Biology Open

Cullin-RING ubiquitin ligases (CRLs) mediate the ubiquitination of numerous protein substrates and target them for proteasomal degradation. The function of CRLs is under tight regulation by Cullin-binding proteins. It has been reported that the Spliceosome-associated protein 130 (SAP130/SF3b-3) binds to several Cullin proteins, yet it remains unknown whether SAP130 plays any role in regulating the function of CRLs. Here, we report that SAP130 overexpression reduces the binding of adaptor protein Skp1 and substrate receptor Skp2 to Cul1, whereas it has no effect on CAND1 binding to Cul1. Overexpression of SAP130 decreases the degradation rate of p27, a protein substrate of the SCF(Skp2) ligase. Interestingly, silencing of SAP130 also inhibits the degradation of p27, suggesting a dual role for SAP130 in the regulation of SCF activity. We hypothesized that the regulatory role of SAP130 could extend to other CRLs; however, overexpression of SAP130 is unable to affect the protein stability of the Cul2 and Cul3 substrates, HIF-1 and NRF-2. SAP130 binds to Cul1, Cul2 and Cul4 with similar affinity, and it binds to Cul3 more strongly. SAP130 localizes in both the nucleus and the cytoplasm. Hence, the inability of SAP130 to regulate Cul2 and Cul3 CRLs cannot be explained by low binding affinity of SAP130 to these cullins or by subcellular sequestration of SAP130. We propose a novel role for SAP130 in the regulation of SCF, whereby SAP130 physically competes with the adaptor protein/F-box protein for Cul1 binding and interferes with the assembly of a functional SCF ligase.

Histone deacetylases (HDACs) and RNA polymerase III (POLR3) play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis in zebrafish. We aim to test the hypothesis that Hdacs and Polr3 cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A) and an inhibitor of Polr3 (ML-60218) synergistically prohibited the expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and ML-60218 produced augmented suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive upregulation of the pro-apoptotic regulator BAX and the cyclin-dependent kinase inhibitor p21(CDKN1A). tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators of exocrine pancreas can be translated into an approach with potential impact on therapeutic response in pancreatic cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity.

This study examined the effects of testosterone (T) on the contractile properties of two sexually dimorphic forelimb muscles and one non-dimorphic muscle in male bullfrogs (Rana catesbeiana, Shaw 1802). The dimorphic muscles in castrated males with testosterone replacement (T+) achieved higher forces and lower fatigability than did castrated males without replaced testosterone (T0 males), but the magnitude of the differences was low and many of the pair-wise comparisons of each muscle property were not statistically significant. However, when taken as a whole, the means of seven contractile properties varied in the directions expected of masculine values in T+ animals in the sexually dimorphic muscles. Moreover, these data, compared with previous data on male and female bullfrogs, show that values for T+ males are similar to normal males and are significantly different from females. The T0 males tended to be intermediate in character between T+ males and females, generally retaining masculine values. This suggests that the exposure of young males to T in their first breeding season produces a masculinizing effect on the sexually dimorphic muscles that is not reversed between breeding seasons when T levels are low. The relatively minor differences in contractile properties between T+ and T0 males may indicate that as circulating T levels rise during breeding season in normal males, contractile properties can be enhanced rapidly to maximal functional levels for breeding success.

Studies of skin wound healing in crocodilians are necessary given the frequent occurrence of cannibalism in intensive farming systems. Air temperature affects tissue recovery because crocodilians are ectothermic. Therefore, the kinetics of skin wound healing in Caiman yacare were examined at temperatures of 33°C and 23°C. Sixteen caiman were selected and divided into two groups of eight maintained at 23°C or 33°C. The studied individuals' scars were photographed after 1, 2, 3, 7, 15 and 30 days of the experimental conditions, and samples were collected for histological processing after 3, 7, 15 and 30 days. Macroscopically, the blood clot (heterophilic granuloma) noticeably remained in place covering the wound longer for the caiman kept at 23°C. Microscopically, the temperature of 23°C slowed epidermal migration and skin repair. Comparatively, new blood vessels, labeled using von Willebrand factor (vWF) antibody staining, were more frequently found in the scars of the 33°C group. The collagen fibers in the dermis were denser in the 33°C treatment. Considering the delayed healing at 23°C, producers are recommended to keep wounded animals at 33°C, especially when tanks are cold, to enable rapid wound closure and better repair of collagen fibers because such lesions tend to compromise the use of their skin as leather.

Seahorses are the vertebrate group with the embryonic development occurring within a special pouch in males. To understand the reproductive efficiency of the lined seahorse, Hippocampus erectus Perry, 1810 under controlled breeding experiments, we investigated the dynamics of reproductive rate, offspring survivorship and growth over births by the same male seahorses. The mean brood size of the 1-year old pairs in the 1(st) birth was 85.4±56.9 per brood, which was significantly smaller than that in the 6(th) birth (465.9±136.4 per brood) (P<0.001). The offspring survivorship and growth rate increased with the births. The fecundity was positively correlated with the length of brood pouches of males and trunk of females. The fecundity of 1-year old male and 2-year old female pairs was significantly higher than that from 1-year old couples (P<0.001). The brood size (552.7±150.4) of the males who mated with females that were isolated for the gamete-preparation, was larger than those (467.8±141.2) from the long-term pairs (P<0.05). Moreover, the offspring from the isolated females had higher survival and growth rates. Our results showed that the potential reproductive rate of seahorses H. erectus increased with the brood pouch development.

Because the actin network in active lamellipodia is continuously assembling at the edge, moving inward and disassembling, there is a question as to how actin-binding proteins and other components are transported to the leading edge and how nascent adhesions are stabilized. Active transport could play a significant role in these functions but the components involved are unknown. We show here that Myosin 1E (a long tailed Myosin 1 isoform) rapidly moves to the tips of active lamellipodia and to actin-rich early adhesions, unlike Myosin 1G, 1B or 1C (short tailed isoforms). Myosin 1E co-localizes with CARMIL, FHOD1, Arp3 and β3-integrin in those early adhesions. But these structures precede stable paxillin-rich adhesions. Myosin 1E movement depends upon actin-binding domains and the presence of an SH3 oligomerization domain. Overexpression of a Myosin 1E deletion mutant without the extreme C-terminal interacting (SH3) domain (Myosin 1EΔSH3) increases edge fluctuations and decreases stable adhesion lifetimes. In contrast, overexpression of Myosin 1E full tail domain (TH1+TH2+TH3/SH3) decreases edge fluctuation. In Myosin 1E knockdown cells, and more prominently in cells treated with Myosin 1 inhibitor, cell-matrix adhesions are also short-lived and fail to mature. We suggest that, by moving to actin polymerization sites and early adhesion sites in active lamellipodia, Myosin 1E might play important roles in transporting not only important polymerizing proteins but also proteins involved in adhesion stabilization.

Non-ionizing radiation at 2.45 GHz may modify the expression of genes that codify heat shock proteins (HSP) in the thyroid gland. Using the enzyme-linked immunosorbent assay (ELISA) technique, we studied levels of HSP-90 and HSP-70. We also used hematoxilin eosin to look for evidence of lesions in the gland and applied the DAPI technique of fluorescence to search for evidence of chromatin condensation and nuclear fragmentation in the thyroid cells of adult female Sprague-Dawley rats. Fifty-four rats were individually exposed for 30 min to 2.45 GHz radiation in a Gigahertz transverse electromagnetic (GTEM) cell at different levels of non-thermal specific absorption rate (SAR), which was calculated using the finite difference time domain (FDTD) technique. Ninety minutes after radiation, HSP-90 and HSP-70 had decreased significantly (P<0.01) after applying a SAR of 0.046±1.10 W/Kg or 0.104±5.10(-3) W/Kg. Twenty-four hours after radiation, HSP-90 had partially recovered and HSP-70 had recovered completely. There were few indications of lesions in the glandular structure and signs of apoptosis were negative in all radiated animals. The results suggest that acute sub-thermal radiation at 2.45 GHz may alter levels of cellular stress in rat thyroid gland without initially altering their anti-apoptotic capacity.

Although approximately 50% of Down Syndrome (DS) patients have heart abnormalities, they exhibit an overprotection against cardiac abnormalities related with the connective tissue, for example a lower risk of coronary artery disease. A recent study reported a case of a person affected by DS who carried mutations in FBN1, the gene causative for a connective tissue disorder called Marfan Syndrome (MFS). The fact that the person did not have any cardiac alterations suggested compensation effects due to DS. This observation is supported by a previous DS meta-analysis at the molecular level where we have found an overall upregulation of FBN1 (which is usually downregulated in MFS). Additionally, that result was cross-validated with independent expression data from DS heart tissue. The aim of this work is to elucidate the role of FBN1 in DS and to establish a molecular link to MFS and MFS-related syndromes using a computational approach. To reach that, we conducted different analytical approaches over two DS studies (our previous meta-analysis and independent expression data from DS heart tissue) and revealed expression alterations in the FBN1 interaction network, in FBN1 co-expressed genes and FBN1-related pathways. After merging the significant results from different datasets with a Bayesian approach, we prioritized 85 genes that were able to distinguish control from DS cases. We further found evidence for several of these genes (47%), such as FBN1, DCN, and COL1A2, being dysregulated in MFS and MFS-related diseases. Consequently, we further encourage the scientific community to take into account FBN1 and its related network for the study of DS cardiovascular characteristics.

Exploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.

The abundance of Myc protein must be exquisitely controlled to avoid growth abnormalities caused by too much or too little Myc. An intriguing mode of regulation exists in which Myc protein itself leads to reduction in its abundance. We show here that dMyc binds to the miR-308 locus and increases its expression. Using our gain-of-function approach, we show that an increase in miR-308 causes a destabilization of dMyc mRNA and reduced dMyc protein levels. In vivo knockdown of miR-308 confirmed the regulation of dMyc levels in embryos. This regulatory loop is crucial for maintaining appropriate dMyc levels and normal development. Perturbation of the loop, either by elevated miR-308 or elevated dMyc, caused lethality. Combining elevated levels of both, therefore restoring balance between miR-308 and dMyc levels, resulted in lower apoptotic activity and suppression of lethality. These results reveal a sensitive feedback mechanism that is crucial to prevent the pathologies caused by abnormal levels of dMyc.

Mammalian sperm are carriers of not only the paternal genome, but also the paternal epigenome in the forms of DNA methylation, retained histones and noncoding RNAs. Although paternal DNA methylation and histone retention sites have been correlated with protein-coding genes that are critical for preimplantation embryonic development, physiological evidence of an essential role of these epigenetic marks in fertilization and early development remains lacking. Two miRNA clusters consisting of five miRNAs (miR-34b/c and miR-449a/b/c) are present in sperm, but absent in oocytes, and miR-34c has been reported to be essential for the first cleavage division in vitro. Here, we show that both miR-34b/c- and miR-449-null male mice displayed normal fertility, and that intracytoplasmic injection of either miR-34b/c- or miR-449-null sperm led to normal fertilization, normal preimplantation development and normal birth rate. However, miR-34b/c and miR-449 double knockout (miR-dKO) males were infertile due to severe spermatogenic disruptions and oligo-astheno-teratozoospermia. Injection of miR-dKO sperm into wild-type oocytes led to a block at the two-pronucleus to zygote transition, whereas normal preimplantation development and healthy pups were obtained through injection of miR-dKO round spermatids. Our data demonstrate that miR-34b/c and miR-449a/b/c are essential for normal spermatogenesis and male fertility, but their presence in sperm is dispensable for fertilization and preimplantation development. © 2015. Published by The Company of Biologists Ltd.

Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM) components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed γ-Tubulin. During interphase, most proteins were arranged at specific distances from the walls of centrioles, resulting in ring staining, often with discernible density masses. Through use of site-specific antibodies, we found the C-terminus of Cep152 to be closer to centrioles than the N-terminus, illustrating the power of 3D-SIM to study protein disposition. Appendage proteins showed rings with multiple density masses, and the number of these masses was strongly reduced during mitosis. At the proximal end of centrioles, Sas-6 formed a dot at the site of daughter centriole assembly, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but Cep135 was associated mostly with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation. Our study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.

Caenorhabditis elegans seam cells divide in the stem-like mode throughout larval development, with the ability to both self-renew and produce daughters that differentiate. Seam cells typically divide asymmetrically, giving rise to an anterior daughter that fuses with the hypodermis and a posterior daughter that proliferates further. Previously we have identified rnt-1 (a homologue of the mammalian cancer-associated stem cell regulator Runx) as being an important regulator of seam development, acting to promote proliferation; rnt-1 mutants have fewer seam cells whereas overexpressing rnt-1 causes seam cell hyperplasia. We isolated the interacting CEH-20/Pbx and UNC-62/Meis TALE-class transcription factors during a genome-wide RNAi screen for novel regulators of seam cell number. Animals lacking wild type CEH-20 or UNC-62 display seam cell hyperplasia, largely restricted to the anterior of the worm, whereas double mutants have many additional seam cells along the length of the animal. The cellular basis of the hyperplasia involves the symmetrisation of normally asymmetric seam cell divisions towards the proliferative stem-like fate. The hyperplasia is completely suppressed in rnt-1 mutants, and rnt-1 is upregulated in ceh-20 and unc-62 mutants, suggesting that CEH-20 and UNC-62 function upstream of rnt-1 to limit proliferative potential to the appropriate daughter cell. In further support of this we find that CEH-20 is asymmetrically localised in seam daughters following an asymmetric division, being predominantly restricted to anterior nuclei whose fate is to differentiate. Thus, ceh-20 and unc-62 encode crucial regulators of seam cell division asymmetry, acting via rnt-1 to regulate the balance between proliferation and differentiation.

The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib) in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-X(L), or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas.

We have analyzed health and physiological aging parameters in male and female Atlantic cod, Gadus morhua, captured in Kattegat, Skagerrak and in Öresund. Gender differences were clearly evident in a number of variables. Males had longer liver telomeres and higher catalase activities than females, while females had higher superoxide dismutase activity, liver somatic index and condition factor. Effects of age were found for males where levels of the antioxidant glutathione and telomere length declined with age, indicating physiological aging. Liver somatic index increased and percentage oxidized glutathione decreased with age. Between-site comparisons of males show that percentage oxidized glutathione and catalase were lowest in Kattegat, whereas protein carbonyls and condition factor were higher in Skagerrak. Females, on the other hand, showed no differences between sites or indications of somatic aging or age-related effects in egg quality, indicating that older and larger female cod are healthy and show no changes in eggs with age. In contrast, males showed indications of physiological aging and lower condition than females. The results emphasize the importance of conserving old mature fish, in particular high egg-productive females, when managing fisheries.

Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo. © 2015. Published by The Company of Biologists Ltd.

Our previous studies in zebrafish development have led to identification of the novel roles of the transient receptor potential melastatin-subfamily member 7 (TRPM7) ion channels in human pancreatic cancer. However, the biological significance of TRPM7 channels in pancreatic neoplasms was mostly unexplored. In this study, we determined the expression levels of TRPM7 in pancreatic tissue microarrays and correlated these measurements in pancreatic adenocarcinoma with the clinicopathological features. We also investigated the role of TRPM7 channels in pancreatic cancer cell invasion using the Matrigel(TM)-coated transwell assay. In normal pancreas, TRPM7 is expressed at a discernable level in the ductal cells and centroacinar cells and at a relatively high level in the islet endocrine cells. In chronic pancreatitis, pre-malignant tissues, and malignant neoplasms, there is variable expression of TRPM7. In the majority of pancreatic adenocarcinoma specimens examined, TRPM7 is expressed at either moderate-level or high-level. Anti-TRPM7 immunoreactivity in pancreatic adenocarcinoma significantly correlates with the size and stages of tumors. In human pancreatic adenocarcinoma cells in which TRPM7 is highly expressed, short hairpin RNA-mediated suppression of TRPM7 impairs cell invasion. The results demonstrate that TRPM7 channels are over-expressed in a proportion of the pre-malignant lesions and malignant tumors of the pancreas, and they are necessary for invasion by pancreatic cancer cells. We propose that TRPM7 channels play important roles in development and progression of pancreatic neoplasm, and they may be explored as clinical biomarkers and targets for its prevention and treatment. © 2015. Published by The Company of Biologists Ltd.

The transcription factor Ets1 is expressed at low levels in epidermal keratinocytes under physiological conditions, but is over-expressed in cutaneous squamous cell carcinoma (SCC). We previously showed that over-expression of Ets1 in differentiated keratinocytes of the skin leads to significant pro-tumorigenic alterations. Here, we further extend these studies by testing the effects of over-expressing Ets1 in the proliferative basal keratinocytes of the skin, which includes the putative epidermal stem cells. We show that induction of the Ets1 transgene in the basal layer of skin during embryogenesis results in epidermal hyperplasia and impaired differentiation accompanied by attenuated expression of spinous and granular layer markers. A similar hyper-proliferative skin phenotype was observed when the transgene was induced in the basal layer of the skin of adult mice leading to hair loss and open sores. The Ets1-mediated phenotype is accompanied by a variety of changes in gene expression including alterations in Notch signaling, a crucial mediator of normal skin differentiation. Finally, we show that Ets1 disrupts Notch signaling in part via its ability to upregulate ΔNp63, an established transcriptional repressor of several of the Notch receptors. Given the established tumor suppressive role for Notch signaling in skin tumorigenesis, the demonstrated ability of Ets1 to interfere with this signaling pathway may be important in mediating its pro-tumorigenic activities.

How morphogen gradients are shaped is a major question in developmental biology, but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to Hh N-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.

Here, we report on the results of an experimental study that assessed the visitation frequency of wild bees to conspecific flowers with different sized floral guides. UV absorbent floral guides are ubiquitous in Angiosperms, yet surprisingly little is known about conspecific variation in these guides and very few studies have evaluated pollinator response to UV guide manipulation. This is true despite our rich understanding about learning and color preferences in bees. Historical dogma indicates that flower color serves as an important long-range visual signal allowing pollinators to detect the flowers, while floral guides function as close-range signals that direct pollinators to a reward. We initiated the work presented here by first assessing the population level variation in UV absorbent floral guides for conspecific flowers. We assessed two species, Rudbeckia hirta and R. fulgida. We then used several petal cut-and-paste experiments to test whether UV floral guides can also function to attract visitors. We manipulated floral guide size and evaluated visitation frequency. In all experiments, pollinator visitation rates were clearly associated with floral guide size. Diminished floral guides recruited relatively few insect visitors. Exaggerated floral guides recruited more visitors than smaller or average sized guides. Thus, UV floral guides play an important role in pollinator recruitment and in determining the relative attractiveness of conspecific flower heads. Consideration of floral guides is therefore important when evaluating the overall conspicuousness of flower heads relative to background coloration. This work raises the issue of whether floral guides serve as honest indicators of reward, since guide size varies in nature for conspecific flowers at the same developmental stage and since preferences for larger guides were found. To our knowledge, these are the first cut-and-paste experiments conducted to examine whether UV absorbent floral guides affect visitation rates and pollinator preference.

Embryonic Stem (ES) cells are able to give rise to the three germ layers of the embryo but are prevented from contributing to the trophoblast. The molecular nature of this barrier between embryonic and trophectodermal cell fates is not clear, but is known to involve DNA methylation. Here we demonstrate that the Nucleosome Remodeling and Deacetylation (NuRD) co-repressor complex maintains the developmental barrier between embryonic and trophectodermal cell fates by maintaining transcriptional silencing of trophectoderm determinant genes in ES cells. We further show that NuRD activity facilitates DNA methylation of several of its target promoters, where it acts non-redundantly with DNA methylation to enforce transcriptional silencing. NuRD-deficient ES cells fail to completely silence expression of the trophectoderm determinant genes Elf5 and Eomes, but this alone is not sufficient to induce transdifferentiation towards the trophectoderm fate. Rather this leaves ES cells capable of activating expression of trophectoderm-specific genes in response to appropriate extracellular signals, enabling them to commit to a trophectodermal cell fate. Our findings clarify the molecular nature of the developmental barrier between the embryonic and trophoblast cell fates, and establish a role for NuRD activity in specifying sites for de novo DNA methylation.

Rainbow smelt (Osmerus mordax) display an impressive ability to acclimate to very cold water temperatures. These fish express both anti-freeze proteins and glycerol in their plasma, liver, muscle and other tissues to avoid freezing at sub-zero temperatures. Maintenance of glycerol levels requires active feeding in very cold water. To understand how these fish can maintain activity at cold temperatures, we explored thermal acclimation by the myotomal muscle of smelt exposed to cold water. We hypothesized that cold-acclimated fish would show enhanced swimming ability due to shifts in muscle contractile properties. We also predicted that shifts in swimming performance would be associated with changes in the expression patterns of muscle proteins such as parvalbumin (PV) and myosin heavy chain (MyHC). Swimming studies show significantly faster swimming by smelt acclimated to 5°C compared to fish acclimated to 20°C when tested at a common test temperature of 10°C. The cold-acclimated fish also had faster muscle contractile properties, such as a maximum shortening velocity (Vmax) almost double that of warm-acclimated fish at the same test temperature. Cold-acclimation is associated with a modest increase in PV levels in the swimming muscle. Fluorescence microscopy using anti-MyHC antibodies suggests that MyHC expression in the myotomal muscle may shift in response to exposure to cold water. The complex set of physiological responses that comprise cold-acclimation in smelt includes modifications in muscle function to permit active locomotion in cold water.

Goldfish have been used for cold acclimation studies, which have focused on changes in glycolytic and oxidative enzymes or alterations in lipid composition in skeletal muscle. Here we examine the effects of cold acclimation on the functional properties of isolated mitochondria and permeabilized fibers from goldfish white skeletal muscle, focusing on understanding the types of changes that occur in the mitochondrial respiratory states. We observed that cold acclimation promoted a significant increase in the mitochondrial oxygen consumption rates. Western blot analysis showed that UCP3 was raised by ∼1.5-fold in cold-acclimated muscle mitochondria. Similarly, we also evidenced a rise in the adenine nucleotide translocase content in cold-acclimated muscle mitochondria compared to warm-acclimated mitochondria (0.96±0.05 vs 0.68±0.02 nmol carboxyatractyloside mg(-1) protein). This was followed by a 2-fold increment in the citrate synthase activity, which suggests a higher mitochondrial content in cold-acclimated goldfish. Even with higher levels of UCP3 and ANT, the effects of activator (palmitate) and inhibitors (carboxyatractyloside and GDP) on mitochondrial parameters were similar in both warm- and cold-acclimated goldfish. Thus, we propose that cold acclimation in goldfish promotes an increase in functional oxidative capacity, with higher mitochondrial content without changes in the mitochondrial uncoupling pathways.

During spermiogenesis, haploid spermatids undergo extensive chromatin remodeling events in which histones are successively replaced by more basic protamines to generate highly compacted chromatin. Here we show for the first time that H3K79 methylation is a conserved feature preceding the histone-to-protamine transition in Drosophila melanogaster and rat. During Drosophila spermatogenesis, the Dot1-like methyltransferase Grappa (Gpp) is primarily expressed in canoe stage nuclei. The corresponding H3K79 methylation is a histone modification that precedes the histone-to-protamine transition and correlates with histone H4 hyperacetylation. When acetylation was inhibited in cultured Drosophila testes, nuclei were smaller and chromatin was compact, Gpp was little synthesized, H3K79 methylation was strongly reduced, and protamines were not synthesized. The Gpp isoform Gpp-D has a unique C-terminus, and Gpp is essential for full fertility. In rat, H3K79 methylation also correlates with H4 hyperacetylation but not with active RNA polymerase II, which might point towards a conserved function in chromatin remodeling during the histone-to-protamine transition in both Drosophila and rat.

Cells recognize and respond to changes in intra- and extracellular mechanical conditions to maintain their mechanical homeostasis. Linear contractile bundles of actin filaments and myosin II known as stress fibres (SFs) mediate mechanical signals. Mechanical cues such as excessive stress driven by myosin II and/or external force may damage SFs and induce the local transient accumulation of SF-repair complexes (zyxin and VASP) at the damaged sites. Using an atomic force microscope mounted on a fluorescence microscope, we applied mechanical damage to cells expressing fluorescently tagged cytoskeletal proteins and recorded the subsequent mobilization of SF-repair complexes. We found that a LIM protein, paxillin, transiently accumulated at the damaged sites earlier than zyxin, while paxillin knockdown did not affect the kinetics of zyxin translocation. The C-terminal half of paxillin, comprising four-tandem LIM domains, can still translocate to damaged sites on SFs, suggesting that the LIM domain is essential for the mechanosensory function of paxillin. Our findings demonstrate a crucial role of the LIM domain in mechanosensing LIM proteins.