Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

γ-Tubulin ring complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC–tethering proteins, including Drosophila Centrosomin (Cnn). Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here, we identify an extreme N-terminal CM1 autoinhibition (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits γ-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phosphomimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to γ-TuRCs is isoform specific and that misregulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is autoinhibited from binding γ-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to γ-TuRCs is regulated.

The lumen of cytoplasmic microtubules is a poorly explored expanse of intracellular space. Although typically represented in textbooks as a hollow tube, studies over several decades have shown that the microtubule lumen is occupied by a range of morphologically diverse components. These are predominantly globular particles of varying sizes which appear to exist either in isolation, bind to the microtubule wall, or form discontinuous columns that extend through the lumenal space. Actin filaments with morphologies distinct from the canonical cytoplasmic forms have also now been found within the microtubule lumen. In this review, we examine the historic literature that observed these lumenal components in tissues from diverse species and integrate it with recent cryo-electron tomography studies that have begun to identify lumenal proteins. We consider their cell and tissue distribution, possible mechanisms of incorporation, and potential functions. It is likely that continuing work in this area will open a new frontier in cytoskeletal biology.

Abstractγ-tubulin-ring-complexes (γ-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within γ-TuRC-tethering proteins, including Drosophila Cnn. Binding promotes microtubule nucleation and is restricted to centrosomes, but the mechanism regulating binding remains unknown. Here we identify an extreme N-terminal “CM1 auto-inhibition” (CAI) domain within the centrosomal isoform of Cnn (Cnn-C) that inhibits γ-TuRC binding. Cnn-C is phosphorylated at centrosomes and we find that phospho-mimicking sites within the CAI domain helps relieve auto-inhibition. In contrast, the testes-specific mitochondrial Cnn-T isoform lacks the CAI domain and can bind strongly to cytosolic γ-TuRCs. Ubiquitously expressing a version of Cnn-C lacking the CAI domain leads to major cell division defects, which appears to be due to ectopic cytosolic microtubule nucleation. We propose that the CAI domain folds back to sterically inhibit the CM1 domain, and that this auto-inhibition is relieved by phosphorylation that occurs specifically at centrosomes.