Biology subjects

Voortman, L. M.

Publications and source records attributed to Voortman, L. M..

10 recordsLinked to original sources

bioRxiv2025

Scaling data analyses in cellular cryoET using comprehensive segmentation

Automation and improved hardware have greatly accelerated the rate of data generation in cryoET. As the field moves towards quantitative cryoET, the scale of the resulting datasets presents a significant challenge for analysis and interpretation. To explore ways of handling datasets comprising thousands of tomograms, we investigated a comprehensive segmentation strategy - assigning an ontology-based identity to every voxel in a dataset - that is based on the sequential application of multiple convolutional neural networks. Using an openly available dataset of over 1800 Chlamydomonas reinhardtii tomograms as a test case, we demonstrate the segmentation of 25 different subcellular features across the full dataset, while requiring only a few seconds of processing time per tomogram. We show how the approach enables the representation of large datasets as searchable databases and propose the usage of ontology-based segmentations for improving two common processing tasks in cryoET. First, we explore context-aware particle picking as a method to retain biological context when selecting particles for subtomogram averaging and other downstream analyses. Secondly, we demonstrate area-selective template matching, where we use segmentation-based masks to avoid redundant computations in template matching and enable >500-fold faster processing in specific cases. To illustrate the utility of the approach, all segmentation results have also been made available online via cryopom.streamlit.app.

molecular biology↗

bioRxiv2024

Correlating Protein Aggregate Structure with Cellular Function in Differentiated Muscle Cells: Discriminating Pathogenic from Non-Pathogenic Forms

Ageing has a major adverse impact on maintaining cellular proteostasis and age-related dysregulation leads to an increase in protein aggregation. Equivalently, the accumulation of aggregated proteins accelerates proteostasis impairment. Accumulation of protein aggregates and impaired proteostasis are hallmarks of ageing-associated neuromuscular disorders and tissue degeneration is predominantly in post-mitotic muscle and neuronal cells. A short alanine expansion mutation in the Poly(A) binding protein nuclear 1 (PABPN1) causes Oculopharyngeal muscular dystrophy (OPMD), a rare age-associated protein aggregation myopathy. PABPN1 is a vital RNA-binding protein but OPMD pathology is limited to skeletal muscles connected to nuclear aggregates. In contrast to the mutant PABPN1, the wild-type PABPN1 forms age-associated non-pathogenic aggregates. We generated an inducible muscle cell models for mutant and wild-type PABPN1 protein aggregation. By combining four different, but complementary, imaging modalities, covering micro- to nanoscale resolutions, we were able to characterise differences in structure and dynamics between pathogenic and non-pathogenic PABPN1 aggregates in differentiated muscle cells. These data allowed us to correlate the structure of aggregates to cellular function, providing important insights into how aggregates lead to cell dysfunction in post-mitotic cells. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/591067v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@22870corg.highwire.dtl.DTLVardef@d0616borg.highwire.dtl.DTLVardef@abe34dorg.highwire.dtl.DTLVardef@d7f359_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Ais: streamlining segmentation of cryo-electron tomography datasets

Segmentation is a critical data processing step in many applications of cryo-electron tomography. Downstream analyses, such as subtomogram averaging, are often based on segmentation results, and are thus critically dependent on the availability of open-source software for accurate as well as high-throughput tomogram segmentation. There is a need for more user-friendly, flexible and comprehensive segmentation software that offers an insightful overview of all steps involved in preparing automated segmentations. Here, we present Ais: a dedicated tomogram segmentation package that is geared towards both high performance and accessibility, available at github.com/bionanopatterning/Ais. In this report, we demonstrate two common processing steps that can be greatly accelerated with Ais: particle picking for subtomogram averaging, and generating many-feature segmentations of cellular architecture based on in situ tomography data. Featuring comprehensive annotation, segmentation, and rendering functionality, as well as an open repository for trained models at aiscryoet.org, we hope that Ais will help accelerate research and dissemination of data involving cryoET.

molecular biology↗

bioRxiv2023

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we exemplify all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate the further adaptation of the technique within the field of cryoEM.

molecular biology↗

bioRxiv2023

Building a super-resolution fluorescence cryomicroscope

Correlating super-resolution fluorescence microscopy with cryo-electron tomography is a recent advancement in the field of cryo-electron microscopy that enables targeted, high-resolution imaging of specific biomolecules of interest. Critical to this approach is that the cryo-correlated light and electron microscopy (cryoCLEM) workflow requires samples to be cryogenically fixed prior to imaging, and thus a fluorescence microscope is required that can maintain the cryogenically preserved state of the sample while also being capable of super-resolution imaging. In this report, we outline the blueprint of a microscope that was designed for single molecule localization microscopy of cryosamples, and we describe the rationale behind its design. All specifications, including a detailed 3d model of the entire assembly, are freely available via ccb.lumc.nl/downloads-231.

molecular biology↗

bioRxiv2023

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A concern in this combined method (called SR-cryoCLEM), however, is whether and how fluorescence imaging prior to cryoEM acquisition is detrimental to sample integrity. In this report, we investigated the effect of high-dose laser light irradiation on apoferritin samples prepared for cryoEM with excitation wavelengths commonly used in fluorescence microscopy, and comparing these samples to controls that were kept in the dark. We found that laser illumination, of equal duration and intensity as used in super-resolution cryomicroscopy and in the presence of high concentrations of fluorescent protein, did not affect the achievable resolution in cryoEM, with final reconstructions reaching resolutions of ~1.8 [A] regardless of the illumination conditions. The finding that super-resolution fluorescence imaging of cryosamples prior to cryoEM data acquisition does not limit the achievable resolution suggests that super-resolution cryo-fluorescence microscopy and in situ structural biology using cryoEM are entirely compatible. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2022

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy (FM) are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude, and demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated microscopy.

molecular biology↗

bioRxiv2022

Multi-scale Assessment of Brain Blood Volume and Perfusion in the APP/PS1 Mouse Model of Amyloidosis

Vascular dysfunction is increasingly recognized to play a role in the development of Alzheimers disease (AD). The relation between vascular dysfunction and the neuropathological amyloid {beta} accumulation characteristic for AD is however unclear. The limited resolution of in vivo imaging techniques, the intricate 3D structure of the microvasculature and the different co-occurring types of amyloid {beta} accumulation in patients hamper studying this relation in patients. Here, we therefore employed the APP/PS1 mouse model, which develops parenchymal amyloid {beta} plaques, to study the effect of parenchymal amyloid {beta} plaques on the structure and function of the vasculature. Blood vessels and amyloid {beta} plaques were fluorescently labeled in vivo with lectin-DyLight594 and methoxy XO4, respectively, in APP/PS1 mice at old age. The brain tissue was cleared post-mortem with the CUBIC clearing protocol, which allowed structural imaging at microscopic resolution of the vessels and plaques in a large 3D volume. Segmentation of the vasculature enabled mapping of the microvascular Cerebral Blood Volume (mCBV), which ranged from 2 % to 5 % in the white matter and the thalamus, respectively. No mCBV differences were observed between APP/PS1 mice and wild type (WT) control mice. The effect of the amyloid {beta} plaques on vascular function was studied in vivo by measuring Cerebral Blood Flow (CBF) and Arterial Transit Time (ATT) with Arterial Spin Labeling (ASL) MRI. Similar to the mCBV findings, no differences were observed in CBF or ATT between APP/PS1 and control mice, indicating that brain vascular morphology and function in this mouse model are preserved in the presence of amyloid {beta} plaques.

neuroscience↗

bioRxiv2022

A transcriptome atlas of leg muscles from healthy human volunteers reveals molecular and cellular signatures associated with muscle location

Skeletal muscles support the stability and mobility of the skeleton but differ in biomechanical properties and physiological functions. The intrinsic factors that regulate muscle-specific characteristics are poorly understood. To study these, we constructed a large atlas of RNA-seq profiles from six leg muscles and two locations from one muscle, using biopsies from 20 healthy young males. We identified differential expression patterns and cellular composition across the seven tissues using three bioinformatics approaches confirmed by large-scale newly developed quantitative immune-histology procedures. With all three procedures, the muscle samples clustered into three groups congruent with their anatomical location. Concomitant with genes marking oxidative metabolism, genes marking fast- or slow-twitch myofibers differed between the three groups. The groups of muscles with higher expression of slow-twitch genes were enriched in endothelial cells and showed higher capillary content. In addition, expression profiles of Homeobox (HOX) transcription factors differed between the three groups and were confirmed by spatial RNA hybridization. We created an open-source graphical interface to explore and visualize the leg muscle atlas (https://tabbassidaloii.shinyapps.io/muscleAtlasShinyApp/). Our study reveals molecular specialization of human leg muscles and provides a novel resource to study muscle-specific molecular features, which could be linked with (patho)physiological processes.

neuroscience↗

bioRxiv2022

Vimentin intermediate filaments organize organellar architecture in response to ER stress

Compartmentalization of organelles in space and time affects their functional state and enables higher order regulation of essential cellular processes. How organellar residence is maintained in a defined area of the cell remains poorly understood. In this study, we uncover a new role for intermediate filaments in the maintenance of organellar architecture and dynamics, which is executed through a functional connection between Vimentin and the ER-embedded ubiquitin ligase ring finger protein 26 (RNF26). While the ubiquitin ligase function of RNF26 promotes perinuclear positioning of endolysosomes, its catalytically inactive mutant I382R preferentially binds Vimentin through the RNF26 C-terminal tail. Loss of either RNF26 or Vimentin redistributes endolysosomes throughout the cytosol and mobilizes ER membranes from the perinuclear ER towards the periphery. Furthermore, RNF26 and Vimentin control changes in ER morphology and organelle compartmentalization during ER stress. Collectively, we define a new function for Vimentin-containing intermediate filaments as anchors of a dynamic interplay between the ER and endosomes, critical to the integrity of the perinuclear ER and corresponding perinuclear endosomal cloud during homeostatic and stress conditions. SynopsisThe perinuclear area hosts a wide variety of cellular organelles, and their interaction with the ER governs essential cellular processes. To spatiotemporally organize endosomes and ER in the perinuclear region, the ER-embedded E3 ubiquitin ligase RNF26 interacts with Vimentin to physically link the perinuclear ER membrane with the intermediate filament cytoskeleton. As a result, Vimentin ensures perinuclear RNF26 retention, which in turn controls the perinuclear location of ER membranes and endosomes, which can be affected during stressed conditions. O_LIVimentin interacts with inactive RNF26 in the ER membrane C_LIO_LIRNF26 by virtue of the Vimentin interaction controls perinuclear organization of ER membranes and the endosomal system C_LIO_LIVimentin immobilizes ER membranes in the perinuclear area C_LIO_LIVimentin and RNF26 compartmentalize organelles in the perinuclear region during ER stress C_LIO_LIWe define a new function of Vimentin intermediate filaments in the control of the perinuclear endosomal and ER organization C_LI

cell biology↗