The Francis Crick Institute

Endomembrane damage of intracellular vesicles triggers signals that activate membrane repair in mammalian cells to restore homeostasis. However, the signals that drive diverse membrane repair recruitment at the individual organelle level are unknown. Here by recording Ca2+ leakage history with a newly developed Ca2+ probe in human macrophages, we discovered that Ca²⁺ leakage serves as a conserved signal that triggers ATG8/LC3 lipidation after different types of sterile membrane damage. The damaged compartments consisted of both single membrane and multilayered membrane structures undergoing extensive membrane remodelling. We show the complexity and acidification of these ATG8/LC3-positive compartments depends on the nature of the membrane damage trigger. Functionally, the formation of these multimembrane ATG8/LC3-positive compartments restricted membrane damage independently of canonical autophagy and the recruitment of ESCRT components CHMP2A/CHMP4B. Altogether, we show that endolysosomal Ca²⁺ leakage triggers non-canonical LC3 lipidation on damaged membranes to promote membrane repair in human macrophages.

Abstract The bone marrow haematopoietic niche is composed of a diverse array of cell types and extracellular matrix components that together support healthy haematopoiesis. However, live imaging of the bone marrow microenvironment is hampered by tissue accessibility limitations. Using intravital imaging through a titanium imaging window, we investigated the dynamics of human haematopoietic cells and mesenchymal stromal cells within an ectopically implanted humanised scaffold in an immunodeficient murine host. These cell populations expand and differentiate over time, accompanied by progressive remodelling of the scaffold. We observe migration of murine endothelial cells into the scaffold, leading to the formation of a vascular network during the initial development of the humanised niche. Subsequently, the dense collagen matrix that makes up the implanted niche is altered and larger gaps form in regions populated by mesenchymal stroma cells. Collectively, our findings demonstrate dynamic remodelling of the extracellular milieu that supports haematopoietic cell development and establish a platform for longitudinal, in vivo investigation of these processes. Altogether, we describe a novel model that aligns with the 3R guiding principles and enables real-time assessment of bone marrow cell dynamics in vivo. Summary statement Ratcliffe and Mian et al. image in vivo dynamics of a bone marrow haematopoietic niche model.

At which developmental stage does the human brain start to differ from other mammals? We uncover human features of telencephalic patterning as early as the fourth post-conception week, comparing the early mouse and human forebrains using single-cell transcriptomics and 3D spatial multi-transcript imaging. In comparison to mice, the human telencephalon is delayed in ventral SHH signaling, accompanied by a human signature of anterior FGF signaling. These observations correlate with a less-resolved patterning and reduced progenitor diversity along the dorsoventral and anteroposterior axes, as well as a human early neurogenic signature. Our complementary approaches reveal a human divergence in allocation of telencephalic progenitor identities, propelled by temporal and qualitative differences in ventro-anterior signals following neural tube closure in the developing forebrain.

Abstract Transforming growth factor-beta (TGF-β), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-β inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-β (mmTGF-β), which potently and specifically inhibits TGF-β activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-β was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-β pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-β. Furthermore, we show that fusion of mmTGF-β to a CD44 binding domain enhances the inhibitory potential of mmTGF-β on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-β for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

Abstract Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.

The hypothalamic-pituitary-gonadal axis (HPG) controls pubertal development, sexual maturation, and fertility. We identified a role of hypothalamic microglia in controlling the HPG axis through receptor activator of nuclear factor κβ (Rank) signaling. Whole-body and microglia Rank depletion led to hypogonadotropic hypogonadism (HH) resulting from an alteration in gonadotropin-releasing hormone (GnRH) neuron function. In addition, we identified rare gene variants of RANK in patients with HH. Transcriptional profiling upon Rank loss revealed defective microglia activation and morphological alterations in the median eminence, decreasing the contacts and engulfment of GnRH terminal projections and impairing GnRH neuronal responses to kisspeptin. Overall, our data uncover the microglia as regulator of GnRH neuronal function through Rank signaling, with potential implications for reproductive maturation and fertility.

Immune responses to parasite infection involve the increased production of basophils and eosinophils. These two myeloid cell types have key roles in type 2 anti-parasite immunity1 and rely on GATA family transcription factors for their specification2,3. The first committed step in basophil and eosinophil production is generation of basophil-eosinophil-mast cell progenitors (BEMPs) from oligopotent erythroid-primed multipotent progenitors (EMPPs). However, it is not well established how immune responses act on progenitors to initiate type 2 myelopoiesis. Here we show that infection with the helminth Heligmosomoides polygyrus increases EMPP commitment to myeloid fate at the expense of erythropoiesis. Uponinfection with H. polygyrus, the IL-33 alarmin accumulated in the bone marrow, causing EMPPs to upregulate the GATA co-factor LMO4 and preferentially differentiate into myeloid cells. LMO4 was sufficient to instruct myeloid fate in EMPPs by interacting with GATA2, displacing the FOG1 co-factor and redistributing GATA binding from megakaryocyte-erythroid-specific to basophil, eosinophil and mast cell (BEM)-specific chromatin. Accordingly, mice carrying a GATA2 mutation that selectively impairs the LMO4-GATA2 interaction were deficient in GATA factor allocation to BEM chromatin, myeloid lineage commitment, basophil and eosinophil production, and parasite control. This identifies LMO4 as an IL-33-regulated master regulator of type 2 myelopoiesis, and transcription factor reallocation as a mechanism of lineage commitment.