Tracking extracellular vesicle (EV) cargo as a platform for studying EVomics, signaling, and targeting <i>in vivo</i>

Nikonorova, Inna A.; Wang, Juan; Cope, Alexander L; Tilton, Peter; Power, Kaiden M; Walsh, Jonathon; Akella, Jyothi S.; Ar, Krauchunas; Shah, Premal; Barr, Maureen M.

doi:10.1101/2021.09.23.461577

Cited by 3 publications

(3 citation statements)

References 95 publications

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“…In hermaphrodites, the parsimonious hypothesis is that NSPF proteins passively diffuse from the spermathecae, where activated sperm reside, to the uterus. The location of NSPF proteins within the uterus is similar to other proteins that males transfer, which originate in newly identified extracellular vesicles (Nikonorova et al, 2022). While the NSPFs do not appear in these vesicles, it is possible that this alternative vesicle type also contributes to seminal fluid proteins, supporting our finding that nematodes employ a distinct mechanism for secreting seminal fluid proteins than either mammals or Drosophila (McGraw et al, 2015).…”

Section: Discussionsupporting

confidence: 76%

A novel sperm-derived seminal fluid protein inCaenorhabditisnematodes

Kasimatis

Rehaluk

Rowe

et al. 2022

Preprint

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Post-insemination molecular interactions involving sperm are an ideal system for linking genotype, phenotype, and fitness. Nematode sperm contain subcellular vesicles known as membranous organelles (MOs) that fuse with the cell membrane upon sperm activation to release their soluble contents into the extracellular space. The second most abundant protein group in the MOs is the conserved Nematode-Specific Peptide family, group F (NSPF) gene family. We hypothesize that these proteins contribute to seminal fluid upon MO fusion and are part of post-insemination reproductive tract dynamics. We used experimental evolution to compete the wildtype allele against a deletion allele in 10 replicate populations. We calculated a significant mean selective disadvantage of 0.1% for the deletion allele, which indicated that the NSPF genes are an evolutionarily important component of male reproductive success. This conclusion was reinforced by qualitative trends from lower powered single-generation male fertility assays. We then characterized the functional location of NSPF proteins during fertilization using whole- worm immunostaining of a His-tagged nspf-1 transgene. Throughout male development, we found that NSPF presence and abundance was correlated with reproductive maturity in males, localizing to the seminal vesicle during L4 and to the spicules throughout adulthood. We confirmed that NSPF proteins are transferred to females during mating and hypothesize that proteins adhere to the spicules during transfer. NSPF proteins localize to the vulva and uterus lumen when transferred to mated females and in unmated adult hermaphrodites. These results suggest that the uterine localization of the NSPF proteins is likely a functional property of both male-derived sperm and self-sperm and not simply incidental to the point of transfer during mating. Our study demonstrates that nematodes use a novel mechanism for generating seminal fluid proteins and shows that the highly abundant NSPF proteins likely have a female-beneficial fitness effect.

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Section: Discussionsupporting

confidence: 76%

A novel sperm-derived seminal fluid protein inCaenorhabditisnematodes

Kasimatis

Rehaluk

Rowe

et al. 2022

Preprint

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“…Western blotting for EV markers (ARF6, TSG101, ALIX) confirmed the presence of these markers in our EV samples, while absence of the cis-Golgi marker GM130 ruled out cellular contamination (Figure S2c). Ciliary proteins like ARL13B, acetylated αtubulin and PC2 were detected in both large and small EV fractions, and KIF13B itself was abundantly present in large EVs (Figure S2c,d), in line with our live cell imaging analysis (Figure 2; Movie 2) and evidence from C. elegans identifying KLP-6 in neuronal ciliary EVs (Nikonorova et al, 2022). Moreover, for the Kif13b -/cells we observed a significant shift in distribution of PC2 and TSG101 from large to small EV fractions, as compared to WT cells (Figure S2c,d), indicating that in 72 h serum-starved cells, KIF13B promotes the release of PC2 and TSG101 in large EVs and that loss of KIF13B causes these proteins to be released via small EVs instead.…”

Section: Kif13b Regulates Release and Composition Of Small Evssupporting

confidence: 83%

Loss of KIF13B causes time-dependent changes in ciliary polycystin-2 levels and extracellular vesicle release

Rezi,

Frei,

Campestre

et al. 2024

Preprint

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The polycystic kidney disease gene product polycystin-2 (PC2) localizes to and is released from primary cilia in extracellular vesicles (EVs). We report that KIF13B regulates ciliary EV release and PC2 levels in kidney epithelial cells in a time-dependent manner and show that KIF13B itself is released from the ciliary tip. In early stages of ciliation, Kif13b-/- cells displayed excessive ciliary accumulation of PC2 and initially released fewer small EVs than control cells. Over time, ciliated Kif13b-/- cells increased their small EV release rate to control levels, however proteomic analysis identified >50 proteins depleted from mutant EV samples. These included the ubiquitin E3 ligase ITCH and palmitoyl transferase ZDHHC5, which localized to primary cilia. Mature Kif13b-/- cilia exhibited aberrant membrane bulges and decreased PC2 and ALIX, an ITCH substrate that negatively regulated ciliary PC2 levels. Our work provides new insight into the mechanisms of ciliary EV release, which is important for regulating ciliary membrane homeostasis and signalling function.

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“…2c ). In addition, tagging the EVs with GFP and polycystin-2 (PKD-2) served as a platform to track release of single EVs released from the cilia of sensory neurons in C. elegans , revealing that a single cilium produces multiple EVs carrying complex and heterogeneous cargo [ 107 ].…”

Section: Loading Evs With Functional Luminal Cargomentioning

confidence: 99%