Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

Background It is becoming clearer that living cells use water/water (w/w) phase separation to form membraneless organelles that exhibit various important biological functions. Currently, it is believed that the specific localization of biomacromolecules, including DNA, RNA and proteins in w/w microdroplets is closely related to their bio-activity. Despite the importance of this possible role of micro segregation, our understanding of the underlying physico-chemical mechanism is still unrefined. Further research to unveil the underlying mechanism of the localization of macromolecules in relation to their steric conformation in w/w microdroplets is needed. Principal findings Single-DNA observation of genome-size DNA (T4 GT7 bacteriophage DNA; 166kbp) by fluorescence microscopy revealed that DNAs are spontaneously incorporated into w/w microdroplets generated in a binary aqueous polymer solution with polyethylene glycol (PEG) and dextran (DEX). Interestingly, DNAs with elongated coil and shrunken conformations exhibit Brownian fluctuation inside the droplet. On the other hand, tightly packed compact globules, as well as assemblies of multiple condensed DNAs, tend to be located near the interface in the droplet. Conclusion and significance The specific localization of DNA molecules depending on their higher-order structure occurs in w/w microdroplet phase-separation solution under a binary aqueous polymer solution. Such an aqueous solution with polymers mimics the crowded conditions in living cells, where aqueous macromolecules exist at a level of 30–40 weight %. The specific positioning of DNA depending on its higher-order structure in w/w microdroplets is expected to provide novel insights into the mechanism and function of membraneless organelles and micro-segregated particles in living cells.

What makes a histone variant a variant: Changing H2A to become H2A.Z

Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the htz1Δ mutant. Furthermore, combining three unique H2A.Z regions (K79 and L81, M6, C-terminal tail) was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant.

The GTPase Arf1 Is a Determinant of Yeast Vps13 Localization to the Golgi Apparatus

VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13′s interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.

Over-representation of potential SP4 target genes within schizophrenia-risk genes

Reduction of Sp4 expression causes age-dependent hippocampal vacuolization and many other intermediate phenotypes of schizophrenia in Sp4 hypomorphic mice. Recent human genetic studies from both the Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) and the Genome-Wide Association Study (GWAS) validated SP4 as a schizophrenia-risk gene over the exome-wide or the genome-wide significance. Truncation of the human SP4 gene has an odds ratio of 9.37 (3.38–29.7) for schizophrenia. Despite successful identification of many schizophrenia-risk genes, it is unknown whether and how these risk genes may interact with each other in the development of schizophrenia. By taking advantage of the specific localization of the GC-boxes bound by SP4 transcription factors, I analyzed the relative abundance of these GC-boxes in the proximal promoter regions of schizophrenia-risk genes. I found that the GC-box containing genes are significantly over-represented within schizophrenia-risk genes, suggesting that SP4 is not only a high-risk gene for schizophrenia, but may also act as a hub of network in the regulation of many other schizophrenia-risk genes via these GC-boxes in the pathogenesis of schizophrenia.

AMPK Localization: A Key to Differential Energy Regulation

As the central node between nutrition signaling input and the metabolic pathway, AMP-activated protein kinase (AMPK) is tightly regulated to maintain energy homeostasis. Subcellular compartmentalization of AMPK is one of the critical regulations that enables AMPK to access proper targets and generate appropriate responses to specific perturbations and different levels of stress. One of the characterized localization mechanisms is RanGTPase-driven CRM1 that recognizes the nuclear export sequence (NES) on the α subunit to translocate AMPK into the cytoplasm. Nuclear localization putatively employs RanGTPase-driven importin that might recognize the nuclear localization signal (NLS) present on the AMPKα2 kinase domain. Nucleo-cytoplasmic shuttling of AMPK is influenced by multiple factors, such as starvation, exercise, heat shock, oxidant, cell density, and circadian rhythm. Tissue-specific localization, which distributes AMPK trimers with different combinations, has also been shown to be vital in maintaining tissue-specific metabolism. Tissue-specific and subcellular distribution of AMPK might be attributed to differences in the expression of the subunit, the stabilization by protein regulators, tissue activity, and the localization of AMPK activators. Considering the importance of AMPK localization in coordinating signaling and metabolism, further research is due to fully elucidate the largely unknown complex mechanism underlying this regulation.

Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion

Pancreatic cancer has a high mortality rate due to metastasis. Whereas KRAS is mutated in most pancreatic cancer patients, controlling KRAS or its downstream effectors has not been succeeded clinically. ARL4C is a small G protein whose expression is induced by the Wnt and EGF-RAS pathways. In the present study, we found that ARL4C is frequently overexpressed in pancreatic cancer patients and showed that its localization to invasive pseudopods is required for cancer cell invasion. IQGAP1 was identified as a novel interacting protein for ARL4C. ARL4C recruited IQGAP1 and its downstream effector, MMP14, to invasive pseudopods. Specific localization of ARL4C, IQGAP1, and MMP14 was the active site of invasion, which induced degradation of the extracellular matrix. Moreover, subcutaneously injected antisense oligonucleotide against ARL4C into tumor-bearing mice suppressed metastasis of pancreatic cancer. These results suggest that ARL4C-IQGAP1-MMP14 signaling is activated at invasive pseudopods of pancreatic cancer cells.

Gastric choristoma of hypopharynx in neonate

Summary Gastric choristoma is composed of the ectopic microscopically normal cells of gastric mucosa and can be located anywhere in the gastrointestinal tract. Choristomas are mostly clinically silent or present with minimal symptoms and are usually dia­gnosed in adulthood. This case report presents a neonate who experienced difficulty breathing, dyspnea, orotracheal intubation and an early surgery in the postnatal period due to a specific localization and size of its tumor. Key words gastric choristoma – benign tumor – heterotophy – neonate

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABAARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.

Immunohistochemistry and Expression Profile of Estrogen Receptor 2 Gene in Different Grade Size Ovarian Follicles of Leizhou Black Ducks

BackgroundEstrogen receptor 2 (ESR2) plays significant biological roles in the reproductive system and ovarian follicle development. This study, therefore, aimed to reveal the expression pattern and cell-specific localization of ESR2 in the ovarian follicles of Leizhou black ducks. MethodFour laying Leizhou black ducks at 43 weeks old were annihilated and different grade-sized follicles were collected for immunohistochemistry and expression profile study. The follicles were grouped into seven (7) as small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF), follicle 5 (F5), follicle 2 (F2), and follicle 1 (F1). ResultsThe qRT/PCR results displayed that ESR2 mRNA was expressed in all follicles with the highest (P < 0.05) level of expression found in F1 compared to other follicles. Immunohistochemistry analysis of the cell-specific localization of ESR2 protein revealed that ESR2 was distributed in both granulosa and theca cells region in all the follicles examined. There was a significantly higher localization of ESR2 protein in the granulosa cells than the theca cells of SWF, SYF, LYF, F2, and F1. Comparatively, ESR2 was highly expressed in the granulosa cells of LYF than in all the other follicles. ConclusionThese results provide theoretical knowledge for the in-depth study of the related biological functions of the ESR2 gene and its application at the cellular level.

Influence of vitreoretinal traction localization on horseshoe tear configuration and risk of rhegmatogenous retinal detachment

Purpose. To evaluate the relationship between the shape of horseshoe tear and the localization of vitreoretinal tractions (VRT) using methods of the peripheral vitreoretinal interface visualization and classify horseshoe tears by shape for surgical planning. Material and methods. We studied horseshoe tears parameters of 52 patients (52 eyes). The localization of VRT was determined by wide-field OCT, the horseshoe tear shape was determined as the ratio of length to width of the tear (l/b) by multispectral laser scanning. The ratio of the obtained data was evaluated by the Spearman correlation analysis. We used Ward's method of hierarchical clustering to classify the horseshoe tears by shape. The obtained data were used to perform YAG- laser excision of the vitreoretinal adhesion zone in patients with rhegmatogenous retinal detachment as part of the combined microinvasive laser-surgical technology. Results. The l/b ratio ranged from 1/4 to 3/1. A strong negative correlation has been revealed between the horseshoe tear shape and the fixation of vitreoretinal tractions (rs -0.95; p <0.05). Horseshoe tears were identified in 4 groups using Ward's method of clustering. Each group corresponded to a specific localization of VRT. The extension of VRT beyond the tear was associated with a high risk of rhegmatogenous retinal detachment. Conclusion. A significant correlation has been found between the studied factors confirm the possibility of using data on the horseshoe tear shape for an approximate evaluation of vitreoretinal adhesion localization. The obtained data allows to determine the exact VRT excision zone when performing combined laser-surgical technology without the need for preoperative wide-field OCT. Key words: rhegmatogenous retinal detachment, wide-field OCT, horseshoe tear, vitreoretinal traction, YAG – laser retinotomy.