Biology subjects

Rosenbaum, J. C.

Publications and source records attributed to Rosenbaum, J. C..

5 recordsLinked to original sources

bioRxiv2023

Xenopus laevis lack the critical sperm factor PLCζ

Fertilization of eggs from the African clawed frog Xenopus laevis is characterized by an increase in cytosolic calcium, a phenomenon that is also observed in other vertebrates such as mammals and birds. During fertilization in mammals and birds, the transfer of the soluble PLC{zeta} from sperm into the egg is thought to trigger the release of calcium from the endoplasmic reticulum (ER). Injecting sperm extracts into eggs reproduces this effect, reinforcing the hypothesis that a sperm factor is responsible for calcium release and egg activation. Remarkably, this occurs even when sperm extracts from X. laevis are injected into mouse eggs, suggesting that mammals and X. laevis share a sperm factor. However, X. laevis lacks an annotated PLCZ1 gene, which encodes the PLC{zeta} enzyme. In this study, we attempted to determine whether sperm from X. laevis express an unannotated PLCZ1 ortholog. We identified PLCZ1 orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in either X. laevis or the closely related Xenopus tropicalis. Additionally, we performed RNA sequencing on testes obtained from adult X. laevis males and did not identify potential PLCZ1 orthologs in our dataset or in previously collected ones. These findings suggest that PLCZ1 may have been lost in the Xenopus lineage and raise the question of how fertilization triggers calcium release and egg activation in these species.

physiology↗

bioRxiv2022

Studying metal-protein interactions using fluorescent protein indicators

Metals are widespread environmental toxins that disrupt normal cellular processes through their interactions with proteins and other macromolecules. In this study, we developed the metalsensitive fluorescent protein mseGFP as a ratiometric reporter capable of binding heavy metals. We found that mseGFP bound mercury and lead tightly but had substantially lower sensitivity to other metals. By comparison, the redox sensor roGFP2 functioned as a ratiometric indicator for transition metals, with the highest sensitivity for copper, followed by nickel and cobalt. mseGFP and roGFP2 could also report metal binding through fluorescence quenching, and we used this effect to measure high affinity interactions for both proteins with copper and iron. Crystal structure analysis of mseGFP complexed with phenylarsine oxide revealed an unexpected mode of heavy metal interaction, with mseGFP binding PAO with 2:2 stoichiometry. Glutathione strongly inhibited most metal interactions with the fluorescent protein reporters, but increased the affinity of arsenic and cadmium for mseGFP. When expressed in HEK293T cells, mseGFP reported uptake of mercury and phenylarsine oxide from surrounding media. Glutathione depletion enhanced binding of phenylarsine oxide to mseGFP in cells, validating the importance of glutathione in modulating metal-protein interactions.

biochemistry↗

bioRxiv2022

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.

developmental biology↗

bioRxiv2022

PIP2 and Ca2+ regulation of TMEM16A currents in excised inside-out patches

The Ca2+ activated Cl- channel formed by transmembrane member 16A (TMEM16A) is broadly expressed and regulates diverse processes. In addition to Ca2+, TMEM16A channels require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open. Like other channels regulated by PI(4,5)P2, TMEM16A-conducted currents recorded in excised patches slowly decay overtime. Here we assessed how intracellular Ca2+ alters the rate of this current rundown, using the channels endogenously expressed in oocytes from the African clawed frog, Xenopus laevis. We found that in excised, inside-out patches, the concentration of applied Ca2+ alters the rate of rundown, with high Ca2+ concentrations speeding rundown by activating membrane associated phospholipase C (PLC). Together, these results clarify our understanding of how Ca2+ regulates both TMEM16A directly, and targets PLC to regulate the membrane PI(4,5)P2 content.

physiology↗

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↗