Biology subjects

Unnersjoe-Jess, D.

Publications and source records attributed to Unnersjoe-Jess, D..

5 recordsLinked to original sources

bioRxiv2024

A Red Fluorescent Lifeact Marker to Study Actin Morphology in Podocytes

F-actin is a major component of the cellular cytoskeleton, responsible for maintaining cell shape, enabling movement and facilitating intracellular transport. In the kidney, glomerular podocytes are highly dependent on their actin cytoskeleton shaping their unique foot processes. Hereditary mutations in actin-binding proteins cause focal segmental glomerulosclerosis, while other organs remain largely unaffected. So far, actin visualization in podocytes has been limited to electron microscopy or indirect immunofluorescent labeling of actin-binding proteins. However, the short F-actin-binding peptide Lifeact enables researchers to study actin dynamics in vitro and in vivo with minimal interference with actin metabolism. Here we introduce a new mouse model with conditional expression of a Lifeact.mScarlet-I fusion protein providing red labeling of actin. Cre recombinase-mediated activity allows cell-specific and mosaic expression in podocytes, enabling selective labeling of individual cells to contrast with non-expressing neighboring cells. Transgenic mice are born healthy and young animals display no kidney-related phenotype. By intravital imaging and super-resolution microscopy, we show subcellular localization of actin to the foot processes in a resolution previously only obtainable by electron microscopy. Our novel mouse line provides the opportunity to study the actin cytoskeleton in podocytes and other cell types by intravital imaging and other conventional light microscopy techniques.

cell biology↗

bioRxiv2023

Correlation of calcium multiphoton with ultrastructural STED imaging of the slit diaphragm in the same glomerulus.

In recent years functional multiphoton imaging of viable mouse tissue and STED imaging of optically cleared tissues allowed new insights into kidney biology. Here, we present a novel workflow where multiphoton imaging of calcium signals in podocytes is, in the same glomerulus, correlated with super-resolved STED imaging and analysis of the slit diaphragm (SD) morphology. Mice expressing the calcium indicator GCaMP3 exclusively in podocytes served as healthy controls and were challenged with two different doses of nephrotoxic serum (NTS). NTS-induced SD architecture damage increased in a dose-dependent manner, whereas intracellular calcium levels exhibited a wide range of variation and showed no correlation with SD damage in the same glomerulus. Our co-imaging protocol is applicable to a broad range of research questions as it is suitable for other tissues and compatible with a variety of reporters and tissue antigens. Translational statementThe combination of multiphoton and super-resolution microscopy identifies the modulation of signaling pathways and resolves the changes in ultrastructure in disease states in the same glomeruli. Using our new method, we can compare functional and morphological data to investigate the degree of correlation to reveal novel underlying pathomechanism. This knowledge can improve our ability to draw conclusions from human kidney biopsies, in which the ultrastructure will be assessed more-often by routine super-resolution microscopy in the future.

cell biology↗

bioRxiv2023

Mechanical forces at the kidney filtration barrier govern spatial orientation of podocyte processes on capillaries

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the very high hydrostatic pressure in glomerular capillaries. Interdigitating cellular processes of podocytes form the slits for fluid filtration. They are connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Using high-resolution microscopy, we show that filtration-slit-generating secondary processes preferentially align along the capillaries longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. The preferential orientation requires maturation in development and is lost in disease states. We demonstrate that loss of proper orientation might contribute to impaired filtration by collapsing of the filtration slits and reducing the mechanical stability of podocyte processes. Together, these data suggest that podocytes sense mechanical strain to utilize circumferential hoop stress balancing the massive mechanical strain generated from fluid flow over the filtration slit.

cell biology↗

bioRxiv2021

Three-dimensional Super-resolved Imaging of Paraffin-embedded Kidney Samples

Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. New technologies, including superresolved nanoscopy and expansion microscopy, as well as new sample preparation techniques, are starting to revolutionize imaging of biopsy specimens. However, our previous approaches for simple and fast three-dimensional imaging of optically cleared samples are to date not compatible with formalin fixed paraffin-embedded (FFPE) tissue, impeding application in clinical routine. Here we provide protocols that circumvent these limitations and allow for three dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. This would increase the feasibility to implement these protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

pathology↗

bioRxiv2021

Deep learning-based segmentation and quantification of podocyte foot process morphology

The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

pathology↗