Biology subjects

Wubbolts, R.

Publications and source records attributed to Wubbolts, R..

4 recordsLinked to original sources

bioRxiv2025

EVtrace: tracing extracellular vesicles-associated proteins in recipient cells using stable isotope labeling

Release and uptake of extracellular vesicles (EVs) is a highly conserved means of communication used by cells across all kingdoms of life. These nano-sized vesicles transfer messages encoded in proteins and nucleic acids and play a role in numerous (patho)physiological processes. A longstanding question in the field is whether interaction and uptake of EVs is a stochastic process or whether specific EV subpopulations target different types of recipient cells. Here we present EVtrace, a proteomics-based approach that accommodates stable isotopic labeling of amino acids in culture (SILAC) to trace back labeled EV-associated proteins in unlabeled recipient cell types. We describe the optimization of EV labeling conditions and EV-cell cultures, and introduce a proteomics data analysis pipeline to confidently identify sparse internalized EV proteins among unlabeled recipient cell proteins. As a proof of concept, we studied the targeting of prostate cancer (PCa) EVs to bone cells and non-bone cells, considering that bone is a common metastatic site of PCa. Using EVtrace we demonstrate that interaction of PCa EVs with recipient cell types was not stochastic, and that different subpopulations of EVs targeted different cells. Proteins of EV subpopulations that were traced in bone cells, but not in non-bone cells, were strongly enriched for pathways involved in cancer progression and metastasis. With super-resolution microscopy we confirmed that EV proteins traced in different targeted cell types co-occurred on specific EV subpopulations. EVtrace is a valuable new tool for EV research because it supports identification of EV subpopulations that are transferred to recipient cells and discloses candidate proteins potentially involved in EV binding/uptake and functional modification of target cells.

molecular biology↗

bioRxiv2025

Visualizing extracellular vesicle-mediated RNA transfer using a novel metabolic labeling approach

Extracellular vesicles (EVs) are key players in intercellular communication, facilitated by the transfer of RNA and other molecular cargo between cells. However, how EV-RNA is processed in recipient cells remains poorly understood, particularly the uptake and intracellular trafficking pathways leading to functional transfer remains elusive. Visualizing EV-RNA in recipient cells may help to address these knowledge gaps. Although EV labeling methods are well-established, tracking endogenous RNA cargo within EVs and in recipient cells is challenging due to its low abundance, requiring highly sensitive labeling methods. Existing RNA labeling methods often lack sensitivity, specificity for donor cell-derived RNA, or are limited to labeling long RNA species. In this study, we propose a novel metabolic labeling approach to track RNA in donor cells, EVs and recipient cells using 5-ethynyl uridine (5-EU). 5-EU is a nucleoside analog of uridine that is incorporated into nascent RNA and can be detected through click chemistry with a fluorescent azide. To enhance RNA labeling efficiency, we overexpressed Uridine-Cytidine Kinase 2 (UCK2) in MDA-MB-231 donor cells, which increased 5-EU incorporation into nascent RNA chains. This approach allowed visualization and quantification of RNA in isolated EVs. Furthermore, co-culturing MDA-MB-231 UCK2+ donor cells expressing a CD63-HaloTag fusion protein with HMEC1-eGFP+ recipient cells enabled visualization and quantification of intercellular transfer of EVs and their endogenous RNA cargo. This novel approach offers a sensitive and specific tool for studying physiological EV-RNA transfer, thereby advancing our understanding of the biological roles of EVs in intercellular communication.

cell biology↗

bioRxiv2025

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

microbiology↗

bioRxiv2020

Excessive E2F transcription in single cancer cells precludes transient cell cycle exit after DNA damage.

E2F transcription factors control the expression of cell cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we now demonstrate in human cancer biopsies that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we deleted the atypical E2F repressors (E2F7/8) in untransformed cells. Individual cells with elevated E2F-activity during S/G2-phase failed to exit the cell cycle after DNA damage and underwent mitosis. In contrast, wild type cells completed S-phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F-target Emi1. Strikingly, many arrested wildtype cells could eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F-transcription fail to exit the cell cycle after DNA damage which potentially causes genomic instability, promotes malignant progression and reduces drug sensitivity.

cancer biology↗