Biology subjects

Birtasu, A. N.

Publications and source records attributed to Birtasu, A. N..

5 recordsLinked to original sources

bioRxiv2025

The native human glomerulus features a slit diaphragmresembling a densely interwoven fishnet

The slit diaphragm (SD), a core element of the glomerular filtration barrier, is essential for renal filtration and disrupted in all forms of glomerulopathy. Using cryo-electron tomography of human kidney tissue, we resolved the near-native SD architecture at unprecedented resolution. The SD comprises crisscrossing strands intersecting at [~]90{degrees}, forming a fishnet-like lattice across the [~]44 nm space between podocyte foot processes. An atomic model based on the Nephrin-Neph1 heterodimer reveals [~]9 nm spacing in humans, compared with 12.3 nm in mice and 15 nm in Drosophila. Our data establish the SD as a conserved fishnet-like assembly from invertebrates to mammals, with the tighter human lattice likely conferring enhanced permselectivity.

physiology↗

bioRxiv2025

In situ structure of a gap junction - stomatin complex

Gap junctions (GJ) are intercellular channels that mediate electrical signals and the transfer of small molecules. GJs are crucial for the functions of the brain, heart and other organs. While structures of purified homomeric GJs are available, we lack in situ structures. In vivo, GJs can form heteromers with different functionalities, and may associate with other proteins. Here, we analyzed Caenorhabditis elegans GJs by cryo-electron tomography and sub-tomogram averaging. We observed hexagonal arrays of GJs at cellular junctions in primary embryonal cell culture that displayed distinct wide and narrow conformations. Moreover, in about 20% of the observed channels, we found a cap-like, cytosolic protein assembly enclosing the channel pore. We propose that the cap-structure is formed by the stomatin UNC-1, which is known to interact with C. elegans GJs, and strengthen this hypothesis by matching AlphaFold3 models of UNC-1 multimers with our GJ average. Furthermore, expressing UNC-1 and the C. elegans innexin UNC-9 in HEK cells resulted in similar structures at cell-cell contacts. UNC-1/stomatin ring assemblies may affect GJ formation or functions like rectification, that might be evolutionarily conserved. Significance StatementGap junction (GJ) channels connect neighboring cells. Structures of (purified) GJs have been studied in vitro, but not in situ. We identified GJ channels in primary Caenorhabditis elegans cells by cryo-electron tomography, and analyzed their structure by sub-tomogram averaging. The channels transverse the membranes of connected cells, and AlphaFold3 (AF3) models of the GJ subunit UNC-9, assuming dodecamers, fit the experimentally obtained surface map well. We observed a cytosolic cap structure on the GJ channels. The stomatin protein UNC-1 is known to physically interact with UNC-9 GJs. AF3 models of UNC-1 hexadecamers fit the cap structure, indicating that it may be formed by UNC-1, providing a first idea how UNC-1 interacts with, and may functionally influence, GJ channels.

neuroscience↗

bioRxiv2025

The slit diaphragm in Drosophila features a bi-layered, fishnet-like architecture

The kidney filters large volumes of blood plasma, relying on the glomerulus for the filtration. The slit diaphragm, a critical component of the glomerulus, is formed between podocytes by the immunoglobulin domain proteins nephrin and Neph1. The molecular architecture of the slit diaphragm has remained elusive for decades. Using cryo-electron tomography on focused ion beam-milled Drosophila nephrocytes, an invertebrate podocyte model, we show that the slit diaphragm adopts a fishnet-like pattern. Comparison of hundreds of slit diaphragm segments reveals that it is bi-layered and highly periodic. Based on the cryo-electron tomography map, we propose four possible models for the arrangement of the Drosophila nephrin ortholog (Sns), and the Drosophila Neph1 ortholog (Kirre), the main components of the slit diaphragm in Drosophila. In each model, precise and consistent homo- and heterophilic interactions between crossing immunoglobulin domains of Sns and Kirre become apparent, with immediate implications for the stability and the assembly of the slit diaphragm. Cryo-electron tomography shows that sns silencing disrupts this fishnet pattern, linking this directly to Drosophila nephrin. After Rab5 silencing, causing Sns mistrafficking and ectopic SD formation, the fishnet pattern appears ectopically as well. Our findings align with observations applying cryo-electron tomography to podocytes in mice, indicating that the molecular architecture is evolutionarily conserved across animals. This highlights the value of the nephrocyte as a podocyte model and establishes a crucial link between the architecture of the slit diaphragm and its function.

biophysics↗

bioRxiv2024

Localization of Albumin with Correlative Super Resolution Light- and Electron Microscopy in the Kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

biophysics↗

bioRxiv2023

The molecular architecture of the kidney slit diaphragm

Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics flexible fitting, we construct a blueprint of the SD, where we describe all interactions. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.

cell biology↗