Biology subjects

Damstra, H. G. J.

Publications and source records attributed to Damstra, H. G. J..

5 recordsLinked to original sources

bioRxiv2025

Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2-induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome-plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.

cell biology↗

bioRxiv2025

Combinatorial protein barcodes enable self-correcting neuron tracing with nanoscale molecular context

Mapping nanoscale neuronal morphology with molecular annotations is critical for understanding healthy and dysfunctional brain circuits. Current methods are constrained by image segmentation errors and by sample defects (e.g., signal gaps, section loss). Genetic strategies promise to overcome these challenges by using easily distinguishable cell identity labels. However, multicolor approaches are spectrally limited in diversity, whereas nucleic acid barcoding lacks a cellfilling morphology signal for segmentation. Here, we introduce PRISM (Protein-barcode Reconstruction via Iterative Staining with Molecular annotations), a platform that integrates combinatorial delivery of antigenically distinct, cell-filling proteins with tissue expansion, multi-cycle imaging, barcode-augmented reconstruction, and molecular annotation. Protein barcodes increase label diversity by >750-fold over multicolor labeling and enable morphology reconstruction with intrinsic error correction. We acquired a [~]10 million {micro}m3 volume of mouse hippocampal area CA2/3, multiplexed across 23 barcode antigen and synaptic marker channels. By combining barcodes with shape information, we achieve an 8x increase in automatic tracing accuracy of genetically labelled neurons. We demonstrate PRISM supports automatic proofreading across micron-scale spatial gaps and reconnects neurites across discontinuities spanning hundreds of microns. Using PRISMs molecular annotation capability, we map the distribution of synapses onto traced neural morphology, characterizing challenging synaptic structures such as thorny excrescences (TEs), and discovering a size correlation among spatially proximal TEs on the same dendrite. PRISM thus supports selfcorrecting neuron reconstruction with molecular context.

neuroscience↗

bioRxiv2021

Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.

cell biology↗

bioRxiv2021

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

cell biology↗

bioRxiv2020

Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells.

When a T cell and an antigen-presenting cell form an immunological synapse, rapid dynein-driven translocation of the centrosome towards the contact site leads to reorganization of microtubules and associated organelles. Currently, little is known about how the regulation of microtubule dynamics contributes to this process. Here, we show that the knockout of KIF21B, a kinesin-4 linked to autoimmune disorders, causes microtubule overgrowth and perturbs centrosome translocation. KIF21B restricts microtubule length by inducing microtubule pausing typically followed by catastrophe. Catastrophe induction with vinblastine prevented microtubule overgrowth and was sufficient to rescue centrosome polarization in KIF21B-knockout cells. Biophysical simulations showed that a relatively small number of KIF21B molecules can restrict microtubule length and promote an imbalance of dynein-mediated pulling forces that allows the centrosome to translocate past the nucleus. We conclude that proper control of microtubule length is important for allowing rapid remodeling of the cytoskeleton and efficient T cell polarization.

cell biology↗