Biology subjects

Roverts, R.

Publications and source records attributed to Roverts, R..

5 recordsLinked to original sources

bioRxiv2023

Osteoblast-induced collagen alignment in a 3D in vitro bone model

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized by hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we developed a 3D cell culture system in which osteogenic cells deposit an oriented collagen matrix, that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenic cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration towards the periphery of the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenesis and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

cell biology↗

bioRxiv2023

A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

Liquid phase electron microscopy (LP-EM) has emerged as a powerful technique for in-situ observation of material formation in liquid. However, monitoring these processes requires the repeated interaction of the electron beam with the aqueous environment leading to the decomposition of water molecules (radiolysis), which affects the formation processes under investigation. Graphenes ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. Using the strategy, the process of interest is initiated when the graphene liquid cells (GLCs) are sealed. This means that the process cannot be imaged at early time points since microscope preparation and initiation of image acquisition at the region of interest are time-consuming. Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the most significant limitations of the graphene liquid cells, while combining the advantages of fluorescence and electron microscopy. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at a selected time point. We demonstrate the workflow first by observing multiple day crystallization processes and highlight its potential by observing a biological process: the complexation of calciprotein particles. With this observation, we show the exciting possibilities for LP-EM in biology.

biochemistry↗

bioRxiv2023

Comparative 3D ultrastructure of Plasmodium falciparum gametocytes

Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. In this study, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure of Plasmodium falciparum gametocytes of both genders and in different stages of development as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome variant. Furthermore, we generate, among other organelles, the first 3D reconstructions of endoplasmic reticulum (ER), Golgi apparatus, and extraparasitic structures in gametocytes. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar membrane of the gametocyte. Public availability of these volumetric electron microscopy resources of wild-type asexual and sexual blood-stage malaria parasites will facilitate reinterrogation of this global dataset with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure of P. falciparum gametocytes in high detail and shed light on the unique organellar biology of these deadly parasites.

microbiology↗

bioRxiv2022

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

cell biology↗

bioRxiv2022

DNA uptake by cell wall-deficient bacteria reveals a putative ancient macromolecule uptake mechanism

Horizontal gene transfer in bacteria is widely believed to occur via three main mechanisms: conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. We present here a new mechanism of DNA uptake that is independent of canonical DNA uptake machineries and is used by bacteria that live without a cell wall. We show that the cell wall-deficient bacteria engulf extracellular material, whereby intracellular vesicles are formed, and DNA is internalized. This mechanism is not specific to DNA, and allows uptake of other macromolecules and even 125 nm lipid nanoparticles (LNPs). Uptake was prevented by molecules known to inhibit eukaryotic endocytosis, suggesting this to be an energy-dependent process. Given that cell wall-deficient bacteria are considered a model for early life forms, our work provides a possible mechanism for primordial cells to acquire new genetic material or food before invention of the bacterial cell wall.

microbiology↗