focal adhesions (FAs) shows that HIT is produced to mechanically peel off FAs that lag behind, and HIT intensity is correlated with the local cell retraction rate. High-level molecular tension was also consistently generated at the cell margin during artificially induced cell front retraction and during keratocyte migration mediated by biotin-streptavidin bonds. Collectively, these experiments provide direct evidence showing that migrating keratocytes concentrate force at the cell rear margin to mediate rear de-adhesion., Graphical Abstract, Highlights • Tension sensor (ITS) mapped and calibrated integrin tension in migratory cells • High-level integrin tension (HIT) above 54 pN was observed in migrating keratocytes • HIT is exclusively and narrowly localized at the cell rear margin • HIT peels off focal adhesions at cell rear and facilitates keratocyte retraction, Functional Aspects of Cell Biology; Biophysics; Biomechanics

Summary Endoplasmic reticulum (ER)-to-Golgi anterograde transport is driven by COPII vesicles mainly composed of a Sec23/Sec24 inner shell and a Sec13/Sec31 outer cage. How COPII vesicles are tethered to the Golgi is not completely understood. We demonstrated here that PAQR3 can facilitate tethering of COPII vesicles to the Golgi. Proximity labeling using PAQR3 fused with APEX2 identified that many proteins involved in intracellular transport are in close proximity to PAQR3. ER-to-Golgi trafficking of N-acetylgalactosaminyltransferase-2 on removal of brefeldin A is delayed by PAQR3 deletion. RUSH assay also revealed that ER-to-Golgi trafficking is affected by PAQR3. The N-terminal end of PAQR3 can interact with the WD domains of Sec13 and Sec31A. PAQR3 enhances Golgi localization of Sec13 and Sec31A. Furthermore, PAQR3 is localized in the ERGIC and cis-Golgi structures, the acceptor sites for COPII vesicles. Taken together, our study uncovers a role for PAQR3 as a player in regulating ER-to-Golgi transport of COPII vesicles., Graphical Abstract, Highlights • ER-to-Golgi trafficking of COPII vesicles is affected by PAQR3 • PAQR3 is localized in the ERGIC and cis-Golgi structures • PAQR3 interacts with the coat proteins Sec13 and Sec31 of COPII vesicles • PAQR3 facilitates tethering of COPII vesicles to the Golgi apparatus, Molecular Biology Experimental Approach; Cell Biology; Functional Aspects of Cell Biology

Summary Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. Here, we show that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. Our findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals., Graphical Abstract, Highlights • Loss of Ssk leads to intestinal barrier dysfunction, dysbiosis, and early-onset death • Restoration of Ssk reverses each of these age-associated phenotypes • Up-regulation of Ssk prevents bacterial translocation upon pathogenic infection • Ssk up-regulation improves barrier integrity, limits dysbiosis, and extends lifespan, Molecular Mechanism of Behavior; Microbiome; Functional Aspects of Cell Biology; Model Organism

Summary We report that p73 is expressed in ovarian granulosa cells and that loss of p73 leads to attenuated follicle development, ovulation, and corpus luteum formation, resulting in decreased levels of circulating progesterone and defects in mammary gland branching. Ectopic progesterone in p73-deficient mice completely rescued the mammary branching and partially rescued the ovarian follicle development defects. Performing RNA sequencing (RNA-seq) on transcripts from murine wild-type and p73-deficient antral follicles, we discovered differentially expressed genes that regulate biological adhesion programs. Through modulation of p73 expression in murine granulosa cells and transformed cell lines, followed by RNA-seq and chromatin immunoprecipitation sequencing, we discovered p73-dependent regulation of a gene set necessary for cell adhesion and migration and components of the focimatrix (focal intra-epithelial matrix), a basal lamina between granulosa cells that promotes follicle maturation. In summary, p73 is essential for ovarian folliculogenesis and functions as a key regulator of a gene network involved in cell-to-cell adhesion and migration., Graphical Abstract, Highlights • p73 is required for murine ovarian folliculogenesis and proper corpus luteum formation • p73 loss leads to defects in progesterone signaling and mammary gland branching • In murine ovaries, p73 is expressed specifically in granulosa cells • p73 regulates components of the granulosa cell focimatrix and migration, Molecular Network; Functional Aspects of Cell Biology; Developmental Biology; Omics

Summary In response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition., Graphical Abstract, Highlights • ULK-101 has improved potency and selectivity when compared with SBI-0206965 • ULK-101 inhibits both the nucleation of autophagic vesicles and turnover • ULK-101 sensitizes KRAS-driven lung cancer cells to nutrient restriction • ULK-101 is a valuable molecular tool to study the function of ULK1 and autophagy, Therapeutics; Functional Aspects of Cell Biology; Cancer

Summary Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1β release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1β forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells., Graphical Abstract, Highlights • NLRP3 inflammasome induces necrotic cell death in the absence of caspase-1/11 • ASC initiates GSDME-dependent pyroptosis via caspase-8 • IL-1α, but not IL-1β, is released during Casp1/11-independent pyroptosis • Pharmacological inhibition of caspase-1 permits IL-1α release during pyroptosis, Immunology; Cell Biology; Functional Aspects of Cell Biology

Summary Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology., Graphical Abstract, Highlights • Two polybasic domains mediate septin 9 interactions with PIs • Human septins have amphipathic helices suitable for membrane binding • Septin 9 polybasic domains mediate the formation of septin higher-order structures • The mutation or depletion of septin polybasic domains induces Golgi fragmentation, Membrane Architecture; Molecular Interaction; Cell Biology; Functional Aspects of Cell Biology

Summary Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3β. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b−/− mice suppressed tumor growth to the same degree as Gsk3a/b−/− mice, whereas Gsk3a−/− mice behaved similarly to wild-type, revealing an important role for GSK-3β in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and β isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy., Graphical abstract, Highlights • Conditional knockouts reveal a non-redundant role for GSK-3α and GSK-3β in T cells. • GSK-3α and GSK-3β act together to enhance PD-1 expression. • GSK-3β depletion alone enhances T cell mediated tumor rejection. • GSK-3β plays an important role in regulating T cell-mediated anti-tumor immunity., Cancer; Cell biology; Functional aspects of cell biology; Immunology

Summary Clinically important broadly reactive B cells evolve during multiple infections, with B cells re-activated after secondary infection differing from B cells activated after a primary infection. Here we studied CD27highCD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct clusters were identified. The largest cluster 0/1 was plasma cell-related, with cells coding for serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. Cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway, and mitochondrial dysfunction. Clusters 2 and 3 showed a transcriptional footprint of T cell help, in line with activation from naive B cells or memory B cells. Our results contribute to the understanding of the parallel B cell activation events that occur in humans after natural primary and secondary infection., Graphical abstract, Highlights • CD27highCD38high plasmablasts from patients with dengue form three clusters • Clusters 0/1 express high levels, and more dengue-specific, antibodies • Clusters 0/1 are reminiscent of extrafollicular activation • Clusters 2 and 3 are metabolically active with an expression footprint of T cell help, Immunology; Cell biology; Functional aspects of cell biology; Systems biology

Summary Using in vivo muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, in vitro cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses Wisp1 expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation., Graphical abstract, Highlights • Widespread myogenic cell communication in muscle during mechanical overload (MOV) • Extracellular vesicles from myogenic progenitors repress Wisp1 in fibrogenic cells • Fibrogenic fate and chemokines are influenced by satellite cells early during MOV • Prior to fusion, satellite cell communication regulates long-term hypertrophy, Cell Biology; Functional Aspects of Cell Biology

Summary Homeostasis in the intestinal epithelium is maintained by Lgr5-positive intestinal stem cells (ISCs) located at the base of the crypt. The function of ISCs is reduced upon aging which leads to a decline of regeneration of the intestinal epithelium. We report that aged intestinal crypts present with an elevated activity of the small RhoGTPase Cdc42. Elevation of Cdc42 activity in young animals by genetic means causes premature ISC aging, whereas pharmacological suppression of elevated Cdc42 activity restores organoid formation potential in vitro. Consistent with a critical role of elevated Cdc42 activity in aged ISCs for a reduced regenerative capacity of aged ISCs, suppression of Cdc42 activity in vivo improves crypt regeneration in aged mice. Thus, pharmacological reduction of Cdc42 activity can improve the regeneration of aged intestinal epithelium., Graphical abstract, Highlights • Intestinal stem cells show high RhoGTPase Cdc42 activity compared to Paneth cells • Cdc42 activity is further increased in aged intestinal stem cells (ISCs) • Attenuation of Cdc42 activity ex vivo or in vivo improves the function of aged ISCs, Cell Biology; Stem Cells Research; Functional Aspects of Cell Biology

Summary Epithelial-to-mesenchymal transition (EMT), an evolutionary conserved phenomenon, has been extensively studied to address the unresolved variable treatment response across therapeutic regimes in cancer subtypes. EMT has long been envisaged to regulate tumor invasion, migration, and therapeutic resistance during tumorigenesis. However, recently it has been highlighted that EMT involves an intermediate partial EMT (pEMT) phenotype, defined by incomplete loss of epithelial markers and incomplete gain of mesenchymal markers. It has been further emphasized that pEMT transition involves a spectrum of intermediate hybrid states on either side of pEMT spectrum. Emerging evidence underlines bi-directional crosstalk between tumor cells and surrounding microenvironment in acquisition of pEMT phenotype. Although much work is still ongoing to gain mechanistic insights into regulation of pEMT phenotype, it is evident that pEMT plays a critical role in tumor aggressiveness, invasion, migration, and metastasis along with therapeutic resistance. In this review, we focus on important role of tumor-intrinsic factors and tumor microenvironment in driving pEMT and emphasize that engineered controlled microenvironments are instrumental to provide mechanistic insights into pEMT biology. We also discuss the significance of pEMT in regulating hallmarks of tumor progression i.e. cell cycle regulation, collective migration, and therapeutic resistance. Although constantly evolving, current progress and momentum in the pEMT field holds promise to unravel new therapeutic targets to halt tumor progression at early stages as well as tackle the complex therapeutic resistance observed across many cancer types., Graphical Abstract, Highlights • Partial EMT phenotype drives key hallmarks of tumor progression • Role of tumor microenvironment in pEMT phenotype via cellular signaling pathways • Engineering 3D in vitro models to study pEMT phenotype • Opportunities and challenges in understanding pEMT phenotype, Functional aspects of cell biology; cancer; bioengineering; tissue engineering

Summary Regulations of cell motility and proliferation are essential for epithelial development and homeostasis. However, it is not fully understood how these cellular activities are coordinated in epithelial collectives. In this study, we find that keratinocyte sheets exhibit time-dependent coordination of collective cell movement and cell cycle progression after seeding cells. Cell movement and cell cycle progression are coordinately promoted by Rac1 in the “early phase” (earlier than ∼30 h after seeding cells), which is not abrogated by increasing the initial cell density to a saturated level. The Rac1 activity is gradually attenuated in the “late phase” (later than ∼30 h after seeding cells), leading to arrests in cell motility and cell cycle. Intact adherens junctions are required for normal coordination between cell movement and cell cycle progression in both early and late phases. Our results unveil a novel basis for integrating motile and proliferative behaviors of epithelial collectives., Graphical Abstract, Highlights • Cell motility and cell cycle progression in keratinocyte sheets are temporally coordinated • Rac1 promotes both cell motility and cell cycle progression in keratinocyte sheets • Arrest of cell motility and cell cycle is associated with Rac1 deactivation • Adherens junction is required for coordinating cell motility and cell cycle, Biological Sciences; Cell Biology; Organization Aspects of Cell Biology; Functional Aspects of Cell Biology

Summary Lipid composition varies among organelles, and the distinct lipid composition is important for specific functions of each membrane. Lipid transport between organelles, which is critical for the maintenance of membrane lipid composition, occurs by either vesicular or non-vesicular mechanisms. In yeast, ceramide synthesized in the endoplasmic reticulum (ER) is transported to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. Here we show that a fraction of Tcb3p, a yeast tricalbin protein, localizes to ER-Golgi contact sites. Tcb3p and their homologs Tcb1p and Tcb2p are required for formation of ER-Golgi contacts and non-vesicular ceramide transport. Absence of Tcb1p, Tcb2p, and Tcb3p increases acylceramide synthesis and subsequent lipid droplet (LD) formation. As LD can sequester excess lipids, we propose that tricalbins act as regulators of ceramide transport at ER-Golgi contact sites to help reduce a potentially toxic accumulation of ceramides., Graphical Abstract, Highlights • Yeast tricalbin Tcb3p localizes at ER-Golgi contact sites • Lack of tricalbins reduces ER-Golgi contacts • Tricalbins regulate non-vesicular ceramide transport • Tricalbin deletion causes both acylceramide and lipid droplet accumulation, Cell Biology; Functional Aspects of Cell Biology

Summary Myeloid derived suppressor cells (MDSCs) are a population of immature myeloid cells that suppress adaptive immune function, yet the factors that regulate their suppressive function in patients with infection remain unclear. We studied MDSCs in patients with leprosy, a disease caused by Mycobacterium leprae, where clinical manifestations present on a spectrum that correlate with immunity to the pathogen. We found that HLA-DR-CD33+CD15+ MDSCs were increased in blood from patients with disseminated/progressive lepromatous leprosy and possessed T cell-suppressive activity as compared with self-limiting tuberculoid leprosy. Mechanistically, we found ER stress played a critical role in regulating the T cell suppressive activity in these MDSCs. Furthermore, ER stress augmented IL-10 production, contributing to MDSC activity, whereas IFN-γ allowed T cells to overcome MDSC suppressive activity. These studies highlight a regulatory mechanism that links ER stress to IL-10 in mediating MDSC suppressive function in human infectious disease., Graphical Abstract, Highlights • Cells with an MDSC phenotype are increased in blood and skin of patients with leprosy • Only MDSCs from patients with leprosy with disseminated infection suppress T cell function • MDSC function is dependent on increased ER stress and IL-10 production • MDSC function can be reversed in the presence of IFN-γ, Biological Sciences; Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Summary Cell-cycle quiescence is a common feature of early germline development in many animal species. In Drosophila germline progenitors (pole cells), both G2/M and G1/S transitions are blocked. G2/M transition is repressed by maternal Nanos through suppression of Cyclin B production. However, the molecular mechanism underlying blockage of G1/S transition remains elusive. We found that repression of miR-10404 expression is required to block G1/S transition in pole cells. Expression of miR-10404, a microRNA encoded within the internal transcribed spacer 1 of rDNA, is repressed in early pole cells by maternal polar granule component. This repression delays the degradation of maternal dacapo mRNA, which encodes an inhibitor of G1/S transition. Moreover, derepression of G1/S transition in pole cells causes defects in their maintenance and their migration into the gonads. Our observations reveal the mechanism inhibiting G1/S transition in pole cells and its requirement for proper germline development., Graphical Abstract, Highlights • Expression of mir-10404 encoded by rDNA region was increased in pgc− pole cells • Increased miR-10404 expression caused premature degradation of maternal dap mRNA • Premature degradation of maternal dap mRNA derepressed G1/S transition in pole cells • Derepression of G1/S transition impaired proper germline development, Cell Biology; Functional Aspects of Cell Biology; Integrative Aspects of Cell Biology

Summary Tgf-β signaling is a major antiproliferative pathway governing different biological functions, including cellular reprogramming. Upon injury, Müller glial cells of zebrafish retina reprogram to form progenitors (MGPCs) essential for regeneration. Here, the significance of Tgf-β signaling for inducing MGPCs is explored. Notably, Tgf-β signaling not only performs a pro-proliferative function but also is necessary for the expression of several regeneration-associated, essential transcription factor genes such as ascl1a, lin28a, oct4, sox2, and zebs and various microRNAs, namely, miR-200a, miR-200b, miR-143, and miR-145 during different phases of retinal regeneration. This study also found the indispensable role played by Mmp2/Mmp9 in the efficacy of Tgf-β signaling. Furthermore, the Tgf-β signaling is essential to cause cell cycle exit of MGPCs towards later phases of regeneration. Finally, the Delta-Notch signaling in collaboration with Tgf-β signaling regulates the critical factor, Her4.1. This study provides novel insights into the biphasic roles of Tgf-β signaling in zebrafish during retinal regeneration., Graphical Abstract, Highlights • Tgf-β signaling is essential for retinal progenitor proliferation and cell cycle exit • pSmad3 binds to 5GC and TIE elements to cause gene activations and repressions • Tgf-β signaling regulates Zebs and various miRNAs for cellular reprograming • Translation of Tgf-β signaling requires Mmp2/Mmp9 activity, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology

Summary Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular “compass” for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway., Graphical Abstract, Highlights • Daple localizes to cell junction, regulates planar cell migration • Localization requires Daple's C-terminal PDZ-binding motif (PBM) • The PBM binds a PDZ module of the polarity determinant PARD3 • The Daple⋅PARD3 interaction is regulated by tyrosine-based signals, Biological Sciences; Cell Biology; Functional Aspects of Cell Biology