Biology subjects

Zagoriy, I.

Publications and source records attributed to Zagoriy, I..

6 recordsLinked to original sources

bioRxiv2022

Genetically encoded multimeric tags for intracellular protein localisation in cryo-EM

Cryo-electron tomography is a powerful label-free tool for visualizing biomolecules in their native cellular context at molecular resolution. However, the precise localisation of biomolecules of interest in the tomographic volumes is challenging. Here, we present a tagging strategy for intracellular protein localisation based on genetically encoded multimeric particles (GEMs). We show the applicability of drug-controlled GEM labelling of endogenous proteins in cryo-electron tomography and cryo-correlative fluorescence imaging in human cells.

cell biology↗

bioRxiv2021

Condensate-mediated reactivation of mumps virus infection under stress

Many viruses can establish long-term persistent infections in the human host and cause chronic diseases. Here we combine cell biology, whole-cell proteomics and cryo-electron tomography to uncover how cellular stress disrupts the host-virus equilibrium in persistent infection and induces viral replication, using a model of negative-stranded RNA viruses, the mumps virus. We show that phosphorylation of the largely disordered viral phosphoprotein coincides with increased partitioning of viral polymerase into pre-formed liquid-like condensates and the formation of a stable replication machinery. By obtaining the first atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events that occur within viral condensates upon stress, together with concerted down-regulation of the host antiviral defense, provide an environment that supports up-regulation of viral replication and constitute a stress-mediated switch that disrupts the host-virus equilibrium in persistent infection. In BriefA multi-scale approach uncovers molecular and structural basis of how cellular stress provokes activation of persistent viral infection mediated by biomolecular condensates.

cell biology↗

bioRxiv2021

Integrated light and electron microscopy continuum resolution imaging of 3D cell cultures

3D cell cultures, in particular organoids, are emerging models to investigate healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nano-scale volume electron microscopy by performing 3D cell cultures in a single carrier amenable to all imaging steps. This allows to follow organoids growth, probe their morphology with fluorescent markers, identify areas of interest and analyze their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures, and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses reveal local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum resolution imaging pipeline is thus suited to foster basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy. HighlightsO_LIEstablishment of 3D cell cultures in sample carriers directly amenable to high-pressure freezing (HPF) C_LIO_LI3D cell cultures in HPF carriers allow drug treatment and live-cell imaging C_LIO_LIMulti-scale imaging of 3D cultures from live-cell light microscopy to volume electron microscopy C_LIO_LIEstablishments of HPF conditions for mouse and patient-derived organoids C_LIO_LIDeep-learning automatic segmentation of ultrastructural detail and quantitative data-mining reveal different subcellular organization associated with epithelium polarity C_LI

cell biology↗

bioRxiv2021

A Modular Platform for Streamlining Automated Cryo-FIB Workflows

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, current preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. The software enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM guided preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

biophysics↗

bioRxiv2020

Nuclear pores constrict upon energy depletion

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

cell biology↗

bioRxiv2019

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery.

cell biology↗