scholarly journals Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice

2021 ◽  
pp. ASN.2020091346
Author(s):  
Favian Hatje ◽  
Uta Wedekind ◽  
Wiebke Sachs ◽  
Desiree Loreth ◽  
Julia Reichelt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Communication Networks ◽  
Super Resolution ◽  
Cell Types ◽  
Mouse Strains ◽  
Cell Type ◽  
Glomerular Cell ◽  
Marker Proteins ◽  
Biochemical Analyses

Background: The glomerulus comprises podocytes, mesangial, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell-types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods: Glomerular cell-types were separated via a novel FACS-sort approach from wildtype or mT/mG mice and the purity validated. Cell-type proteomes were compared between strains, ages, and sex. TimMEP was applied to the podocyte-targeting immunologic THSD7A-associated membranous nephropathy model. Results: TimMEP enabled protein-biochemical analyses of podocytes, mesangial, and endothelial cells derived from reporter-free mice and allowed the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were identified and protein-based potential communication networks and phosphorylation patterns outlined. The analysis detected cell-type specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response chiefly in podocytes, which bulk glomerular analyses could not detect. Combination of proteomics with super-resolution imaging revealed a specific loss of slit-diaphragm but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. Conclusion: TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.

scholarly journals Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

2020 ◽  
Author(s):  
Favian A. Hatje ◽  
Markus M. Rinschen ◽  
Uta Wedekind ◽  
Wiebke Sachs ◽  
Julia Reichelt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
List Type ◽  
Cell Type ◽  
Isolation Method ◽  
Glomerular Cell ◽  
Filtration Barrier ◽  
Kidney Glomerulus ◽  
Antibody Labeling

ABSTRACTPurposeThe kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP).MethodsGlomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis.ResultsTimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli.ConclusionGlobally applicable timMEP abolishes the need for costly, time- and animal-consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.Key messagesA tripartite isolation method for mesangial, endothelial and podocyte cell-types from reporter-free mice.Generation of bulk cell-type samples and primary co-cultures for biochemical and protein-based analyses.Strain and transgene-dependent expression of proteins among glomerular cell-types, including protein profiles, intra-glomerular communication machineries, and reporter-dependent artefacts.Disease specific time-resolved resolution of glomerular cell-type’s response to injury.

scholarly journals Connectivity characterization of the mouse basolateral amygdalar complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Houri Hintiryan ◽  
Ian Bowman ◽  
David L. Johnson ◽  
Laura Korobkova ◽  
Muye Zhu ◽  
...  
Keyword(s):  
Granular Cell ◽  
Cell Types ◽  
Projection Neurons ◽  
Cell Type ◽  
Connectivity Map ◽  
Analysis Techniques ◽  
Domain Specific ◽  
Cell Type Specific ◽  
Unique Domain

AbstractThe basolateral amygdalar complex (BLA) is implicated in behaviors ranging from fear acquisition to addiction. Optogenetic methods have enabled the association of circuit-specific functions to uniquely connected BLA cell types. Thus, a systematic and detailed connectivity profile of BLA projection neurons to inform granular, cell type-specific interrogations is warranted. Here, we apply machine-learning based computational and informatics analysis techniques to the results of circuit-tracing experiments to create a foundational, comprehensive BLA connectivity map. The analyses identify three distinct domains within the anterior BLA (BLAa) that house target-specific projection neurons with distinguishable morphological features. We identify brain-wide targets of projection neurons in the three BLAa domains, as well as in the posterior BLA, ventral BLA, posterior basomedial, and lateral amygdalar nuclei. Inputs to each nucleus also are identified via retrograde tracing. The data suggests that connectionally unique, domain-specific BLAa neurons are associated with distinct behavior networks.

Cytoplasmic, nuclear, membrane-bound and secreted [35S]methionine-labelled polypeptide pattern in differentiating fibroblast stem cells in vitro

1989 ◽  
Vol 92 (2) ◽  
pp. 231-239
Author(s):  
P.I. Francz ◽  
K. Bayreuther ◽  
H.P. Rodemann
Keyword(s):  
Protein Fraction ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Specific Marker ◽  
Human Skin Fibroblast ◽  
Nuclear Fraction ◽  
Cell Type ◽  
Marker Proteins ◽  
Cell Type Specific ◽  
Membrane Bound

Methods for the selective enrichment of various subpopulations of the human skin fibroblast cell line HH-8 have been developed. These methods permit the selection of homogeneous populations of the three mitotic fibroblast cell types MF I, II and III, and the four postmitotic cell types PMF IV, V, VI and VII. These seven cell types exhibit differentiation-dependent and cell-type-specific patterns of [35S]methionine-labelled polypeptides in total soluble cytoplasmic and nuclear proteins, also in membrane-bound proteins, and in secreted proteins. In the differentiation sequence MF II-MF III-PMF IV - PMF V - PMF VI 14 cell-type-specific marker proteins have been found in the cytoplasmic and nuclear fraction, also 24 cell-type-specific marker proteins have been found in the membrane-bound protein fraction, and 11 cell-type-specific marker proteins in the secreted protein fraction. Markers in spontaneously arising and experimentally selected or induced populations of a single fibroblast cell type were found to be identical.

scholarly journals Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection

2020 ◽  
Author(s):  
Samina Momtaz ◽  
Belen Molina ◽  
Luwanika Mlera ◽  
Felicia Goodrum ◽  
Jean M. Wilson
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Biosynthetic Pathway ◽  
Cell Types ◽  
Endocytic Pathway ◽  
Specific Cell ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Increased Risk ◽  
Cell Type Specific

AbstractHuman cytomegalovirus (HCMV), while highly restricted for the human species, infects an unlimited array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies that incorporate viral products including dense bodies and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). Cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in endothelial cells were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that virus-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. Virus containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non-canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV.ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with increased risk vascular disease. HCMV infects many cells in the human and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact the outcome of infection.

scholarly journals A Single-Cell Transcriptome Atlas of the Mouse Glomerulus

2018 ◽  
Vol 29 (8) ◽  
pp. 2060-2068 ◽  
Author(s):  
Nikos Karaiskos ◽  
Mahdieh Rahmatollahi ◽  
Anastasiya Boltengagen ◽  
Haiyue Liu ◽  
Martin Hoehne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Single Cell ◽  
Mesangial Cells ◽  
Transcriptional Profiling ◽  
Wild Type Mouse ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Marker Genes ◽  
Glomerular Cell

Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown.Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

2007 ◽  
Vol 293 (6) ◽  
pp. H3325-H3332 ◽  
Author(s):  
Derek B. J. Bone ◽  
James R. Hammond
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Nucleoside Transporter ◽  
Active Metabolites ◽  
Equilibrative Nucleoside Transporter ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells ◽  
Nucleobase Transport

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a Vmaxof 3.4 ± 1 pmol·μl−1·s−1, with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs ( Km= 96 ± 37 μM; Vmax= 1.6 ± 0.3 pmol·μl−1·s−1) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine ( Ki= 19 ± 7 μM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK( 15 ) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na+or H+. PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

scholarly journals Rickettsia rickettsii Infection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

2010 ◽  
Vol 78 (6) ◽  
pp. 2599-2606 ◽  
Author(s):  
Elena Rydkina ◽  
Loel C. Turpin ◽  
Sanjeev K. Sahni
Keyword(s):  
Endothelial Cells ◽  
Host Response ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Cell Type ◽  
Rickettsia Rickettsii ◽  
Specific Host ◽  
Cell Type Specific ◽  
Cox 2 ◽  
Microvascular Endothelial

ABSTRACT Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-κB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-κB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E2 after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

scholarly journals Bulk Tissue Cell Type Deconvolution with Multi-Subject Single-Cell Expression Reference

2018 ◽  
Author(s):  
Xuran Wang ◽  
Jihwan Park ◽  
Katalin Susztak ◽  
Nancy R. Zhang ◽  
Mingyao Li
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Tissue Cell ◽  
Specific Gene ◽  
Rna Seq ◽  
Cell Type ◽  
Cell Type Specific ◽  
Cell Expression

AbstractWe present MuSiC, a method that utilizes cell-type specific gene expression from single-cell RNA sequencing (RNA-seq) data to characterize cell type compositions from bulk RNA-seq data in complex tissues. When applied to pancreatic islet and whole kidney expression data in human, mouse, and rats, MuSiC outperformed existing methods, especially for tissues with closely related cell types. MuSiC enables characterization of cellular heterogeneity of complex tissues for identification of disease mechanisms.

scholarly journals Profiling of Primary and Mature miRNA Expression in Atherosclerosis Associated Cell Types

2021 ◽  
Author(s):  
Pierre R. Moreau ◽  
Vanesa Tomas Bosch ◽  
Maria Bouvy-Liivrand ◽  
Kadri Õunap ◽  
Tiit Örd ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Umbilical Vein ◽  
Cell Types ◽  
Integrative Approach ◽  
Dependent Manner ◽  
Cell Type ◽  
A Cell ◽  
Cell Type Specific

Objective: Atherosclerosis is the underlying cause of most cardiovascular diseases. The main cell types associated with disease progression in the vascular wall are endothelial cells, smooth muscle cells, and macrophages. Although their role in atherogenesis has been extensively described, molecular mechanisms underlying gene expression changes remain unknown. The objective of this study was to characterize microRNA (miRNA)-related regulatory mechanisms taking place in the aorta during atherosclerosis: Approach and Results: We analyzed the changes in primary human aortic endothelial cells and human umbilical vein endothelial cell, human aortic smooth muscle cell, and macrophages (CD14+) under various proatherogenic stimuli by integrating GRO-seq, miRNA-seq, and RNA-seq data. Despite the highly cell-type-specific expression of multi-variant pri-miRNAs, the majority of mature miRNAs were found to be common to all cell types and dominated by 2 to 5 abundant miRNA species. We demonstrate that transcription contributes significantly to the mature miRNA levels although this is dependent on miRNA stability. An analysis of miRNA effects in relation to target mRNA pools highlighted pathways and targets through which miRNAs could affect atherogenesis in a cell-type-dependent manner. Finally, we validate miR-100-5p as a cell-type specific regulator of inflammatory and HIPPO-YAP/TAZ-pathways. Conclusions: This integrative approach allowed us to characterize miRNA dynamics in response to a proatherogenic stimulus and identify potential mechanisms by which miRNAs affect atherogenesis in a cell-type-specific manner.

scholarly journals Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Julio D Perez ◽  
Susanne tom Dieck ◽  
Beatriz Alvarez-Castelao ◽  
Georgi Tushev ◽  
Ivy CW Chan ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Large Population ◽  
Cell Types ◽  
Mrna Localization ◽  
Gabaergic Neurons ◽  
Local Translation ◽  
Single Neurons ◽  
Cell Type ◽  
Cell Type Specific

Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell-types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated mRNA abundances in the soma and dendrites of single glutamatergic and GABAergic neurons. We found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. We observed more cell type-specific features among somatic transcriptomes than their associated dendritic transcriptomes. Finally, using cell-type specific metabolic labelling of isolated neurites, we demonstrated that the processes of Glutamatergic and, notably, GABAergic neurons were capable of local translation, suggesting mRNA localization and local translation is a general property of neurons.

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