Biology subjects

Krepelka, P.

Publications and source records attributed to Krepelka, P..

3 recordsLinked to original sources

bioRxiv2026

Automated cryo-volume EM for high-resolution 3D imaging and in situ structural analysis of cells and tissues

Cryo-volume electron microscopy (CVEM) enables three-dimensional imaging of biological ultrastructure in a near-native state but has been limited by low image contrast and charging artifacts that hinder data interpretation and complicate automation of data acquisition. Here we present an experimental and computational workflow that combines orthogonal cryo-SEM imaging, spot-geometry optimized O+ plasma-FIB milling, dedicated acquisition-control routines, and dedicated image alignment procedure. The workflow enables autonomous acquisition of volumetric datasets from vitrified cells and tissues at [~]15-20 nm isotropic resolution. In addition, sub-volume averaging of 113 nuclear pore complexes extracted from CVEM dataset of Cos-7 cell yielded its reconstruction at 9.4 nm resolution. Together, these results establish CVEM as a robust platform for autonomous high-resolution volumetric imaging and structural analysis of vitrified biological specimens.

Cell Biology↗

bioRxiv2026

Inhibition of Mitochondrial Respiration Fragments ER Architecture and Remodels Organelle Contact Sites, as Revealed by FIB-SEM

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic {beta}-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures--a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H2O2-induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process. Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope-mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in {beta}-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727587v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4f63b5org.highwire.dtl.DTLVardef@1b3dc8org.highwire.dtl.DTLVardef@7527fcorg.highwire.dtl.DTLVardef@1944f2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi

The globally distributed marine alga Emiliania huxleyi produces reflective calcite disks (coccoliths) that increase the albedo of ocean water and thus reduce the heat absorption in the ocean, which cools the Earths climate. The population density of E. huxleyi is restricted by nucleocytoplasmic large DNA viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane decorated with numerous transmembrane proteins. The virions are prone to deformation, and parts of their capsids deviate from the icosahedral arrangement. EhV-201 virions infect E. huxleyi by using their fivefold vertex to bind to a host cell and fuse the viruss inner membrane with the plasma membrane. Whereas the replication of EhV-201 probably occurs in the nucleus, virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. Upon the completion of genome packaging, the capsids change conformation, which enables them to acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which allows the continuous release of virions by exocytosis. Our results provide insight into how EhVs bypass the surface protective layers of E. huxleyi and exploit the organelles of an infected cell for progeny assembly.

molecular biology↗