Proneural-mesenchymal transition (PMT) is a phenotypic alteration and contributes to the malignant progression of glioblastoma (GBM). Macrophages, as a main infiltrating component of the tumor immune microenvironment (TIM), control the biological processes of PMT; however, the mechanisms driving this process remain largely unknown. Here, the overall landscape of tumor and nontumor cells was described by scMulti-omics technology. Then, we demonstrated that chitinase-3-like protein 1 (CHI3L1) played a critical role in maintaining mesenchymal (MES) status and reprogramming macrophage phenotype using C57BL/6 and NSG mice models derived from PN20 cells. Mechanistically, osteopontin (OPN)/ITGB1 maintained the activation of nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways by establishing a positive feedback loop with the CHI3L1-STAT3 axis, resulting in PMT. CHI3L1 enhanced the phosphorylation, nuclear localization, and transcriptional activity of STAT3 via directly binding its coiled-coil domain (CCD). Importantly, we screened and validated that hygromycin B (HB), an inhibitor of the STAT3-CCD domain, disrupted the CHI3L1-STAT3 interaction, thereby reducing the tumor burden in vitro and in vivo.

Cellular stresses, particularly endoplasmic reticulum (ER) stress induced by ER-to-Golgi transport blockade, trigger Golgi-independent secretion of cytosolic and transmembrane proteins. However, the molecular mechanisms underlying this unconventional protein secretion (UPS) remain largely elusive. Here, we report that an ER tubulovesicular structure (ER tubular body [ER-TB]), shaped by the tubular ER-phagy receptors ATL3 and RTN3L, plays an important role in stress-induced UPS of transmembrane proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Correlative light-electron microscopy analyses demonstrate the formation of ER-TB under UPS-inducing conditions in HEK293 and HeLa cells. Individual gene knockdowns of ATL3 and RTN3 inhibit ER-TB formation and the UPS of trafficking-deficient ΔF508-CFTR. Combined supplementation of ATL3 and RTN3L induces ER-TB formation and UPS. ATL3 also participates in the SARS-CoV-2-associated convoluted membrane formation and Golgi-independent trafficking of SARS-CoV-2 spike protein. These findings suggest that ER-TB serves a common function in mediating stress-induced UPS, which participates in various physiological and pathophysiological processes.

Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are genetic drivers in various cancers, including non-small cell lung cancer (NSCLC). However, the regulatory mechanisms underlying the progression of NSCLC driven by oncogenic KRAS mutants are incompletely understood. Here, we show that ubiquitin specific peptidase 25 (USP25) impedes ring finger protein 31 (RNF31)-mediated linear ubiquitination of KRAS oncogenic mutants (KRAS) independently of its deubiquitinase activity, which facilitates the plasma membrane (PM) localization and the downstream oncogenic signaling of KRAS. Importantly, knockout (KO) of USP25 effectively suppresses tumor growth and RAS signaling in KRAS-driven autochthonous NSCLC mouse models and xenograft models, which is restored by additional deletion or inhibition of RNF31. Notably, knockin of USP25 in KRas-driven NSCLC models fails to inhibit cancer progression and reconstitution of USP25 into USP25 KO A549 cells restores tumor growth. These findings identify previously uncharacterized roles of USP25 and RNF31 in oncogenic KRAS-driven NSCLC progression and provide potential therapeutic targets for KRAS-related cancers.

Plants induce the expression of the florigen FLOWERING LOCUS T (FT) in response to seasonal changes. FT is expressed in a distinct subset of phloem companion cells in Arabidopsis. Using tissue-specific translatome analysis, we discovered that the FT-expressing cells also express FLOWERING PROMOTING FACTOR 1 (FPF1)-LIKE PROTEIN 1 (FLP1), specifically under long-day conditions with the red/far-red ratio of natural sunlight. The master regulator of FT, CONSTANS (CO), is essential for FLP1 expression, suggesting that FLP1 is involved in the photoperiod pathway. We show that FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 also facilitates inflorescence stem elongation. Our cumulative evidence suggests that the small FLP1 protein acts as a mobile signal like FT. Taken together, FLP1 accelerates flowering in parallel with FT and orchestrates flowering and stem elongation during the reproductive transition.

The neural crest is a highly plastic stem cell population that represents an exception to the germ layer theory. Despite being of ectodermal origin, cranial neural crest cells can differentiate into skeletal derivatives typically formed by mesoderm. Here, we report that SMAD2/3-mediated transforming growth factor β (TGF-β) signaling enhances neural crest developmental potential in the chicken embryo. Our results show that TGF-β signaling modulates neural crest axial identity and directly controls the gene circuits that support skeletal differentiation. Cooperation between TGF-β and low levels of WNT signaling in the embryonic head activates cranial-specific cis-regulatory elements. Activation of TGF-β signaling reprogrammed trunk neural crest cells into adopting an anterior identity and led to the development of an improved protocol for the generation of human cranial neural crest cells. Our findings indicate TGF-β signaling is required for the specification of cranial neural crest cells, endowing them with the potential to give rise to the craniofacial skeleton.

Nucleotide-binding site, leucine-rich repeat (NLR) proteins activate a robust immune response on recognition of pathogen invasion. However, the function and regulatory mechanisms of NLRs during Puccinia striiformis f. sp. tritici (Pst) infection in wheat remain elusive. Here, we identify an ankyrin (ANK) repeat and tetratricopeptide repeat (TPR)-containing protein, TaANK-TPR1, which plays a positive role in the regulation of wheat resistance against Pst and the immune response of NLR. TaANK-TPR1 targets the NLR protein TaRPP13L1 (Recognition of PeronosporaParasitica 13-like 1) to facilitate its homodimerization and cell death to enhance the resistance of wheat against Pst. Meanwhile, TaANK-TPR1 binds to the TGACGT motif (methyl jasmonate-responsive element) of the TaRPP13L1 promoter and activates TaRPP13L1 transcription. Both TaANK-TPR1 and TaRPP13L1 respond to jasmonic acid (JA) signaling via the TGACGT element. Importantly, overexpressing TaRPP13L1 confers robust rust resistance without impacting important agronomic traits in the field. These findings identify a regulatory mechanism of NLR protein and provide targets for improving crop disease resistance.

Regulation of immunity is critical to balance growth and defense signaling. In this issue of Developmental Cell, Zhou et al. investigate two antagonistic receptors, SYR1 and SYR2, that control the growth-defense trade-off in plants. This work advances our understanding of the plant receptor kinase network in terms of molecular signaling and evolution of a feedback-based regulatory mechanism.

The concept that mechanical cell competition may contribute to tumor cell expansion has been widely discussed. However, whether this process could occur during natural tumor progression, as well as its underlying mechanisms and clinical implications, remains largely unknown. In this study, we observed that self-seeded tumor cell lines of human oral cancer, SCC9- and SCC25-seeded cells, exhibited a mechanical competitive advantage, outcompeted neighboring cells, and became "winner" cells. Mechanical compression-induced calcium influx activates myosin II in "loser" cells, leading to apoptotic nuclear breakdown and subsequent clearance. N-cadherin/Rac1/PAK1/myosin light-chain kinase (MLCK)-controlled myosin II inactivation endows cells with resistance to mechanical stress and superior cellular flexibility, thus providing a cell competition advantage to self-seeded cells. The activation of the N-cadherin/Rac1/PAK1/MLCK/myosin II signaling axis is associated with drug resistance. Together, these results suggest that N-cadherin/Rac1/PAK1/MLCK signaling-induced myosin II inactivation enables tumor cells to acquire resistance to mechanical stress and a competitive advantage. Our study also provides insights into drug resistance from a stress-sensitivity perspective.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

The plant funiculus anchors the developing seed to the placenta within the inner dorsal pod strands of the silique wall and directly transports nutrients to the seeds. The lignified vasculature critically supports nutrient transport through the funiculus. However, molecular mechanisms underlying lignified secondary cell wall (SCW) biosynthesis in the funiculus remain elusive. Here, we show that the transcription factor ZINC FINGER PROTEIN2 (ZFP2) represses SCW formation in the cortex cells that surround the vasculature. This function is essential for efficient nutrient loading into the seeds. Notably, ZFP2 directly acts on the SCW transcription factor NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) to repress cortex cell lignification, providing a mechanism of how SCW biosynthesis is restricted to the vasculature of the funiculus to ensure proper seed loading in Arabidopsis.

Chromatin domains delimited by CTCF can restrict the range of enhancer action. However, disruption of some domain boundaries results in mild gene dysregulation and phenotypes. We tested whether perturbing a domain with multiple developmental regulators would lead to more severe outcomes. We chose a domain with three FGF ligand genes-Fgf3, Fgf4, and Fgf15-that control different murine developmental processes. Heterozygous deletion of a 23.9-kb boundary defined by four CTCF sites led to ectopic interactions of the FGF genes with enhancers active in the brain and induced FGF expression. This caused orofacial clefts, encephalocele, and fully penetrant perinatal lethality. Loss of the single CTCF motif oriented toward the enhancers-but not the three toward the FGF genes-recapitulated these phenotypes. Our works shows that small sequence variants at particular domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation in development and disease.

Biomolecular condensates perform diverse physiological functions. Previous work showed that VASP, a processive actin polymerase, forms condensates that assemble and bundle actin. Here, we show that this behavior does not require proteins with specific polymerase activity. Specifically, condensates composed of Lamellipodin, a protein that binds actin but is not an actin polymerase, were also capable of assembling actin filaments. To probe the minimum requirements for condensate-mediated actin bundling, we developed an agent-based computational model. Guided by its predictions, we hypothesized that any condensate-forming protein that binds filamentous actin could bundle filaments through multivalent crosslinking. To test this, we added a filamentous-actin-binding motif to Eps15, a condensate-forming protein that does not normally bind actin. The resulting chimera formed condensates that facilitated efficient assembly and bundling of actin filaments. Collectively, these findings broaden the family of proteins that could organize cytoskeletal filaments to include any filamentous-actin-binding protein that participates in protein condensation.

Macrophages possess the capacity to degrade extracellular matrix (ECM), but the specific roles of different cytoskeletal structures in controlling this process are incompletely understood. Here, we report that the inward flow of actin stress fibers delivers endocytosed ECM for lysosomal elimination, replenishing the pool of enzymes for extracellular ECM hydrolysis in actin-rich podosomes. Vimentin deficiency disrupted the balance between stress fibers and podosomes, impairing ECM degradation through integrin CD11b in THP-1 macrophages. In lung adenocarcinoma patient samples, M2-type macrophages exhibit a tighter podosome organization, surrounded by compact vimentin filaments, than M1-type. In vitro experiments verified that the invasion ability of A549 lung carcinoma cells was enhanced when accompanied by wild type, but not vimentin knockout M2-type THP-1, macrophages. Subcutaneous injections of macrophages and tumor cells in nude mice showed that vimentin in macrophages can reduce tumor collagen fibers. Together, our findings provide insights into the cytoskeletal dynamics governing macrophage ECM degradation.

We previously demonstrated that long-term trained immunity (TRIM) involves adaptations that imprint innate immune memory in long-lived myelopoiesis precursors and their progeny, monocytes/macrophages and neutrophils, which thereby acquire enhanced responsiveness to future challenges. Here, we show that a distinct component of myeloid biology, osteoclastogenesis, can also undergo innate immune training. Indeed, β-glucan-induced TRIM was associated with an increased osteoclastogenesis bias in the bone marrow and an expansion of monocytes/osteoclast progenitors in the periphery, resulting in aggravated severity of experimental periodontitis and arthritis. In the setting of trained inflammatory osteoclastogenesis, we observed transcriptomic rewiring in synovial myeloid cells of arthritic mice, featuring prominent upregulation of the transcription factor melanogenesis-associated transcription factor (MITF). Adoptive transfer of splenic monocytes from β-glucan-trained mice to naive recipients exacerbated arthritis in the latter in a strictly MITF-dependent manner. Our findings establish trained osteoclastogenesis as a maladaptive component of TRIM and potentially provide therapeutic targets in inflammatory bone loss disorders.

Despite limited translational capacity, senescent cells trigger inflammation by upregulating the translation and secretion of proinflammatory factors. In this issue of Developmental Cell, Kim et al. identify that altered autophagy and SFPQ-dependent EIF4H splicing during senescence redirects translation to promote inflammation, informing therapeutic strategies for cancer and other age-related diseases.

In this issue of Developmental Cell, Tang et al. identify CD74 neutrophils as a subpopulation promoted by IL-8 in the tumor microenvironment of non-small lung cancer (NSCLC). These CD74 neutrophils support anti-tumor T cell responses and enhance the efficacy of anti-PD-1 immunotherapy and targeted therapy in NSCLC.

Diets composed of chemically pure components (holidic diets) are useful for determining the metabolic roles of individual nutrients. For the model organism Drosophila melanogaster, existing holidic diets are unable to support the rapid growth characteristic of the larval stage. Here, we use a nutrient co-optimization strategy across more than 50 diet variants to design HolFast, a holidic medium tailored to fast larval growth and development. We identify dietary amino acid ratios optimal for developmental speed but show that they compromise survival unless vitamins and sterols are co-optimized. Rapid development on HolFast is not improved by adding fatty acids, but it is dependent upon their de novo synthesis in the fat body via fatty acid synthase (FASN). HolFast outperforms other holidic diets, supporting rates of growth and development close to those of yeast-based diets and, under germ-free conditions, identical. HolFast has wide applications in nutritional and metabolic studies of Drosophila development.

TMC1, a unique causative gene associated with deafness, exhibits variants with autosomal dominant and recessive inheritance patterns. TMC1 codes for the transmembrane channel-like protein 1 (TMC1), a key component of the mechano-electrical transduction (MET) machinery for hearing. However, the molecular mechanism of Ca regulation in MET remains unclear. Calcium and integrin-binding protein 2 (CIB2), another MET component associated with deafness, can bind with Ca. Our study shows that TMC1-CIB2 complex undergoes a Ca-induced conformational change. We identified a vertebrate-specific binding site on TMC1 that interacts with apo CIB2, linked with hearing loss. Using an ex vivo mouse organotypic cochlea model, we demonstrated that disruption of the calcium-binding site of CIB2 perturbs the MET channel conductivity. After systematically analyzing the hearing loss variants, we observed dominant mutations of TMC1 cluster around the putative ion pore or at the binding interfaces with CIB2. These findings elucidate the molecular mechanisms underlying TMC1-linked hearing loss.

Most eukaryotes inherit only maternal mitochondria. The reasons for paternal mitochondrial elimination and the impacts of persistent paternal mitochondria on animals remain elusive. We show that undegraded paternal mitochondria in autophagy-deficient C. elegans embryos are gradually excluded from germ blastomeres through asymmetric partitioning during cell divisions. The embryonic cortical flow drives anterior-directed movements of paternal mitochondria and contributes to their asymmetric apportioning between two daughter blastomeres. By contrast, autophagosome-enclosed paternal mitochondria cluster around and segregate with centrosomes during mitosis and are rapidly degraded through lysosomes concentrated near centrosomes. Failure to exclude persistent paternal mitochondria from the germ blastomere at first cleavage causes their enrichment in the descendant endomesodermal (EMS) blastomere, leading to elevated reactive oxygen species levels, elongated EMS lineage durations, and increased embryonic lethality, which antioxidant treatments can suppress. Thus, regulated paternal mitochondrial distribution away from germ blastomeres is a fail-safe mechanism, protecting embryo development and maternal mitochondrial inheritance.

Deubiquitinating enzymes play crucial roles in various cellular activities, yet their involvement in central nervous system (CNS) vascularization and barrier function remains elusive. Canonical Wnt signaling is essential for proper CNS vascularization and barrier maintenance. Using a loss-of-function screening for Wnt-signaling activity, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a key regulator in brain endothelial cells (BECs). Endothelium-specific Usp9x knockout mice exhibit reduced Wnt-signaling activity, compromising CNS vascularization and barrier function during development. Activation of Wnt signaling rescues these defects. Mechanistically, we identified β-catenin as a direct substrate of USP9X, with USP9X catalyzing K48 polyubiquitin chains to stabilize β-catenin. In pathological mouse models of impaired CNS vascular barrier function, including intracerebral hemorrhage and an oxygen-induced retinopathy, loss of Usp9x intensifies barrier disruption, accentuating defects. This finding implicates USP9X as a critical regulator of CNS vascularization and barrier function through Wnt signaling, offering insights into CNS disease implications.

Pulmonary vascular remodeling (PVR), encompassing microvascular loss and muscularization, contributes to multiple respiratory diseases. c-Kit cells exhibit differentiation potential into both endothelial cells (ECs) and smooth muscle cells. The potential role of lung c-Kit cell differentiation in PVR, however, remains unclear. Lung c-Kit cells increase in pulmonary hypertension patients and in the SU5416/hypoxia (SuHx)-induced PVR mouse model. Employing genetic lineage tracing and single-cell RNA sequencing (scRNA-seq), we elucidate that lung-resident c-Kit cells display an aerocyte and venular endothelial differentiation in the SuHx model. Ablation of tissue-resident c-Kit cells exacerbates PVR. We identify an Nr2f2-expressing c-Kit cell subgroup, which exhibitsvenous EC differentiation and increases during PVR. Notably, the elevation of Nr2f2 in c-Kit cells via AAV enhances differentiation and mitigates PVR. These findings underscore the protective role of lung tissue-resident c-Kit cells in PVR, achieved by differentiating into mature ECs. Targeting NR2F2 expression in c-Kit cells emerges as a promising strategy for reversing PVR.