Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

Adenanthera pavonina, a potential plant-based protein resource: Seed protein composition and immunohistochemical localization of trypsin inhibitors.

Adenanthera pavonina, an underutilized tropical tree, is being promoted as an alternative food source for meeting the nutritional needs of human and animals. In this study, we have shown that trypsin inhibitors as one of the predominant proteins in the seeds of A. pavonina. DE-52 column chromatography resulted in the identification of four peaks with trypsin inhibitor activity. SDS-PAGE and immunoblot analyses revealed DE-52 peaks A and B were enriched in 17 and 15 kDa proteins and these proteins cross-reacted against soybean trypsin inhibitor antibodies. Simulated gastric fluid digestion revealed that the 15-17 kDa proteins are resistant to pepsin digestion. Roasting the seeds lowered the trypsin inhibitor activity while boiling intact seeds elevated the enzyme activity. However, the trypsin inhibitor activity was completely abolished when the seeds were boiled without their seed coats. Immunohistochemical detection and confocal microscopy demonstrated that trypsin inhibitors were localized in the cell cytosol.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

Spatial transcriptomics analysis of uterine gene expression in enhancer of zeste homolog 2 conditional knockout mice†.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

Aptamers with Tunable Affinity Enable Single-Molecule Tracking and Localization of Membrane Receptors on Living Cancer Cells.

Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

The vasotocinergic system and its role in the regulation of stress in birds.

The regulation of stress in birds includes a complex interaction of neural systems affecting the hypothalamic-pituitary-adrenal (HPA) axis. In addition to the hypothalamic paraventricular nucleus, a structure called the nucleus of the hippocampal commissure likewise affects the output of pituitary stress hormones and appears to be unique to avian species. Within the anterior pituitary, the avian V1a and V1b receptors were found in corticotropes. Based on our studies with central administration of hormones in the chicken, corticotropic releasing hormone (CRH) is a more potent ACTH secretagogue than arginine vasotocin (AVT). In contrast, when applied peripherally, AVT is more efficacious. Co-administration of AVT and CRH peripherally, resulted in a synergistic stimulation of corticosterone release. Data suggest receptor oligomerization as one possible mechanism. In birds, vasotocin receptors associated with stress responses include the V1a and V1b receptors. Three-dimensional, homology-based structural models of the avian V1aR were built to test agonists and antagonists for each receptor that were screened by molecular docking to map their binding sites on each receptor. Additionally, binding affinity values for each available peptide antagonist to the V1aR and V1bR were determined. An anterior pituitary primary culture system was developed to determine how effective each antagonist blocked the function of each receptor in culture when stimulated by a combination of AVT/CRH administration. Use of an antagonist in subsequent in vivo studies identified the V1aR in regulating food intake in birds. The V1aR was likewise found in circumventricular organs of the brain, suggesting a possible function in stress.

Peripheral membrane associations of matrix metalloproteinases.

Water soluble matrix metalloproteinases (MMPs) have been regarded as diffusing freely in the extracellular matrix. Yet multiple MMPs are also observed at cell surfaces. Their membrane-proximal activities include sheddase activities, collagenolysis, bacterial killing, and intracellular trafficking reaching as far as the nucleus. The catalytic domains of MMP-7 and MMP-12 bind bilayers peripherally, each in two different orientations, by presenting positive charges and a few hydrophobic groups to the surface. Related peripheral membrane associations are predicted for other soluble MMPs. The peripheral membrane associations may support pericellular proteolysis and endocytosis. The isolated soluble domains of MT1-MMP can also associate with membranes. NMR assays suggest transient association of the hemopexin-like domains of MT1-MMP and MMP-12 with lipid bilayers. Peripheral association of soluble MMP domains with bilayers or heparin sulfate proteoglycans probably concentrates them near the membrane. This could increase the probability of forming complexes with membrane-associated proteins, such as those targeted for proteolysis. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death.

Atherosclerotic plaque destabilization is the major determinant of most acute coronary events. Smooth muscle cell (SMC) death contributes to plaque destabilization. Here, we describe a novel antiapoptotic mechanism in vascular SMCs that involves interaction of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with apurinic/apyrimidinic endonuclease 1 (Ape1), the major oxidized DNA repair enzyme. GAPDH down-regulation potentiated H2O2-induced DNA damage and SMC apoptosis. Conversely, GAPDH overexpression decreased DNA damage and protected SMCs against apoptosis. Ape1 down-regulation reversed the resistance of GAPDH-overexpressing cells to DNA damage and apoptosis, which indicated that Ape1 is indispensable for GAPDH-dependent protective effects. GAPDH bound Ape1 in the SMC nucleus, and blocking (or oxidation) of GAPDH active site cysteines suppressed GAPDH/Ape1 interaction and potentiated apoptosis. GAPDH up-regulated Ape1 via a transcription factor homeobox protein Hox-A5-dependent mechanism. GAPDH levels were reduced in atherosclerotic plaque SMCs, and this effect correlated with oxidative stress and SMC apoptosis. Thus, we demonstrated that nuclear GAPDH/Ape1 interaction preserved Ape1 activity, reduced DNA damage, and prevented SMC apoptosis. Suppression of SMC apoptosis by maintenance of nuclear GAPDH/Ape1 interactions may be a novel therapy to increase atherosclerotic plaque stability.-Hou, X., Snarski, P., Higashi, Y., Yoshida, T., Jurkevich, A., Delafontaine, P., Sukhanov, S. Nuclear complex of glyceraldehyde-3-phosphate dehydrogenase and DNA repair enzyme apurinic/apyrimidinic endonuclease I protect smooth muscle cells against oxidant-induced cell death.

Diencephalic and septal structures containing the avian vasotocin receptor (V1aR) involved in the regulation of food intake in chickens, Gallus gallus.

Recently, it was found that the avian central vasotocin receptor (V1aR) is associated with the regulation of food intake. To identify V1aR-containing brain structures regulating food intake, a selective V1aR antagonist SR-49059 that induced food intake was administrated intracerebroventricularly in male chickens followed by detection of brain structures using FOS immunoreactivity. Particularly, the hypothalamic core region of the paraventricular nucleus, lateral hypothalamic area, dorsomedial hypothalamic nucleus, a subnucleus of the central extended amygdalar complex [dorsolateral bed nucleus of the stria terminalis], medial septal nucleus and caudal brainstem [nucleus of the solitary tract] showed significantly increased FOS-ir cells. On the other hand, the supraoptic nucleus of the preoptic area and the nucleus of the hippocampal commissure of the septum showed suppressed FOS immunoreactivity in the V1aR antagonist treatment group. Further investigation revealed that neuronal activity of arginine vasotocin (AVT-ir) magnocellular neurons in the supraoptic nucleus, preoptic periventricular nucleus, paraventricular nucleus and ventral periventricular hypothalamic nucleus and most likely corticotropin releasing hormone (CRH-ir) neurons in the nucleus of the hippocampal commissure were reduced following the antagonist treatment. Dual immunofluorescence labeling results showed that perikarya of AVT-ir magnocellular neurons in the preoptic area and hypothalamus were colabeled with V1aR. Within the nucleus of the hippocampal commissure, CRH-ir neurons were shown in close contact with V1aR-ir glial cells. Results of the present study suggest that the V1aR plays a role in the regulation of food intake by modulating neurons that synthesize and release anorectic neuropeptides in the avian brain.

Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer.

Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we reported that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241-5253]. In general, various studies established inhibition of cell proliferation by SPRY in cancer. The mechanisms by which SPRY regulates cell proliferation in CRC are investigated. We demonstrate, for the first time, suppression of SPRY2 augmented EGF-dependent oncogenic signaling, however, surprisingly decreased cell proliferation in colon cancer cells. Our data suggest that cell cycle inhibitor p21(WAF1/CIP1) transcriptional activity being regulated by SPRY2. Indeed, suppression of SPRY2 significantly increased p21(WAF1/CIP1) mRNA and protein expression as well as p21(WAF1/CIP1) promoter activity. Conversely, overexpressing SPRY2 triggered a decrease in p21(WAF1/CIP1) promoter activity. Concurrent down-regulation of both SPRY1 and SPRY2 also increased p21(WAF1/CIP1) expression in colon cancer cells. Increased nuclear localization of p21(WAF1/CIP1) in SPRY2 downregulated colon cancer cells may explain the inhibition of cell proliferation in colon cancer cells. Underscoring the biological relevance of these findings in SPRY1 and SPRY2 mutant mouse, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression and nuclear localization of p21(WAF1/CIP1) and decreased cell proliferation. In CRC, the relationship of SPRY with p21 may provide unique strategies for cancer prevention and treatment. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen.

Distribution of the vasotocin type 4 receptor throughout the brain of the chicken, Gallus gallus.

The vasopressin 1a receptor (V1aR) has been shown to have a wide distribution throughout the mammalian brain and pituitary gland and mediates a number of physiological functions as well as social behavior following the binding of its agonist, vasopressin. The avian receptor homologous to the V1aR is the vasotocin 4 receptor (VT4R). Its mRNA distribution has been documented in brain regions of two species of songbird; however, its complete protein distribution in the brain has not been published to date for any avian species. The present work utilizes an antibody made to a sequence of the chicken VT4R to map its distribution from the olfactory bulbs to the caudal end of the brainstem in Gallus gallus. Unexpectedly, immunoreactivity (ir) for the VT4R was found not only in neurons but also in glia located in 10 circumventricular organs (CVOs), olfactory bulbs, hippocampus, and septum. Use of a second antibody made against vimentin provided evidence that some dual-labeled glial cells were tanycytes and radial glia. Additionally, the VT4R was identified in nuclei related to motor function, including the oculomotor complex and motor nucleus of the fourth, fifth, sixth, seventh, tenth, and twelfth cranial nerves. Possible functions for the VT4R are suggested that should have relevance not only to avian species but to other vertebrates because most classes, except for mammals, use vasotocin as the natural ligand for that receptor.

Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12.

Matrix metalloproteinases (MMPs) regulate tissue remodelling, inflammation and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here we demonstrate the binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labelled membrane mimics. Loops project from the ß-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.

Neuroendocrine regulation of stress in birds with an emphasis on vasotocin receptors (VTRs).

The neuroendocrine stress response of vertebrates, particularly mammals, comprises at least two types of neuropeptide containing neurons, corticotropin-releasing hormone (CRH) and vasopressin (VP) neurons, and four receptors [CRH receptor one (CRH-R1) and two (CRH-R2) and VP receptor 1a (V1aR) and 1b (V1bR)]. The avian neuropeptide CRH, a 41-amino acid peptide, has been shown to have the same amino acid sequence as humans while nonapeptide neurohormone arginine-vasotocin (AVT) is regarded as highly conserved having a single amino acid substitution compared to mammalian arginine vasopressin. Similar to mammals, birds have two receptor subtypes (CRH-R1 and CRH-R2) for CRH, however, four vasotocin receptors have been identified. Less is known about the functions of the four avian vasotocin receptors compared to homologous ones found in mammals and other vertebrate classes. Recently, chicken vasotocin receptor two (VT2R) and four (VT4R) have been characterized utilizing immunocytochemistry and an imposed stress test. The purpose of this review is to present evidence that the VT2R and VT4R are involved in the avian stress response and that the cephalic lobe of the anterior pituitary appears specialized for this function as it contains the major population of corticotropes and necessary neuroendocrine receptors to respond to stressors impacting avian species.

Subpallial and hypothalamic areas activated following sexual and agonistic encounters in male chickens.

Male sexual and agonistic behaviors are controlled by the common social behavior network, involving subpallial and hypothalamic brain areas. In order to understand how this common network generates different behavioral outcomes, induction of FOS protein was used to examine the patterns of neuronal activation in adult male chickens following interaction with a female or a male. Males were subjected to one of the following treatments: handling control, non-contact interaction with a female, contact interaction with a live female, a taxidermy female model or another male. The number of FOS-immunoreactive (FOS-ir) cells, and the area and immunostaining density of individual cells were quantified in the medial preoptic nucleus (POM), medial extended amygdala (nucleus taeniae of the amygdala, TnA, and dorsolateral and ventromedial subdivisions of the medial portion of the bed nucleus of stria terminalis, BSTM1 and BSTM2, respectively), lateral septum (SL), hypothalamic paraventricular nucleus (PVN), bed nucleus of the pallial commissure (NCPa) and ventrolateral thalamic nucleus (VLT). An increase in FOS-ir cells following appetitive sexual behavior was found in BSTM2 and NCPa. Copulation augmented FOS-ir in POM, SL, VLT, and PVN. Intermale interactions increased FOS-ir in all examined brain regions except the TnA and BSTM. Within the SL, copulatory and agonistic behavior activated spatially segregated cell groups. In the PVN, different social behaviors induced significant changes in the distribution of FOS-ir cell sizes suggesting activation of heterogeneous subpopulations of cells. Collectively, behavioral outcomes of male-female and male-male interactions are associated with a combination of common and site-specific patterns of neural activation.

Immunohistochemical characterization of chicken pituitary cells containing the vasotocin VT2 receptor.

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.

Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3',5'-cyclic adenosine monophosphate production.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.