Clupper, M. | bioRxiv Science

bioRxiv2021

Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

cell biology↗

bioRxiv2020

A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. This disrupts the balance of postsynaptic excitatory and inhibitory signaling, causing levamisole hypersensitivity. We also examined other genes that resulted in a levamisole hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

genetics↗