Biology subjects

Frangakis, A. S.

Publications and source records attributed to Frangakis, A. S..

16 recordsLinked to original sources

bioRxiv2026

Cryo-electron tomographic analysis of anti-nephrin-mediated podocytopathy

Nephrotic kidney diseases are characterized by proteinuria caused by dysfunction of the glomerular filtration barrier. Here, we perform an ultrastructural analysis of anti-nephrin-mediated podocytopathy by visualizing the near-native slit diaphragm in situ across defined stages of disease progression using cryo-electron tomography. Our analysis shows that at early disease stages punctuate cell-cell membrane approximations appear, with basal membrane apposition between adjacent podocyte foot processes, while the initially planar, fishnet slit diaphragm progressively bends into a dome-shaped configuration towards the urinary space. At later stages, slit diaphragm disappearance coincides with the appearance of endosomal and vesicular structures and extensive cytoskeletal reorganization. These cryo-electron tomography-based observations provide a structural framework for understanding how antibody binding to nephrin translates into podocyte architectural failure.

immunology↗

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

Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs

The third intracellular loop (ICL3) of the {beta}1-adrenergic receptor ({beta}1AR) plays a critical role in regulating G protein coupling, yet the structural basis has remained unclear due to truncations of ICL3 in all available structures of the {beta}1AR in complex with Gs or a G protein mimetic nanobody. To address this, we used cell-free cotranslational insertion of full-length human {beta}1AR into nanodiscs and determined its cryo-EM structure in complex with Gs. In this structure, ICL3 extends transmembrane helix 5, resulting in enhanced interactions with Gs and in a slight rotation of the engaged G protein. This repositioning enables new polar interactions between Gs, ICL2 and helix 8, while ICL1 and helix 8 form additional contacts with G{beta}. These structural insights, supported by mutational analysis, demonstrate that ICL3 enhances G protein activation and downstream cAMP signaling by promoting more extensive interactions between the receptor and the heterotrimeric G protein.

biochemistry↗

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

Conformational plasticity across phylogenetic clusters of RND multidrug efflux pumps and its impact on substrate specificity

Antibiotic efflux plays a key role for the multidrug resistance in Gram-negative bacteria 1-3. Multidrug efflux pumps of the resistance nodulation and cell division (RND) superfamily function as part of cell envelope spanning systems and provide resistance to diverse antibiotics 4,5. Here, we identify two phylogenetic clusters of RND proteins with conserved binding pocket residues. Based on the characterisation of one representative of each cluster, K. pneumoniae OqxB and E. coli AcrB, we show that the transfer of a single conserved residue between both clusters alters the resistance against a panel of structurally unrelated drugs. The substitution is not only associated with changes in the binding pocket architecture, but also alters the equilibrium between the conformational states of the transport cycle. We show that AcrB and OqxB adopt fundamentally different apo states that suggest different mechanisms of initial substrate binding and might determine the differences between the substrate preferences of both pumps. The observed conformational heterogeneity between different RND clusters is suggested to be phylogenetically conserved and might play a role for the diversification of the resistance phenotype between homologous RND multidrug efflux pumps.

biochemistry↗

bioRxiv2024

Camera Paths, Modeling, and Image Processing Tools for ArtiaX

The enhancement of biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectrum of applications. Recently, we have extended the capabilities of UCSF ChimeraX to address the specific demands of cryo-electron tomography. Here, we introduce the evolution of our existing plugin, ArtiaX, designed to generate models that facilitate particle selection, define camera recording paths, and execute particle selection routines. In particular, diverse models can be generated and populated with putative particle positions and orientations. A specifically tailored coarse grained algorithm was developed to rectify overlaps, as encountered in template matching, employing a rapid and efficient approach. In addition, models can be used to drive the camera position, thereby simplifying the process of movie creation. The plugin incorporates fundamental image filtering options for the on-the-fly analysis of tomographic data and also provides compatibility of particle lists with RELION-5 star files. Collectively, this update of ArtiaX comprehensively encompasses essential tools for the analysis and visualization of electron tomograms. It retains its hallmark attributes of speed, reliability, and user-friendliness, fostering seamless human-machine interaction.

bioinformatics↗

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↗

bioRxiv2024

Structural characterization of an essential lipoprotein of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a human pathogen causing atypical community-acquired pneumonia. It serves as a model for a minimal cell and is notable for its absent cell wall, minimal genome, and use of antigenic variation to evade the host immune response. Here, we report the structures of the essential surface proteins Mpn444 and Mpn436 at 3.74 [A] and 3.65 [A] resolution, and the molecular architecture of the Mpn444 homotrimeric complex. We show that both proteins include a peptidyl-prolyl isomerase (PPIase) domain and a chaperone-like domain. In vitro PPIase activity assays suggest that Mpn444 and Mpn436 function as extracellular foldases in Mycoplasma species. Furthermore, both proteins are conserved across multiple Mycoplasma species. We built a composite model integrating previously reported interactions from crosslinking and cryo-ET data and we conclude that Mpn444 is responsible for the extracellular folding of nascent protein chains. Our work underscores the potential of Mpn444 and Mpn436 as a target for the development of novel strategies to treat mycoplasma infections. Author SummaryIn this study, we discovered that the essential proteins Mpn444 and Mpn436 of Mycoplasma pneumoniae act as extracellular PPIases. We solved the structures of Mpn444, Mpn436, and Mpn444s homotrimer by single-particle cryo-EM analysis, marking them as the first experimentally resolved lipoproteins from Mycoplasma pneumoniae. The structures contain a peptidyl-prolyl isomerase (PPIase) domain as well as a chaperone-like domain, and the proteins show PPIase activity and bind unfolded proteins in vitro. By combining our structural data with previously published cryo-electron tomography and crosslinking data from M. pneumoniae cells, we suggest that Mpn444, and potentially Mpn436, works in concert with the Sec translocon and the membrane-bound ribosome. This finding is particularly important as the extracellular folding of bacterial proteins has been a significant gap in our understanding.

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↗

bioRxiv2023

P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

Lipid acquisition and transport are fundamental processes in all organisms. Here, we investigate the lipid uptake and delivery mechanism of the minimal model organism Mycoplasma pneumoniae. We show that the essential protein P116 can transport lipids between liposomes independently and without ATP consumption. Our structural data and molecular dynamics simulations reveal the mechanism by which the N-terminal region of P116 perturbs the membrane, the lipid transfer route, and the regulation of membrane binding by the cargo mass within P116s large hydrophobic cavity. When adequately filled with cargo, P116 undergoes a rapid conformational change that modulates membrane binding. Taken together, our results show that Mycoplasma developed one integrated lipid uptake and delivery machinery that simplifies the complex multi-protein pathways used by higher developed organisms.

molecular biology↗

bioRxiv2022

ArtiaX: An Electron Tomography Toolbox for the Interactive Handling of Sub-Tomograms in UCSF ChimeraX

Cryo-electron tomography analysis involves the selection of macromolecular complexes to be used for subsequent sub-tomogram averaging and structure determination. Here, we describe a plugin developed for UCSF ChimeraX that allows for the display, selection, and editing of particles within tomograms. Positions and orientations of selected particles can be manually set, modified and inspected in real-time, both on screen and in virtual reality, and exported to various file formats. The plugin allows for the parallel visualization of particles stored in several meta data lists, in the context of any 3D image that can be opened with UCSF ChimeraX. The particles are rendered in user-defined colors or using colormaps, such that individual classes or groups of particles, cross-correlation coefficients or other types of information can be highlighted to the user. The implemented functions are fast, reliable and intuitive, exploring the broad range of features in UCSF ChimeraX. They allow for a fluent human-machine interaction, which enables an effective understanding of the sub-tomogram processing pipeline, even for non-specialist users.

biophysics↗

bioRxiv2022

The immunodominant protein P116 is a passive transporter of cholesterol and other essential lipids

Many human pathogens need to extract lipids from their environment for survival and proliferation. How this is accomplished on a molecular level is largely unknown1. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213) from Mycoplasma pneumoniae, a human pathogen responsible for approximately 30% of community-acquired human pneumonia2. Single-particle cryo-electron microscopy of P116 at 3.3 [A] resolution reveals a homodimer with a core domain presenting a previously unseen fold. This fold creates a large cavity of [~]18,000 [A]3 with a hydrophobic internal surface that is accessible to solvent. Within the cavity ligands with a length of 10-19 [A] and a width of 4 [A] could be observed. These ligands were identified as the essential lipids phosphatidylcholine, sphingomyelin and cholesterol using mass spectrometry. When the cavity is emptied, the protein undergoes an extensive conformational change that can no longer accommodate lipids. When emptied P116 is incubated with high-density lipoproteins (HDLs) a net transfer of cholesterol is demonstrated by a radioactivity experiment and cryo-electron microscopy resolves a complex between P116 and HDL. Taken together, our results reveal the mechanism by which P116 extracts essential lipids from the host environment and possibly then delivers them into the membrane by a wringing movement. This mechanism may be precedential for other cholesterol-auxotrophic bacteria.

microbiology↗

bioRxiv2020

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes

BackgroundThe coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. MethodsInfection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. ResultsWe show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. ConclusionsThe demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThis study demonstrates that human cardiac myocytes are permissive for SARS-CoV-2 infection. C_LIO_LIThe study documents that SARS-CoV-2 undergoes a full replicatory circle and induces a cytotoxic response in cardiomyocytes. C_LIO_LIInfection was confirmed in two cardiac tissue models, including living human heart slices. C_LI What are the Clinical Implications?O_LIThe study may provide a rational to explain part of the cardiotoxicity observed in COVID-19 patients C_LIO_LIThe demonstration of direct cardiotoxicity induced by SARS-CoV-2 warrants an in depth further analysis of cardiac tissue of COVID-19 patients and a close monitoring for putative direct cardiomyocyte injury. C_LIO_LIThe established models can be used to test novel therapeutic approaches targeting COVID-19. C_LI

cell biology↗

bioRxiv2018

The Desmosome is a Mesoscale Lipid Raft-Like Membrane Domain

Desmogleins are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. Molecular modeling predicts that this G to R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography (cryo-ET) indicates that the lipid bilayer within the desmosome is ~10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via establishment of a specialized mesoscale lipid raft-like membrane domain.

cell biology↗

bioRxiv2018

The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important lipid intermediate and signaling lipid at the branch point of these pathways and constantly monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. Here, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for the selective binding to membranes containing PA over phosphatidylserine (PS). The insertion of the AH into the hydrophobic core of the membrane renders Opi1 sensitive to the lipid acyl chain composition as an important factor contributing to the regulation of membrane biogenesis. Based on these findings, we rationally designed the membrane binding properties of Opi1 to control its responsiveness in the physiological context. Using extensive molecular dynamics (MD) simulations, we identified two PA-selective three-finger grips that tightly bind the phosphate headgroup, while interacting less intimately and more transiently with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

biochemistry↗