Biology subjects

Sharp, K. G.

Publications and source records attributed to Sharp, K. G..

3 recordsLinked to original sources

bioRxiv2022

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+ activated Cl- channel TMEM16A. To do so, fertilization activates a phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLC{gamma} and PLC{beta}. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLC{gamma}, or inhibitors of Gq/11 pathways, used to stop activation of PLC{beta}. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

physiology↗

bioRxiv2022

Large RNP granules in C. elegans oocytes have distinct phases of RNA binding proteins

The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the C. elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In C. elegans, when oogenesis undergoes an extended meiotic arrest, germ granule proteins and other RNA binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find the RNA binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules MEG-3 is not required for the condensation of PGL-1 or other RNA binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.

cell biology↗

bioRxiv2022

A transcriptional network required for Toxoplasma gondii tissue cyst formation is dispensable for long-term persistence

Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEG{Delta}ROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with {Delta}ROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in {Delta}ROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and {Delta}ROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.

microbiology↗