Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

Pluripotent embryonic stem cells (ESCs) have a shortened cell cycle that enables their rapid proliferation. The ESC-specific miR-290 and miR-302 microRNA families promote proliferation whereas let-7 microRNAs inhibit self-renewal and promote cell differentiation. Lin28 suppresses let-7 expression in ESCs. Here, to gain further insight into mechanisms controlling ESC self-renewal we explore the molecular and cellular role of the let-7 target Trim71 (mLin41). We show that Trim71 associates with Argonaute2 (Ago2) and microRNAs and represses expression of Cdkn1a, a cyclin-dependent kinase inhibitor that negatively regulates the G1–S transition. We identify protein domains required for Trim71 association with Ago2, localization to P-bodies, and for repression of reporter mRNAs. Trim71 knockdown prolongs the G1 phase of the cell cycle and slows ESC proliferation, a phenotype that was rescued by depletion of Cdkn1a. Thus, we demonstrate Trim71 is a factor that facilitates the G1–S transition to promote rapid ESC self-renewal.

β-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. β-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of β-catenin in Sertoli cells during testis formation, we either deleted β-catenin or expressed a constitutively active form of β-catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Müllerian duct regression. β-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized β-catenin mutants, we then investigated the relationship of Wt1 and β-catenin in Sertoli cells and found Wt1 inhibits β-catenin signaling in these cells during testis development. Wt1 deletion resulted in upregulation of β-catenin expression in Sertoli cells both in vitro and in vivo. Our study indicates that Sertoli cell expression of β-catenin is dispensable for testis development. However, the suppression of β-catenin signaling in these cells is essential for proper testis formation and Wt1 is a negative regulator of β-catenin signaling during this developmental process.

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/ BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2 ¶-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation downregulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoterassociated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.

Endometriosis is an estrogen-dependent inflammatory disorder among reproductive-aged women associated with pelvic pain, anxiety, and depression. Pain is characterized by central sensitization; however, it is not clear if endometriosis leads to increased pain perception or if women with the disease are more sensitive to pain, increasing the detection of endometriosis. Endometriosis was induced in mice and changes in behavior including pain perception, brain electrophysiology, and gene expression were characterized. Behavioral tests revealed that mice with endometriosis were more depressed, anxious and sensitive to pain compared to sham controls. Microarray analyses confirmed by qPCR identified differential gene expression in several regions of brain in mice with endometriosis. In these mice, genes such as Gpr88, Glra3 in insula, Chrnb4, Npas4 in the hippocampus, and Lcn2 in the amygdala were upregulated while Lct, Serpina3n (insula), and Nptx2 (amygdala) were downregulated. These genes are involved in anxiety, locomotion, and pain. Patch clamp recordings in the amygdala were altered in endometriosis mice demonstrating an effect of endometriosis on brain electrophysiology. Endometriosis induced pain sensitization, anxiety, and depression by modulating brain gene expression and electrophysiology; the effect of endometriosis on the brain may underlie pain sensitization and mood disorders reported in women with the disease.

Overactive WNT/beta-catenin signaling has been found in many forms of cancer in human patients. Mouse models with mutations in different components of the WNT/beta-catenin signaling pathway have been generated to mimic tumorigenesis in humans. Mice with mutations that result in overactive WNT/beta-catenin signaling developed tumors in some tissues, such as digestive tract, skin, and ovary, but they failed to develop tumors in other tissues, such as mammary gland, liver, kidney, and primordial germ cells. To investigate whether overactive beta-catenin signaling is capable of inducing Sertoli cell tumorigenesis in testes, we generated Ctnnb1(tm1Mmt/+);Tg(AMH-cre)1Flor male mice that express a constitutively active form of beta-catenin specifically in Sertoli cells. No tumors were observed at 4 mo of age, but 70% of the mutant males developed Sertoli cell tumors at 8 mo of age. At 1 yr of age, more than 90% of the mutant males developed tumors. No instances of extratesticular spread of the tumors were found in the mutant mice. These studies show a causal link between overactive WNT/beta-catenin signaling and Sertoli cell tumor development and provide a novel mouse model for the study of Sertoli cell tumor biology.

Single molecule fitting-based superresolution microscopy achieves sub-diffraction-limit image resolution but suffers from a need for long acquisition times to gather enough molecules. Several methods have recently been developed that analyze high molecule density images but most are only applicable to two dimensions. In this study, we implemented a high-density superresolution localization algorithm based on compressed sensing and a biplane approach that provides three-dimensional information about molecules, achieving super-resolution imaging at higher molecule densities than those achieved using the conventional single molecule fitting method.