Prostaglandins limit nuclear actin to control nucleolar function during oogenesis.

Prostaglandins (PGs), locally acting lipid signals, regulate female reproduction, including oocyte development. However, the cellular mechanisms of PG action remain largely unknown. One cellular target of PG signaling is the nucleolus. Indeed, across organisms, loss of PGs results in misshapen nucleoli, and changes in nucleolar morphology are indicative of altered nucleolar function. A key role of the nucleolus is to transcribe ribosomal RNA (rRNA) to drive ribosomal biogenesis. Here we take advantage of the robust, in vivo system of Drosophila oogenesis to define the roles and downstream mechanisms whereby PGs regulate the nucleolus. We find that the altered nucleolar morphology due to PG loss is not due to reduced rRNA transcription. Instead, loss of PGs results in increased rRNA transcription and overall protein translation. PGs modulate these nucleolar functions by tightly regulating nuclear actin, which is enriched in the nucleolus. Specifically, we find that loss of PGs results in both increased nucleolar actin and changes in its form. Increasing nuclear actin, by either genetic loss of PG signaling or overexpression of nuclear targeted actin (NLS-actin), results in a round nucleolar morphology. Further, loss of PGs, overexpression of NLS-actin or loss of Exportin 6, all manipulations that increase nuclear actin levels, results in increased RNAPI-dependent transcription. Together these data reveal PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.

Cardiac injury modulates critical components of prostaglandin E2 signaling during zebrafish heart regeneration.

The inability to effectively stimulate cardiomyocyte proliferation remains a principle barrier to regeneration in the adult human heart. A tightly regulated, acute inflammatory response mediated by a range of cell types is required to initiate regenerative processes. Prostaglandin E2 (PGE2), a potent lipid signaling molecule induced by inflammation, has been shown to promote regeneration and cell proliferation; however, the dynamics of PGE2 signaling in the context of heart regeneration remain underexplored. Here, we employ the regeneration-competent zebrafish to characterize components of the PGE2 signaling circuit following cardiac injury. In the regenerating adult heart, we documented an increase in PGE2 levels, concurrent with upregulation of cox2a and ptges, two genes critical for PGE2 synthesis. Furthermore, we identified the epicardium as the most prominent site for cox2a expression, thereby suggesting a role for this tissue as an inflammatory mediator. Injury also drove the opposing expression of PGE2 receptors, upregulating pro-restorative ptger2a and downregulating the opposing receptor ptger3. Importantly, treatment with pharmacological inhibitors of Cox2 activity suppressed both production of PGE2, and the proliferation of cardiomyocytes. These results suggest that injury-induced PGE2 signaling is key to stimulating cardiomyocyte proliferation during regeneration.

Nuclear Actin: From Discovery to Function.

While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses induce the nuclear localization and alter the structure of actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. Here, we explore the early discovery of actin in the nucleus and discuss the forms and functions of nuclear actin in both normal and disease contexts. Anat Rec, 301:1999-2013, 2018. © 2018 Wiley Periodicals, Inc.

Multiple Pools of Nuclear Actin.

While nuclear actin was reported ~50 years ago, it's in vivo prevalence and structure remain largely unknown. Here, we use Drosophila oogenesis, that is, follicle development, to characterize nuclear actin. We find that three different reagents-DNase I, anti-actin C4, and anti-actin AC15-recognize distinct pools of nuclear actin. DNase I labels monomeric or G-actin, and, during follicle development, G-actin is present in the nucleus of every cell. Some G-actin is recognized by the C4 antibody. In particular, C4 nuclear actin colocalizes with DNase I to the nucleolus in anterior escort cells, follicle stem cells, some mitotic follicle cells, and a subset of nurse cells during early oogenesis. C4 also labels polymeric nuclear actin in the nucleoplasm of the germline stem cells, early cystoblasts, and oocytes. The AC15 antibody labels a completely distinct pool of nuclear actin from that of DNase I and C4. Specifically, AC15 nuclear actin localizes to the chromatin in the nurse and follicle cells during mid-to-late oogenesis. Within the oocyte, AC15 nuclear actin progresses from localizing to puncta surrounding the DNA, to forming a filamentous cage around the chromosomes. Together these findings reveal that nuclear actin is highly prevalent in vivo, and multiple pools of nuclear actin exist and can be recognized using different reagents. Additionally, our localization studies suggest that nuclear actin may regulate stemness, nucleolar structure and function, transcription, and nuclear structure. Such findings call for further studies to explore the prevalence, diversity, and functions of nuclear actin across tissues and organisms. Anat Rec, 301:2014-2036, 2018. © 2018 Wiley Periodicals, Inc.

Fascin regulates nuclear actin during Drosophila oogenesis.

Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.

DNMT3B7 expression promotes tumor progression to a more aggressive phenotype in breast cancer cells.

Epigenetic changes, such as DNA methylation, have been shown to promote breast cancer progression. However, the mechanism by which cancer cells acquire and maintain abnormal DNA methylation is not well understood. We have previously identified an aberrant splice form of a DNA methyltransferase, DNMT3B7, expressed in virtually all cancer cell lines but at very low levels in normal cells. Furthermore, aggressive MDA-MB-231 breast cancer cells have been shown to express increased levels of DNMT3B7 compared to poorly invasive MCF-7 cells, indicating that DNMT3B7 may have a role in promoting a more invasive phenotype. Using data gathered from The Cancer Genome Atlas, we show that DNMT3B7 expression is increased in breast cancer patient tissues compared to normal tissue. To determine the mechanism by which DNMT3B7 was functioning in breast cancer cells, two poorly invasive breast cancer cell lines, MCF-7 and T-47D, were stably transfected with a DNMT3B7 expression construct. Expression of DNMT3B7 led to hypermethylation and down-regulation of E-cadherin, altered localization of ß-catenin, as well as increased adhesion turnover, cell proliferation, and anchorage-independent growth. The novel results presented in this study suggest a role for DNMT3B7 in the progression of breast cancer to a more aggressive state and the potential for future development of novel therapeutics.

Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.