Cell Structure and Function

Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.

Coleoid cephalopods (cuttlefish, octopus, and squid) are marine mollusks with elaborate nervous systems that support a diverse repertoire of complex behaviors. These include the neural control of the color, pattern, and texture of the skin, facilitating both adaptive camouflage and innate patterning that may reflect internal state. The development of transgenic cephalopods expressing fluorescent proteins, optogenetic actuators, and reporters of neural activity would contribute a new and important technology to cephalopod biology. The generation of transgenic cephalopods, however, has remained a major challenge. Here, we report the development of stable transgenic dwarf cuttlefish (Ascarosepion bandense) expressing ubiquitous nuclear-localized mScarlet, a red fluorescent protein. We evaluated multiple strategies for transgenesis, and established cuttlefish lines using both CRISPR and the transposons Sleeping Beauty and Minos. The stable expression of transgenes enabled live imaging of cell dynamics during embryonic development. The Minos transposon emerged as the most efficient transgenesis strategy and is adaptable to promoters and transgenes of choice. These strategies now enable the generation of diverse genetic tools for mechanistic studies of cephalopod biology.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Transgenic GAL4 lines are widely used in Drosophila to label and manipulate specific cell types, but the resulting reporter expression patterns do not always represent ongoing driver activity. We generated a collection of neuropeptide T2A-GAL4 knock-ins and characterized their expression by native-fluorescence imaging using UAS-CD8::GFP and a Brp-miRFP680 knock-in, eliminating the need for immunostaining. During characterization of these lines, we observed cases in which adult GFP patterns did not match the on-going GAL4 activity, as well as cases in which GAL4 produced adverse cellular effects. Using temperature-sensitive GAL80, we found that adult GFP expression in certain neurons driven by CCHa2-T2A-GAL4 and Dsk-T2A-GAL4 depended on GAL4 activity before eclosion and was not reproduced by adult-only induction. Several GAL4 lines also showed dominant developmental phenotypes. Heterozygous Burs-T2A-GAL4 animals, for example, displayed wing-expansion defects that were fully suppressed by GAL80. Severe wing defects were not accompanied by a reduction in the number of reporter-labeled neurons before eclosion, but the morphology of these neurons were affected. These observations illustrate how developmental driver history and GAL4-associated cellular effects can influence reporter patterns in the adult nervous system.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Targeted protein turnover of the transcription factor C/EBP is enhanced by the Tribbles (Trib) pseudokinase family of adaptor proteins, which bind to a conserved degron motif in C/EBP substrate and recruit a cognate COP1 E3 ligase to ubiquitinate C/EBP for degradation. We identified a conserved degron in the Drosophila C/EBP ortholog Slbo and fused this motif to a fluorescent protein (GFP or RFP) under the control of a constitutive promoter. Transgenic flies expressing this fluorescent protein (FP)::degron transgene, which we call the Fluorescent Ubiquitination-based Trbl Turnover Indicator (or FUTTI) sensor, showed reduced levels of fluorescence in response to increased levels of Drosophila Tribbles (Trbl) and conversely, increased levels when Trbl activity was reduced. Mutations in the Trbl conserved pseudokinase substrate binding pocket and in a divergent PEST-like motif in the C-terminal tail region disrupted Trbl activity in the FUTTI assay. These data indicate that FUTTI will be a valuable tool to identify sites of Trbl activity in vivo and the critical features of the protein required for function.

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX < 10%) and high (IRX > 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B'', which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Abstract The canonical WNT signaling pathway guides cell growth throughout the human lifespan. In adults, WNT plays an essential role in tissue homeostasis by maintaining tissue stem cells. In the intestine, the WNT transcription factor, TCF4, is necessary for stem cell maintenance. Aberrant activation of WNT signaling is generally accepted as an initiating event in colon cancer, leading to constitutive TCF4 activity. To investigate the role of this key transcription factor in global gene regulation, we abrogated TCF4 expression in colon cancer cells. Loss of TCF4 function resulted in the over-expression of the WNT transcription factor, LEF1. LEF1 was transcriptionally competent and over-compensated for TCF4 (TCF7L2) silencing in a WNT reporter assay. TCF4 enlisted the transcriptional repressor CtBP1, and bound the LEF1 promoter, indicative of direct repression. TCF4 and LEF1 drove different transcriptional programs, with TCF4 favoring MYC and cell cycle progression, while LEF1 favored immune-related gene signatures. TCF4 was the primary regulator of MYC expression, yet also the mediator of LEF1 repression, suggestive of competition centered on TCF4 transcriptional output despite the {beta}-catenin saturated colon cancer nucleus. We conclude that TCF4 has dual activator-repressor activity in colon cancer.

Stromal cell composition has been proposed as a potential biomarker in ductal carcinoma in situ (DCIS) for assessing the risk of subsequent recurrence or progression to invasive breast cancer (IBC). However, the characteristics of the tumor microenvironment within DCIS associated with these outcomes remain poorly understood. Here, we perform spatial transcriptomic analyses of 49 primary DCIS specimens from patients with no recurrence, DCIS recurrence, or recurrence as IBC. Cell mapping reveals spatially organized stromal cell gradients within DCIS, with enrichment of TREM2+ macrophages and myofibroblastic cancer-associated fibroblasts (myCAFs) in tumor-proximal niches. Notably, the abundance of TREM2+ macrophages near tumor nests is associated with subsequent recurrence as IBC. Moreover, ligand-receptor pair analysis identifies GRN--SORT1, SPP1--CD44, and LGALS9--CD44 interactions as spatially plausible signaling interactions between TREM2+ macrophages and epithelial cells. Finally, these findings are validated at the protein level by multiplex immunostaining of serial sections. This study characterizes the spatial landscape of the DCIS tumor microenvironment associated with subsequent recurrence, providing a basis for developing biomarkers of disease progression and identifying potential therapeutic targets.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

Nucleosomes regulate chromatin folding, accessibility, and factor recruitment. Current models primarily attribute these functions to histone tail modifications, while the core is largely viewed as a structural scaffold. Yet subtle changes within the nucleosome core can produce profound functional consequences, and the mechanisms underlying these effects remain unclear. Here, we describe nucleosome core allostery as a fundamental principle of chromatin regulation that amplifies the impact of minimal nucleosome variations. Leveraging natural differences between H2A.Z variants, we show that the nucleosome core encodes distinct conformational dynamics that propagate allosterically, thereby controlling nucleosome accessibility and recognition by chromatin factors. As a result, a single buried amino acid substitution alone is sufficient to reprogram nucleosome dynamics and bias cell identity. Our findings establish the nucleosome core as an allosteric regulatory module and provide a generalizable framework for how subtle variation within nucleosomes is amplified into diverse biological outcomes in development and disease.

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata can cause respiratory irritation in beach users, yet their environmental drivers and temporal dynamics remain poorly understood. Here, we use a dynamical-systems approach to investigate how environmental conditions shape bloom dynamics from long-term time series of algal abundance and abiotic variables. Using the global modelling framework GPoM, we reconstruct nonlinear ordinary differential equation systems and identify candidate models capable of reproducing key features of the observed dynamics, including chaotic behaviour. Topological and statistical analyses reduce the candidate models to two systems that best capture the observed distributions and dynamical patterns. These models reveal distinct combinations of environmental conditions associated with bloom occurrence and amplitude, providing a framework for interpreting bloom trajectories and a potential basis for short-term prediction.

NF-{kappa}B must remain bound to chromatin long enough to activate innate immune genes, but what stabilizes this transcriptionally productive state is unknown. Here, we identify the Integrator complex as an essential determinant of NF-{kappa}B chromatin residence in mouse and human cells. Integrator associated with NF-{kappa}B and co-occupied its genomic targets. Single-molecule tracking revealed that Integrator markedly prolonged the chromatin residence of RELA molecules in a low-mobility state. Catalytically inactive INTS11-E203Q fully restored NF-{kappa}B-dependent transcription, separating this chromatin-retention function from RNA cleavage. Conditional Ints11 deletion abolished TLR4-dependent dendritic-cell activation in adult mice and impaired LPS-induced transcription in mouse embryonic fibroblasts. Loss of Integrator in human cancer cells similarly suppressed TNF-induced inflammatory gene expression. These findings uncover a noncatalytic function for Integrator that stabilizes NF-{kappa}B at enhancers, complementing its established catalytic control of RNA polymerase II pause release. By coupling signal recognition at enhancers to productive elongation, Integrator provides a molecular solution to a central challenge of multicellularity: converting transient extracellular cues into coherent gene-expression programs across complex chromatin landscapes.

Subcellular mass spectrometry imaging (MSI) has required specialized instrumentation and approaches. Additionally, data analysis has required expensive proprietary software, or code-based open-source tools. These barriers render spatial metabolomics less accessible than peer spatial omics techniques (transcriptomics, proteomics). Here we demonstrate subcellular MSI on standard commercial hardware, analyzed using code-free, open-source software. An inexpensive, fully reversible modification of a stock DESI source takes the platform past its typical 5-10 m pixels to routine 2x2 m pixels, and 1x1 m in proof-of-principle experiments. At a tissue-glass boundary imaged at 2x2 m, tissue-specific ions showed no measurable spillover onto the adjacent glass within the {+/-}4.4 m uncertainty of the edge position. To make these data interpretable, we extend MSI.EAGLE, an open-source, vendor-agnostic application with two new methods: i) Spatial UMAP preserves tissue architecture alongside subcellular detail, where conventional clustering breaks apart at high resolution; ii) Histology Fit performs landmark-free co-registration, and recovers synthetic misalignments of up to 8 m to within one pixel of a reference registration. Together, these enable previously high-burden spatial metabolomics experiments. In mouse brain tissue, we demonstrate genuine subcellular signal by resolving mitochondrial cardiolipin into perinuclear puncta, quantifying cytoplasmic enrichment across 6,073 cells. Cell identities are assigned to MSI pixels using histology-predicted transcriptomics, linking cell provenance to metabolism in 2D space directly. Finally, we recover cell-type-specific metabolomes consistent with the transcriptomic identities. This workflow allows cellular and subcellular spatial metabolomics linked to cell provenance, with only stock MSI hardware, open-source GUI-based software, and common histology techniques, putting it within reach of many researchers and laboratories.

Understanding when species diversification and phenotypic evolution are coupled remains a central challenge in macroevolution. Such coupling may depend on the traits considered, phenotypic integration, or the geographic context in which diversification occurs. Here, we investigate these relationships in Ithomiini, a diverse Neotropical butterfly tribe whose diversification is strongly associated with the Andes-Amazonia system. We quantified forewing and hindwing size and shape for 1,618 individuals representing 291 species using geometric morphometrics, and combined these data with phylogenetic comparative methods, diversification-rate estimates, and spatial analyses of species distributions. Evolutionary rate shifts were largely trait- and wing-specific, indicating limited coupling between forewing and hindwing evolution. Across the phylogeny, wing size evolution and extant wing size were not associated with species diversification, nor was hindwing shape evolution. By contrast, forewing shape evolutionary rates showed a positive association with diversification. Spatial analyses revealed that this coupling was geographically concentrated in the Andes, where high diversification rates coincided with elevated forewing size and shape diversity and increased forewing evolutionary rates. Hindwing diversity, by contrast, more closely tracked phylogenetic diversity and showed weaker or opposing relationships with diversification. Our results show that diversification and phenotypic evolution in Ithomiini are neither generally coupled nor fully independent, but linked in trait-specific and geographically structured ways. More broadly, they highlight the need to decompose complex phenotypes and integrate spatial context when testing macroevolutionary coupling.

Chemical modifications of RNA provide an additional regulatory layer essential for development. N6A methyladenosine (m6A) is the most abundant internal mRNA mark, but how site specificity, stoichiometry, and cellular context together determine mRNA fate remains unclear. Here, we define these rules in cardiogenesis by combining single-molecule nanopore direct RNA sequencing (DRS) with a CRISPRdCas13 epitranscriptomic editing platform in human induced pluripotent stem cells and a conditional Mettl14 cardiac knockout in mice, integrated with multiomics, to map m6A sites and quantify stoichiometry. We uncover a bimodal m6A code in which low versus high modification ratios are associated with opposite mRNA and protein outcomes. We further show that Mettl14 loss causes developmental cardiomyopathy and embryonic lethality by disrupting the glycolysis to oxidative phosphorylation (OXPHOS) metabolic switch, and identify an m6A YY1 (Yin Yang 1) axis that post transcriptionally controls OXPHOS genes. Finally, targeted installation of m6A at defined sites using CRISPR/dCas13 reveals that m6A effects on YY1 are strongly locus and context dependent. Together, these findings establish m6A as a site and stoichiometry dependent regulatory code interpreted by cellular context to direct cardiac metabolic maturation. These principles are likely to extend to other developmental and physiological contexts in which precise control of metabolic state and gene dosage is required.

High-grade serous ovarian cancer progresses within a lipid-rich ascites microenvironment that engages stress-adaptation programs supporting malignant growth, yet how failed stress resolution influences tumor progression remains incompletely defined. Here, we show that unresolved lysosomal stress restrains tumor progression by sustaining tumor cell-intrinsic STING activity and reorganizing antitumor immunity. We identify BLTP3A as a lysosomal stress-resolution factor in ovarian cancer and show that the common germline missense variant BLTP3A M1098T increases stress-induced lysosomal damage. Patients carrying M1098T had longer overall survival and tumors enriched for cytotoxic T cell-myeloid neighborhoods. In syngeneic tumors, Bltp3a loss or orthologous variant expression delayed progression and generated Cxcl9+ antigen-presenting macrophage niches; M1098T-expressing human tumors similarly promoted interferon-responsive myeloid and T-cell enrichment in humanized mice. Mechanistically, ascites-induced lysosomal stress activated a STING-ATF4-BLTP3A feedback program. BLTP3A dysfunction disrupted CASM-associated lysosomal stress resolution, resulting in prolonged STING activity and sustained tumor-derived IFN-{lambda} without increased cGAMP. Tumor-cell STING and IFN-{lambda} were required for immune remodeling and tumor control. Thus, BLTP3A-dependent lysosomal stress resolution constrains the persistence of stress-induced STING signaling, whereas failure of this response sustains a tumor-intrinsic inflammatory program that organizes protective antitumor immunity.

Microglia, the immune cells of the brain parenchyma, play a pivotal role in neurodevelopment and neuroprotection. Human stem cell-derived microglia-like cells (MGL) offer a valuable, human-relevant system to study microglia in vitro. However, their cell states and intercellular interactions within complex 3-dimensional (3D) tissue contexts in vitro remain underexplored. Here, we analyze the gene expression profiles of MGL generated by two differentiation protocols in conventional 2D cultures. We show that co-culture with neural organoids promotes a more mature, in vivo-like MGL phenotype but this signature is lost during long-term co-culture. By varying media compositions, we modulate 2D MGL states toward more active or more homeostatic identities, which may enhance the similarity to primary microglia identities upon subsequent co-culture. Moreover, forced expression of the microglial lineage regulator SALL1 shifts MGL away from a general myeloid state toward a more defined pre-microglial fate. SALL1 overexpression within a 3D neural organoid environment further amplifies the shift toward a homeostatic developmental microglia population. Collectively, our transcriptomic analysis delineates human MGL states in 2D and 3D contexts, refines existing differentiation protocols toward more authentic MGL, and establishes a framework to investigate the intricate microglia-neural tissue interplay.