Biology subjects

Psathaki, O. E.

Publications and source records attributed to Psathaki, O. E..

10 recordsLinked to original sources

bioRxiv2025

Localisation of Apicomplexa motor Myosin A with axial nanometric precision using graphene energy transfer

Migration in Apicomplexan parasites, a phylum that includes Plasmodium spp. responsible for malaria and the zoonotic Toxoplasma, is explained by the linear migration motor model. This model predicts that Myosin A is confined beneath the plasma membrane and above the inner membrane complex (IMC), a membranous barrier separating the cell membrane and the cytoplasm. Cumulative data, using mutant cell lines and biochemical data support this model. Paradoxically, a proof of the precise localization of Myosin A is still lacking due to limitations in resolving the motor with nanometric precision within the space comprising the membrane and the IMC. Here, we implement graphene energy transfer (GET), a novel axial nanometric ruler with a resolution of [~]1 nm, to determine the relative axial position of Myosin A and IMC1 (a reference protein defining IMC position). Using GET, we estimate the IMC dimensions with precision matching electron microscopy (EM) data and the added advantage of identifying specific bauplan proteins. We complement these measurements with 2D STED microscopy in MIRA confiners, uExMIC, and cryo-immunolabeling. Our data present the first direct localization of Myosin A populations with nanometric resolution in the third dimension, which is compatible with the linear motor model. One sentence summaryUsing the novel GET method, this study presents the first direct localization of Myosin A populations in Toxoplasma gondii with 1-4 nm axial precision, supporting the linear model.

biophysics↗

bioRxiv2025

Multi-scale mitochondrial cristae remodeling links Opa1 downregulation to reduced OXPHOS capacity in aged hearts

Aging is closely associated with cardiovascular diseases, the leading cause of mortality worldwide. Mitochondrial dysfunction is a hallmark of cardiovascular aging because it generates most of the hearts ATP at the cristae, specialized sub-compartments where OXPHOS takes place. In this study, we used multiple-scale electron microscopy approaches to evaluate age-related mitochondrial and cristae ultrastructural alterations in human and mouse hearts. We found that aged patients hearts displayed reduced cristae density as seen by TEM, even before any significant decline in the expression of cristae-shaping proteins. Similarly, a multi-scale approach that included SBF-SEM and TEM showed that in aged mices hearts cristae undergo ultrastructural remodeling processes, resulting in a decrease in cristae density and width. Electron tomography suggests an apparent decline in cristae connectivity, and an increase in fenestration size. These changes were linked to Opa1 downregulation, accompanied by reduced OXPHOS maximal respiration, but unrelated to alterations in the levels of OXPHOS core subunits and ATP synthase assembly. Altogether, this indicates that alterations in cristae structure alone are sufficient to impair oxidative metabolism, which highlights its potential as an early signal of cardiac aging, even before noticeable changes in mitochondrial morphology occur.

cell biology↗

bioRxiv2024

Pex3 promotes formation of peroxisome-peroxisome and peroxisome-lipid droplet contact sites

Peroxisomes are ubiquitous organelles that mediate central metabolic functions, such as fatty acid {beta}-oxidation, as well as diverse tissue- and organism-specific processes. Membrane contact sites, regions of close apposition with other organelles for direct communication, are central to several aspects of their life cycle. Pex3 is a conserved multifunctional peroxisomal transmembrane protein involved in the insertion of peroxisomal membrane proteins, in pexophagy, and in the formation of membrane contact sites. Here, we show that elevated Pex3 levels in Saccharomyces cerevisiae induce the formation of peroxisome clusters surrounded by lipid droplets, mediated by peroxisome-peroxisome and peroxisome-lipid droplet contact sites. This clustering occurs independently of Pex3 partners in other processes, Pex19, Inp1, and Atg36. The cytosolic domain of Pex3 binds peroxisomes, suggesting a direct role in homotypic contact site formation. Lipid droplet-peroxisome contact sites require the lipid droplet-localized triacylglycerol lipase Tgl4, which is enriched along with other lipases at this interface. Pex3 overexpression in Drosophila melanogaster similarly alters peroxisome and lipid droplet morphology and promotes contact site formation. Together, our results offer novel molecular insights into homotypic peroxisome contact sites and peroxisome-lipid droplet contact sites across species.

cell biology↗

bioRxiv2023

Rapid in-EPON CLEM for everyone: Combining fast and efficient labeling of self-labeling enzyme tags with EM resistant Janelia Fluor dyes

Correlative light and electron microscopy (CLEM) allows to link light microscopy (LM) of living cells to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between the LM and EM modalities due to chemical and physical distortions. Post-embedding CLEM enables precise registration of fluorescent structures directly on thin resin sections. However, in-resin CLEM techniques require fluorescent markers withstanding EM sample preparation. Most fluorescent proteins lose their fluorescence during EM sample preparation. Synthetic dyes present an alternative as their photostability and brightness exceed those of fluorescent proteins. Together with self-labeling enzymes (SLE) as protein tags, these fluorophores can be used to precisely label cellular structures of interest. By applying SLE labelling for post-embedding CLEM, we compared Janelia Fluor dyes and TMR to identify most suitable fluorophores. Epithelial cells expressing HaloTag fusion proteins were stained with various ligand-conjugated dyes, and fluorescence preservation was quantified after conventional room temperature sample preparation with embedding in EPON. The results obtained show that only the red dyes TMR, JF549, JFX549 and JFX554 retain their fluorescence in resin, with JFX549 and JFX554 yielding best signal intensity and signal-to-background ratio during in-resin super-resolution microscopy. Since all red dyes possess an oxygen atom within their xanthene structure, our results indicate that this might be a crucial feature making them more tolerant to sample preparation for electron microscopy. Our work reports a rapid in-resin CLEM approach that combines fast and efficient labeling of SLE tags with EM-compatible fluorophores, and serve as benchmarks for experimental planning and future engineering of fluorophores for CLEM.

molecular biology↗

bioRxiv2023

Structure and regulation of GSDMD pores at the plasma membrane of pyroptotic cells

Gasdermin D (GSDMD) executes inflammatory cell death pyroptosis by permeabilizing the plasma membrane (PM). We introduce polymer-supported PM (PSPM) to gain access to the cytoplasmic side of the PM with imaging techniques while preserving the native PM complexity and lipid microenvironment. By combining PSPM with DNA-PAINT super-resolution microscopy we visualized, for the first time, GSDMD nanostructures directly at the PM of pyroptotic cells. We resolved diverse macromolecular architectures with ring-and arc-shaped GSDMD oligomers that enable PM permeabilization. The pyroptotically-inactive mutant GSDMD-C192A (human C191A) still interacts with the PM however fails to form pores. GSDMD expression levels affect pore density but not permeabilization ability. Finally, we identified the local PI(3,4,5)P3 concentration as a key regulatory element of PM permeabilization. Increase in PI(3,4,5)P3 levels in the PM during pyroptosis facilitates growth into large ring-shaped pores. Using molecular dynamics (MD) simulations, we identified the mechanism by which PI(3,4,5)P3 stabilizes the GSDMD assembly.

biophysics↗

bioRxiv2023

Resolving exit strategies of mycobacteria by combining high-pressure freezing with 3D-correlative light and electron microscopy

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of vacuolar and cytosolic mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach that integrates both methodologies with almost native ultrastructural preservation is still lacking at the moment. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.

cell biology↗

bioRxiv2022

Early endosomes act as local exocytosis hubs to repair endothelial membrane damage

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of membrane required for wound repair are poorly understood. Here, we show that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, we demonstrate that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal SNARE VAMP2, which is crucial for efficient membrane repair. Thus, the here identified Ca2+-evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

cell biology↗

bioRxiv2022

Single molecule analyses of Salmonella translocated effector proteins reveal targeting to and dynamics in host cell endomembranes

Bacterial pathogens deliver proteins in temporal and spatial coordinated manner to manipulate mammalian host cells. The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. The pathogen resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV). By Salmonella- induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector molecules translocated by the SPI2-encoded type III secretion system (SPI2-T3SS) into host cells. A subset of these effectors is associated with, or integral in SCV and SIF membranes. It remained to be determined how SPI2-T3SS effectors reach their subcellular destination, and how these effectors interact with endomembranes remodeled by Salmonella. We deployed self-labeling enzyme (SLE) tags as novel approach to label translocated effector proteins in living host cells, and analyzed their dynamics on single molecule level. We found that SPI2-T3SS effector proteins diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differed between various effector proteins investigated and was dependent on membrane architecture of SIF. In the early infection, we observed host endosomal vesicles associated with Salmonella effector proteins. Effector-positive vesicles continuously fused with SCV and SIF membranes, providing a route of effector delivery by SPI2-T3SS translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This novel mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

microbiology↗

bioRxiv2021

SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids

Several studies have pointed to retinal involvement in COVID-19 disease, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell-derived retinal organoids to study retinal infection by the SARS-CoV-2 virus. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID-19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS-CoV-2 infection of retinal organoids, indicating that SARS-CoV-2 infects retinal cells in an ACE2-dependent manner. These results suggest a retinal involvement in COVID-19 and emphasize the need to monitor retinal pathologies as potential sequelae of "long COVID".

molecular biology↗

bioRxiv2020

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis towards limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single cell comparison to human fetal and adult myogenic progenitors reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

bioengineering↗