Biology subjects

Mahamid, J.

Publications and source records attributed to Mahamid, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

ColabSeg: An interactive tool for editing, processing, and visualizing membrane segmentations from cryo-ET data

Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting seg-mentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, cleaning, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training dataset for fully automated convolutional neural network (CNN)-based segmentation.

bioinformatics↗

bioRxiv2023

Structural insights into context-dependent inhibitory mechanisms of chloramphenicol in cells

Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied peptidyl transfer center (PTC) binder, and growing evidence suggests it inhibits translation in a nascent peptide sequence-dependent manner. How such inhibition on the molecular scale translates to action on the cellular level remains unclear. Here, we employ cryo-electron tomography to visualize the impact of Cm inside the bacterium Mycoplasma pneumoniae. By resolving cellular Cm-bound ribosomes to 3.0 [A], we provide atomic detail on Cms coordination and interaction with natural nascent peptides and tRNAs in the PTC. We find that Cm leads to accumulation of translation elongation states that indicate ongoing futile accommodation cycles, and to extensive ribosome collisions. We thus suggest that beyond its inhibition of protein synthesis, the action of Cm may involve activation of cellular stress responses. This work exemplifies how in-cell structural biology advances understanding of mechanisms of action for extensively-studied antibiotics.

biophysics↗

bioRxiv2023

The distance between the plasma membrane and the actomyosin cortex acts as a nanogate to control cell surface mechanics

Animal cell shape changes are controlled by the actomyosin cortex, a peripheral actin network tethered to the plasma membrane by membrane-to-cortex attachment (MCA) proteins. Previous studies have focused on how myosin motors or actin turnover can generate the local deformations required for morphogenesis. However, how the cell controls local actin nucleation remains poorly understood. By combining molecular engineering with biophysical approaches and in situ characterization of cortical actin network architecture, we show that membrane-to-cortex tethering determines the distance between the plasma membrane and the actomyosin cortex at the nanoscale of single actin nucleators. In turn, the size of this gap dictates actin filament production and the mechanical properties of the cell surface. Specifically, it tunes formin activity, controlling actin bundling and cortical tension. Our study defines the membrane-to-cortex distance as a nanogate that cells can open or close by MCA proteins to control the activity of key molecules at the cell surface.

biophysics↗

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↗

bioRxiv2022

Cryo-electron tomography reveals enrichment and identifies microtubule lumenal particles in neuronal differentiation

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) structures and organizations of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons and pluripotent P19 cells. We obtain in-cell 3D maps of several lumenal particles from the respective cells and detect structural features that are common to all cell-types, underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggests a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggest a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.

cell biology↗

bioRxiv2022

Convolutional networks for supervised mining of molecular patterns within cellular context

Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (Deep Picker in Context), an open-source deep-learning framework for supervised structure segmentation and macromolecular complex localization in cellular cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles and cytosol. By comparing our method to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally-distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, by applying pre-trained networks to a HeLa cell dataset, we demonstrate that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate exploitation by the community.

bioinformatics↗

bioRxiv2022

Mechanism of RNA Polymerase I selection by transcription factor UAF

Pre-ribosomal RNA is selectively transcribed by RNA Polymerase (Pol) I in eukaryotes. The yeast transcription factor Upstream Activating Factor (UAF) represses Pol II transcription and mediates Pol I preinitiation complex (PIC) formation during the early stages of transcription initiation at the 35S ribosomal RNA gene. To unravel the DNA recognition and Pol I selection mechanisms of UAF, we determined the structure of UAF bound to native promoter DNA and transcription factor TBP. We found that UAF recognizes DNA using a hexameric histone-like scaffold with markedly different interactions than the nucleosome and the histone-fold-rich TFIID. UAF strategically sequesters TBP from DNA and Pol II/III-specific factors, and positions it for Core Factor binding, supporting Pol I recruitment. Our findings therefore reveal the molecular basis of Pol I selection for ribosome biogenesis. As well, they reveal an unexpected potential within the histone fold as a motif for specific protein-DNA interactions inside the cell.

molecular biology↗

bioRxiv2021

Visualizing translation dynamics at atomic detail inside a bacterial cell

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

biochemistry↗

bioRxiv2021

Determinants shaping the nanoscale architecture of the mouse rod outer segment

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long-range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

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↗

bioRxiv2021

DISCA: high-throughput cryo-ET structural pattern mining by deep unsupervised clustering

Cryo-electron tomography directly visualizes heterogeneous macromolecular structures in complex cellular environments, but existing computer-assisted sorting approaches are low-throughput or inherently limited due to their dependency on available templates and manual labels. We introduce a high-throughput template-and-label-free deep learning approach that automatically discovers subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Diverse structures emerging from sorted subsets enable systematic unbiased recognition of macro-molecular complexes in situ.

bioinformatics↗

bioRxiv2021

Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM

Over the last decade, single-particle electron cryo-microscopy has become one of the main techniques contributing to the growing library of high-resolution structures of macromolecules and their assemblies. For a full understanding of molecular mechanisms, however, it is important to place them into the broader context of a cell. Traditionally, this context can be visualized in 3D by electron cryo-tomography, and more recently, has also been studied by template matching of 2D images of cells and viruses. A current limitation of the latter approach is the high computational cost that limits the throughput and widespread adoption of this method. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells and demonstrate that it can function as a versatile tool for in situ visual proteomics and in situ structure determination. We compare the results with 3D template matching of tomograms acquired on identical sample locations. We identify strengths and weaknesses of both techniques which offer complementary information about target localization and identity.

cell biology↗

bioRxiv2021

Liquid-to-solid phase transition of oskar RNP granules is essential for their function in the Drosophila germline

Asymmetric localization of oskar RNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation of the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of specific proteins. Genetic modification of scaffold granule proteins, or tethering the intrinsically disordered region of human Fused in Sarcoma to oskar mRNA, allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate localization and expression of a maternal RNA that instructs germline formation.

cell biology↗

bioRxiv2021

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before. SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

cell biology↗

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↗

bioRxiv2020

Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.7 A inside cells

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 [A]. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.

biophysics↗