[
International Worm Meeting,
2021]
Human autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited ciliopathies and a common cause of end-stage renal failure. ADPKD is caused by mutations in PKD1 and PKD2, which encode the polycystin-1 (PC1) 11 transmembrane receptor and TRP polycystin-2 (PC2) channel. In humans and C. elegans, the polycystins function in a sensory capacity, localize to primary/sensory cilia, and are shed from cells in tiny extracellular vesicles (EVs), suggesting an ancient function. EVs are tiny (100-200 nm) membrane-bound vesicles that deliver lipids, nucleic acids, and proteins to neighboring cells. Cilia are important sites for EV release and interaction. The C. elegans polycystins LOV-1/PC1 and PKD-2/PC1 regulate male mating behaviors. During mating, C. elegans males transfer PKD-2::GFP-labeled EVs to the hermaphrodite vulva. Isolated EVs trigger male tail chasing behavior: PKD-2 location on ciliary EVs is essential for this EV bioactivity. A major gap in our knowledge is the in vivo functions of the polycystins in cilia and EVs.I have developed an arsenal of CRISPR-based, LOV-1 reporters and mutants for studying endogenous localization and function of LOV-1 and PKD-2 in male-specific ciliated EV releasing neurons (CEMs, RnBs, HOB). I am testing two hypotheses: (1) LOV-1 acts as an adhesion G protein-coupled receptor (GPCR) with PKD-2-dependent and -independent functions and (2) LOV-1 autoproteolytic cleavage plays an instrumental role in LOV-1 function. Using CRISPR-generated, N-Terminal (NTM) and C-terminal (CTM) fluorescent-protein fusions of LOV-1/PC1, I observed subcellular localization differences between NTM LOV-1 and CTM LOV-1 GPS cleavage products in dendrites, cilia, and EVs. PKD-2 and CTM LOV-1 (but not NTM LOV-1) colocalize, suggesting functional association. GPS cleavage is required for LOV-1 localization to cilia and release in EVs. In addition, preliminary data suggests the release of polycystin-containing EVs is a regulated process and our ability to detect environmental released FP-tagged EV cargoes is not the result of protein overexpression. Future efforts are aimed at determining the relationship between LOV-1 and PKD-2, the function of polycystins in EVs, the cellular targets of polycystin-carrying EVs, and the role of polycystin-carrying EVs in animal-animal communication.
Nikonorova, Inna, Cope, Alexander, Barr, Maureen, Power, Kaiden, Walsh, Jonathon, Wang, Juan, Shah, Premal, Akella, Jyothi
[
International Worm Meeting,
2021]
Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies. However, the EV field lacks a fundamental understanding of biogenesis, cargo content, signaling, and target interactions. EVs that are transmitted by cilia represent a particular challenge due to small volume of the organelle. Here, we used our established C. elegans system to determine the composition and explore the function of ciliary EVs. We took advantage of the fact that C. elegans releases ciliary EVs from 21 male-specific neurons and 6 core IL2 neurons into environment and thus provides a great platform for discovery of evolutionarily conserved ciliary EV cargo. To collect ciliary EVs we developed a biochemical enrichment procedure based on buoyant density centrifugation and high-resolution fractionation. Using fluorescent-tagged EV cargo PKD-2::GFP and superresolution microscopy we tracked ciliary EVs in the collected fractions and identified two populations of PKD-2 carrying EVs that differ in their densities. Proteomic analysis of the PKD-2 EV-enriched fractions revealed 2,888 proteins of C. elegans EVome that likely originate from multiple tissues. Top candidates were validated via generation of transgenic or CRISPR reporters and visualization of EV release using super-resolution microscopy. This strategy revealed that the male reproductive system is a major source of non-ciliary EVs. To extract ciliary EV cargoes, we integrated our dataset with published transcriptomic data. We identified new ciliary EV cargo involved in nucleotide binding and RNA interference, suggesting that environmentally-released ciliary EVs may also carry nucleic acids. Our work serves as a springboard for discoveries in the EV field and will help shed light on the contribution of ciliary EVs to the pathophysiology of abnormal EV signaling, including ciliopathies, cancer, and neurodegenerative diseases.
[
European Worm Meeting,
2006]
Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.