Biology subjects

Christenson, L. K.

Publications and source records attributed to Christenson, L. K..

4 recordsLinked to original sources

bioRxiv2023

Exomap1 mouse: a transgenic model for in vivo studies of exosome biology

Exosomes are small extracellular vesicles (sEVs) of [~]30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size ([~]80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute [~]15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of [~]50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.

cell biology↗

bioRxiv2022

Maturation of cortical endoplasmic reticulum clusters in the mouse oocyte: changes at fertilization.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum specializations (cortical ER clusters) thought be essential for egg activation. In examination of cortical ER clusters, we observed they were tethered to previously unreported fenestrae within the cortical actin layer. Further, studies demonstrated sperm preferentially bind to plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global maturational change in volume, shape, and calreticulin content that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males demonstrating that gamete fusion plays an important role in ER cluster maturation. In addition to these global changes seen at sites distant to the sperm, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection. Summary StatementSperm-oocyte interactions stimulate global changes in cortical endoplasmic reticulum cluster structure as well as localized responses at the sperm binding site.

developmental biology↗

bioRxiv2020

Loss of Cnot6l impairs inosine RNA modifications in mouse oocytes

Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of Cnot6l, a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during MI entry. Knockout of Btg4, an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in oocytes from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.

developmental biology↗

bioRxiv2018

ADAR1 A-to-I RNA alters codon usage

BackgroundFully grown mammalian oocytes and eggs are transcriptionally quiescent, and therefore have a unique RNA environment in which cellular processes depend on post-transcriptional regulation. RNA editing of adenosines into inosines (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a common post-transcriptional gene regulatory mechanism, yet it has not been systematically studied in oocytes.\n\nResultsA genome-wide RNA editing analysis of transcriptionally active growing oocytes from postnatal day 12 (PND12) mice, fully grown germinal vesicle (GV) oocytes, and transcriptionally quiescent metaphase II (MII) eggs indicates an abundant amount of A-to-I editing of mRNA transcripts. Editing of mRNA was greatest in GV oocyte and MII eggs compared to the PND12 immature oocytes, this was consistent with ADAR1 levels within these cells. Compared to somatic tissues, oocytes exhibited a different pattern of RNA editing, with a high proportion of RNA edits occurring in the coding regions. These edits resulted in nucleotide substitutions that were enriched at the third nucleotide of the codon (wobble position). Codon usage can affect mRNA stability and translation efficiency.\n\nConclusionsRNA editing in mouse oocytes is distinct from RNA editing in somatic cells due to increased frequencies of coding sequence RNA edits. We provide evidence in support of a previously unreported phenomenon of selective ADAR1 editing of the codon wobble position. Editing of the wobble position has the potential to fine tune post-transcriptional gene regulation through altering codon usage. This important observation advances our current understanding of RNA editing in mammalian cells.

developmental biology↗