ABSTRACT
Infantile hemangioma (IH) is the most common tumor in children and a paradigm for pathological vasculogenesis, angiogenesis and regression. Propranolol is the mainstay of treatment for IH. It inhibits hemangioma vessel formation via a ß-adrenergic receptor independent off-target effect of its R(+) enantiomer on the endothelial specific transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18). Transcriptomic profiling of patient-derived hemangioma stem cells uncovered the mevalonate pathway (MVP) as a target of R(+) propranolol. Loss of SOX18 function confirmed R(+) propranolol mode of action on the MVP. Functional validation in preclinical IH models revealed that statins - targeting the MVP - are potent inhibitors of hemangioma vessel formation. We propose a novel SOX18-MVP-axis as a central regulator of IH pathogenesis and suggest statin repurposing to treat IH. Our findings reveal novel pleiotropic effects of beta-blockers and statins acting on the SOX18-MVP axis to disable an endothelial specific program in IH, which may impact other scenarios involving pathological vasculogenesis and angiogenesis.
ABSTRACT
Insoluble cytoplasmic aggregate formation of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases including Amyotrophic Lateral Sclerosis. TDP-43 localizes predominantly in the nucleus, arranging itself into dynamic condensates through liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, contributing to the transition of liquid-like biomolecular condensates into solid-like aggregates. However, to date it has been a challenge to study the dynamics of this process in vivo. We demonstrate through live imaging that human TDP-43 undergoes nuclear condensation in spinal motor neurons in a living animal. RNA-binding deficiencies as well as post-translational modifications can lead to aberrant condensation and altered TDP-43 compartmentalization. Single-molecule tracking revealed an altered mobility profile for RNA-binding deficient TDP-43. Overall, these results provide a critically needed in vivo characterization of TDP-43 condensation, demonstrate phase separation as an important regulatory mechanism of TDP-43 accessibility, and identify a molecular mechanism of how functional TDP-43 can be regulated.
Subject(s)
DNA-Binding Proteins , Motor Neurons , RNA-Binding Proteins , Animals , Humans , Mice , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/genetics , Biomolecular Condensates/metabolism , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Homeostasis , Motor Neurons/metabolism , Mutation , Protein Binding , Protein Processing, Post-Translational , RNA/metabolism , RNA/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
Advanced strategies to interconvert cell types provide promising avenues to model cellular pathologies and to develop therapies for neurological disorders. Yet, methods to directly transdifferentiate somatic cells into multipotent induced neural stem cells (iNSCs) are slow and inefficient, and it is unclear whether cells pass through a pluripotent state with full epigenetic reset. We report iNSC reprogramming from embryonic and aged mouse fibroblasts as well as from human blood using an engineered Sox17 (eSox17FNV). eSox17FNV efficiently drives iNSC reprogramming while Sox2 or Sox17 fail. eSox17FNV acquires the capacity to bind different protein partners on regulatory DNA to scan the genome more efficiently and has a more potent transactivation domain than Sox2. Lineage tracing and time-resolved transcriptomics show that emerging iNSCs do not transit through a pluripotent state. Our work distinguishes lineage from pluripotency reprogramming with the potential to generate more authentic cell models for aging-associated neurodegenerative diseases.
Subject(s)
Neural Stem Cells , Humans , Animals , Mice , Aging , Epigenomics , Gene Expression Profiling , HMGB Proteins , SOXF Transcription Factors/geneticsABSTRACT
Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.
Subject(s)
Endothelial Cells , Lymphatic Vessels , Humans , Mice , Animals , Endothelial Cells/metabolism , Lymphatic Vessels/metabolism , Gene Expression Regulation , Endothelium, Vascular , Transcription Factors/metabolism , Lymphangiogenesis/genetics , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolismABSTRACT
Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of beta blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSCs) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial cell differentiation. As our previous work implicated the transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small-molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with efficacy similar to that seen with the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical, and quantitative molecular imaging assays, we show that R(+) propranolol directly interfered with SOX18 target gene trans-activation, disrupted SOX18-chromatin binding dynamics, and reduced SOX18 dimer formation. We propose that the R(+) enantiomers of widely used beta blockers could be repurposed to increase the efficiency of current IH treatment and lower adverse associated side effects.
Subject(s)
Atenolol/pharmacology , Hemangioma , Neoplastic Stem Cells/metabolism , Neovascularization, Pathologic , Propranolol/pharmacology , Animals , Hemangioma/blood supply , Hemangioma/drug therapy , Hemangioma/metabolism , Humans , Mice , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.
Subject(s)
Glomerulonephritis/genetics , Hypotrichosis/genetics , Lymphedema/genetics , SOXF Transcription Factors/genetics , Telangiectasis/genetics , Transcription, Genetic , Animals , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Gene Expression Regulation , Gene Regulatory Networks , Genes, Reporter , Glomerulonephritis/metabolism , Glomerulonephritis/pathology , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Hypotrichosis/metabolism , Hypotrichosis/pathology , Luciferases/genetics , Luciferases/metabolism , Lymphedema/metabolism , Lymphedema/pathology , MEF2 Transcription Factors/genetics , MEF2 Transcription Factors/metabolism , Mice , Mutation , SOXF Transcription Factors/metabolism , Single Molecule Imaging , Telangiectasis/metabolism , Telangiectasis/pathologyABSTRACT
Single molecule localisation microscopy (SMLM) is a powerful tool that has revealed the spatial arrangement of cell surface signalling proteins, producing data of enormous complexity. The complexity is partly driven by the convolution of technical and biological signal components, and partly by the challenge of pooling information across many distinct cells. To address these two particular challenges, we have devised a novel algorithm called K-neighbourhood analysis (KNA), which emphasises the fact that each image can also be viewed as a composition of local neighbourhoods. KNA is based on a novel transformation, spatial neighbourhood principal component analysis (SNPCA), which is defined by the PCA of the normalised K-nearest neighbour vectors of a spatially random point pattern. Here, we use KNA to define a novel visualisation of individual images, to compare within and between groups of images and to investigate the preferential patterns of phosphorylation. This methodology is also highly flexible and can be used to augment existing clustering methods by providing clustering diagnostics as well as revealing substructure within microclusters. In summary, we have presented a highly flexible analysis tool that presents new conceptual possibilities in the analysis of SMLM images.
ABSTRACT
Protein-protein interactions at the plasma membrane mediate transmembrane signaling. Dual-channel fluorescence cross-correlation spectroscopy (dc-FCCS) is a method with which these interactions can be quantified in a cellular context. However, factors such as incomplete maturation of fluorescent proteins, spectral crosstalk, and fluorescence resonance energy transfer (FRET) affect quantification. Some of these can be corrected or accounted for during data analysis and/or interpretation. Here, we experimentally and analytically demonstrate that it is difficult to correct the error caused due to FRET when applying dc-FCCS to measure binding affinity or bound molecular concentrations. Additionally, the presence of dark fluorescent proteins due to incomplete maturation introduces further errors, which too cannot be corrected in the presence of FRET. Based on simulations, we find that modalities such as pulse-interleaved excitation FCCS do not eliminate FRET-induced errors. Finally, we demonstrate that the detrimental effect of FRET can be eliminated with careful experimental design when applying dc-FCCS to quantify protein-protein interactions at the plasma membrane of living cells.
ABSTRACT
Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.
ABSTRACT
Cell wall mannans of Candida albicans mask ß-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling.
Subject(s)
Candida/metabolism , Glucans/metabolism , Mannans/metabolism , HumansABSTRACT
Fungi in the Candida genus are the most common fungal pathogens. They not only cause high morbidity and mortality but can also cost billions of dollars in healthcare. To alleviate this burden, early and accurate identification of Candida species is necessary. However, standard identification procedures can take days and have a large false negative error. The method described in this study takes advantage of hyperspectral confocal fluorescence microscopy, which enables the capability to quickly and accurately identify and characterize the unique autofluorescence spectra from different Candida species with up to 84% accuracy when grown in conditions that closely mimic physiological conditions.
Subject(s)
Candida/classification , Microscopy, Fluorescence/methods , Optical Imaging/methods , Species SpecificityABSTRACT
Candida species are the cause of many bloodstream infections through contamination of indwelling medical devices. These infections account for a 40% mortality rate, posing a significant risk to immunocompromised patients. Traditional treatments against Candida infections include amphotericin B and various azole treatments. Unfortunately, these treatments are associated with high toxicity, and resistant strains have become more prevalent. As a new frontier, light-activated phenylene ethynylenes have shown promising biocidal activity against Gram-positive and -negative bacterial pathogens, as well as the environmental yeast Saccharomyces cerevisiae In this study, we monitored the viability of Candida species after treatment with a cationic conjugated polymer [poly(p-phenylene ethynylene); PPE] or oligomer ["end-only" oligo(p-phenylene ethynylene); EO-OPE] by flow cytometry in order to explore the antifungal properties of these compounds. The oligomer was found to disrupt Candida albicans yeast membrane integrity independent of light activation, while PPE is able to do so only in the presence of light, allowing for some control as to the manner in which cytotoxic effects are induced. The contrast in killing efficacy between the two compounds is likely related to their size difference and their intrinsic abilities to penetrate the fungal cell wall. Unlike EO-OPE-DABCO (where DABCO is quaternized diazabicyclo[2,2,2]octane), PPE-DABCO displayed a strong propensity to associate with soluble ß-glucan, which is expected to inhibit its ability to access and perturb the inner cell membrane of Candida yeast. Furthermore, treatment with PPE-DABCO unmasked Candida albicans ß-glucan and increased phagocytosis by Dectin-1-expressing HEK-293 cells. In summary, cationic phenylene ethynylenes show promising biocidal activity against pathogenic Candida yeast cells while also exhibiting immunostimulatory effects.
Subject(s)
Alkynes/pharmacology , Antifungal Agents/pharmacology , Ethers/pharmacology , beta-Glucans/pharmacology , Candida albicans/drug effects , Candida albicans/genetics , HEK293 Cells , Humans , Lectins, C-Type/metabolismABSTRACT
The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on ß-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or "masked," from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of ß-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of ß-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1-binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host-Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.
Subject(s)
Antifungal Agents/pharmacology , Candida albicans/drug effects , Cell Wall/drug effects , Echinocandins/pharmacology , Lipopeptides/pharmacology , beta-Glucans/immunology , Candida albicans/immunology , Candida albicans/metabolism , Carbohydrate Conformation , Caspofungin , Cell Wall/metabolism , Cell Wall/ultrastructure , Dendritic Cells/metabolism , Dendritic Cells/microbiology , Humans , Lectins, C-Type , Ligands , beta-Glucans/chemistry , beta-Glucans/metabolismABSTRACT
Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs) that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (â¼80 nm) on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to 4 h. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal recognition, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of <30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.
ABSTRACT
To understand the process of innate immune fungal recognition, we developed computational tools for the rigorous quantification and comparison of receptor recruitment and distribution at cell-cell contact sites. We used these tools to quantify pattern recognition receptor spatiotemporal distributions in contacts between primary human dendritic cells and the fungal pathogens C. albicans, C. parapsilosis and the environmental yeast S. cerevisiae, imaged using 3D multichannel laser scanning confocal microscopy. The detailed quantitative analysis of contact sites shows that, despite considerable biochemical similarity in the composition and structure of these species' cell walls, the receptor spatiotemporal distribution in host-microbe contact sites varies significantly between these yeasts. Our findings suggest a model where innate immune cells discriminate fungal microorganisms based on differential mobilization and coordination of receptor networks. Our analysis methods are also broadly applicable to a range of cell-cell interactions central to many biological problems.