Biology subjects

Bebelman, M. P.

Publications and source records attributed to Bebelman, M. P..

3 recordsLinked to original sources

bioRxiv2025

The axonal ER couples translation and secretion machineries for local delivery of axonal transmembrane proteins to promote axonal development

Neuronal development and function rely on the polarized sorting of transmembrane proteins (TMPs). Most TMPs follow a conventional secretory pathway confined to the cell soma, where ER-translated TMPs exit the ER through ERES to reach the Golgi, prior to PM delivery. We recently found that the axonal ER regulates local translation; however, how this is linked to secretion remains unknown. Here, we find that axonally translated TMPs exit the ER and reach the axonal PM via an ERES-dependent/Golgi-independent route. Intriguingly, we uncover a feedback loop linking translation and secretion at axonal ERES. HDLBP-dependent TMP synthesis regulates ERES formation, and axonal ERES in turn modulate TMP translation. Subsequently, the NRZ-SEC22B tethering complex links local TMP ER exit to PM delivery, dependent on ER-PM contacts. Axonal ERES and these associated molecular players support axon growth and bouton assembly. We propose a novel mechanism in which coupling of axonal TMP synthesis and secretion is essential for neuronal development.

cell biology↗

bioRxiv2024

Hypoxia and TNF-alpha modulate extracellular vesicle release from human induced pluripotent stem cell-derived cardiomyocytes

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte-derived EV (CM-EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that express NanoLuc-tetraspanin reporters. These modified hiPSC-CMs allow for robust quantification of CM-EV secretion from small numbers of cells without the need for time-consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM-EV biogenesis and secretion under basal and stress-associated conditions. We observed that EV biogenesis is context-dependent in hiPSC-CMs. Nutrient starvation decreases CM-EV secretion while hypoxia increases the production of CM-EVs in a nSmase2-dependent manner. Moreover, the inflammatory cytokine TNF- increased CM-EV secretion through a process involving NLRP3 inflammasome activation and mTOR signaling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC-CMs upon MI-associated stressors.

cell biology↗

bioRxiv2023

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad- spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIII{beta} as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions.

cell biology↗