Project description:Current spatial transcriptomics methods identify cell types and states in a spatial context but lack morphological information. Electron microscopy, in contrast, provides structural details at nanometer resolution without decoding the diverse cellular states and identity. STEM address this limitation by correlating multiplexed error-robust FISH with electron microscopy from adjacent tissue sections. Using STEM to characterize demyelinated lesions in mice, we were able to bridge spatially resolved transcriptional data with morphological information on cell identities. This approach allowed us to link the morphology of foamy microglia and interferon-response microglia with their transcriptional signatures.
Project description:We performed whole genome gene expression profiling in bronchial biopsies from PCD patients. We used the Quality Threshold clustering algorithm to identify groups of genes that revealed highly correlated RNA expression patterns in the biopsies. The largest cluster contained 372 genes and was significantly enriched for genes related to cilia. The database and literature search showed that 16250 genes in this cluster were known cilia genes, strongly indicating that the remaining 21022 genes were likely to be new cilia genes. The tissue expression pattern of the 210 new cilia genes and the 162 known genes was consistent with the presence of motile cilia in a given tissue. Analysis of the upstream promotor sequences revealed evidence for RFX transcription factors binding site motif in both subgroups. Total RNA obtained from 6 primary ciliary dyskinesia patients and 9 control individuals
Project description:Current spatial transcriptomics methods provide molecular and spatial information but no morphological readout. Here, we present STEM - a method that correlates multiplexed error-robust FISH with electron microscopy from neighboring tissue sections of the same sample. STEM links transcriptional and spatial organization of single cells with ultrastructural morphology of the tissue in vivo. Using STEM to characterize demyelinated white-matter lesions allowed us to link morphology of myelin-laden foamy microglia to transcriptional signature. Moreover, we revealed that interferon-response microglia have unique morphology and are enriched near CD8 T-cells.
Project description:Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using cryo-electron tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that cryo-electron tomography can be applied to human disease by directly imaging patient samples.
Project description:To find and fuse with the egg, mammalian sperm must complete an arduous voyage through the female reproductive tract. This odyssey is powered by the sperm tail, a specialized motile cilium. Mammalian sperm tails are reinforced at the molecular scale with sperm-specific microtubule inner proteins (sperm-MIPs), but the identities of these sperm-MIPs are unknown. Here, we report high-resolution cryo-electron microscopy structures of bovine sperm doublet microtubules (DMTs), allowing us to identify many sperm-MIPs. We also resolve structures of singlet MTs in the endpiece, revealing MIPs shared between singlet and doublet MTs. We demonstrate that at least two sperm-MIPs bind and stabilize MTs in vitro. Our structures shed light on ciliary diversity across cell types and provide structural frameworks for understanding molecular underpinnings of male infertility
Project description:Approximately 80% of clinically clearly diagnosed patients suffering from primary ciliary dyskinesia (PCD) cannot be assigned to a specific gene defect. Despite extensive research on PCD and despite the increasing number of PCD genes and knowledge about their sites of action as e.g structural component or cytoplasmic pre-assembly factor, the biology of motile cilia and the pathomechanism leading to PCD is largely unknown. The aim of this study is to identify novel PCD related genes and processes relevant for motile cilia function.
We will perform exome sequencing, aiming on the analysis of family trios. In these families, the diagnosis of PCD is secured, but the underlying gene defects has so far not been identified.
Project description:Primary ciliary dyskinesia (PCD) is a disorder affecting motile cilia. An early accurate diagnosis helps prevent lung damage and preserve lung function. To make a diagnostic assessment, one of the commonly used methods that allows for the examination of ciliary ultrastructure is transmission electron microscopy (TEM). This allows for a quantitative assessment of ciliary components to identify defects associated with PCD. Heavy metal staining is required to provide a contrast when imaging cilia in the TEM. One of the most commonly used stains is uranyl acetate (UA). UA can be applied to cellular material before embedding (en bloc), or to ultrathin sections of embedded samples (grid staining). UA is radioactive and, due to growing safety concerns and restrictions by government bodies, universities and hospitals, it is essential to find a suitable alternative. We show UA-zero (UAZ), when used en bloc, provides a high contrast and is a suitable replacement for UA. PCD diagnostic experts, having reviewed ciliary cross-sections stained with UAZ en bloc, are confident that the staining and PCD defects are readily detectable similar to samples that have been stained with UA.
Project description:Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.