Bleached Area Research Articles

Disrupted hair follicle activity in cattle grazing endophyte-infected tall fescue in the summer insulates core body temperatures

ABSTRACT Two experiments were conducted to characterize and evaluate rough hair-coats of cattle grazing endophyte (Neotyphodium coenophialum)-infected tall fescue (Lolium arundinaceum) during the summer and their effect on body temperature. In Exp. 1, hair was clipped in bleached areas over the rumps of 5 steers grazing endophyte-infected fescue in the summer to determine percentages in 5 length intervals for hair emerged during decreasing day lengths (fall and winter) and those emerged during increasing day lengths (spring and summer). In Exp. 2, rectal temperature data from 7 grazing experiments were compiled to compare linear relationships between rectal temperature and both ambient temperature and temperature-humidity indices, and between cattle with rough, transitional (i.e., some hair shedding), and sleek hair-coats. Frequency distributions of hair-length intervals in Exp. 1 differed (P 20 mm; however, 80% of the hairs were emerged during long day lengths rather than short day lengths. For Exp. 2, linear increases in rectal temperatures as mean ambient temperature and temperature-humidity indices increased were greater for cattle with rough and transitional hair coats than for cattle with sleek hair coats. From these experiments, it was concluded that rough hair coats on cattle grazing endophyte-infected tall fescue are composed predominately of hair emerged during long-day lengths in the late spring and summer. Growing to excessive hair-lengths, these rough hair coats insulate elevated core body temperature to intensify hyperthermia triggered by ergot alkaloid-induced vasoconstriction.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression¹. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells. ² (,)³

Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

First Report of Cane Blight on Blackberry Caused by Diaporthe eres in Croatia.

A cane disease of blackberry (Rubus sp.) cv. Thornfree was observed in May and June 2010 in two growing regions in the eastern part of Slavonia in Croatia. Symptoms consisted of bleached areas between and around cane nodes with some canes showing wilt symptoms. Infected areas were covered with numerous, black pycnidia immersed in the epidermal tissue. Disease occurrence in orchards growing cv. Thornfree ranged between 1 and 15%. Thirty disease samples were collected, disinfected (1 min in 70% ethanol and 2 min in 1% NaOCl), and placed in a moist chamber for 4 days. Fungal sporulating structures were then picked off and placed on potato dextrose agar (PDA). Fungal isolates obtained were identified as a Phomopsis sp., the conidial state of Diaporthe (3), on the basis of cultural and morphological characteristics. Alpha conidia were unicellular, hyaline, fusiform, sometimes tapering toward one or both ends, biguttulate (sometimes with several guttules), and 5.2 to 9.7 × 1.4 to 2.7 μm (average 6.5 × 2.1 μm). Beta conidia were hyaline, aseptate, filiform, hamate, and 16.6 to 28.2 × 0.5 to 1.5 μm (average 24.0 × 1.1 μm). The teleomorph was not observed. Biomolecular analysis was performed to identify the fungal species by sequencing the internal transcribed spacer (ITS) region spanning ITS 1, 5.8S rDNA, and ITS 2 of two isolates (Phk1 and Phk2). The amplified product was sequenced (GenLab-Enea, Rome, Italy) and a BLAST search of the NCBI nucleotide database was performed. Sequences from Phk1 and Phk2 (GenBank Accession Nos. HQ533144 and HQ533143, respectively) were identical to authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514, BPI 748435, and CBS 109767) ITS sequences in GenBank. Fungal isolates for pathogenicity tests were grown on PDA at 25°C for 7 days (12 h light/dark regimen). Inoculations were made on 30 to 40 cm long green shoots of potted plants of the blackberry cv. Thornfree. One-centimeter long wounds were made with a sterile scalpel and mycelia of D. eres were placed in the wounds. Inoculation sites were covered with a piece of wet cotton wool and aluminum foil to retain moisture. Three replications of 10 plants each were inoculated and these plus 10 control plants (inoculated with plugs of PDA only) were maintained in a growth chamber at 25°C. After 25 days, lesions developed on all 30 inoculated plants that averaged 15 mm long and control plants remained symptomless. D. eres was reisolated from inoculated plants, thus completing Koch's postulates. Phomopsis spp. have previously been reported on blackberry canes in Serbia (1) and Yugoslavia (2,4), however, to our knowledge, this is the first report of the occurrence of D. eres (anamorph P. oblonga) on blackberry in Croatia.

Dye tracer and morphophysical properties to observe water flow in a Gleyic Luvisol

The understanding of the preferential water flow and solute transport is important with regard to losses of nutrients and pesticides that affect the quality of the groundwater or surface water resources. Experiments using the brilliant blue dye tracer, a tension infiltrometer (TI) and a double square infiltrometer (DI) were carried out in the experimental field site located around 15 km southeast of the city of Rostock (North-Eastern Germany) on arable land in a Pleistocene lowland landscape where corn (Zea mays L.) and barley (Hordeum spp.) had been cultivated. One day after dye the infiltration, a pit was dug and vertical profiles were prepared in the TI and DI sites to assess the dye pathways in the subsoil of a Gleyic Luvisol. We wanted to examine if the mottled red and white (bleached) colour-pattern of the Gleyic Luvisol subsoil resulting from temporally stagnant water could be related to flow paths as visualized by dye tracing and if the soil colour could be related to other physical soil properties. Biogenic soil structures were the main transport routes conducting water and solutes into great depth in short time. These pathways had lower bulk density and less cone resistance than the adjacent red or white (bleached) areas of the Gleyic Luvisol subsoil. The red areas were involved in transport because their water contents increased after as compared to before infiltration. However, the measured physical soil properties did not differ between white and red areas. We assume that red areas participate in transport at least by imbibing water from the adjacent biogenic flow paths.

NOS inhibition synchronizes calcium oscillations in human adipose tissue‐derived mesenchymal stem cells by increasing gap‐junctional coupling

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.

Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues.

There is a rapidly growing interest in the advanced analysis of histological data and the development of appropriate detection technologies in particular for mapping of nanoparticle distributions in tissue in nanomedicine applications. We evaluated photothermal (PT) scanning cytometry for color-coded imaging, spectral identification, and quantitative detection of individual nanoparticles and abnormal cells in histological samples with and without staining. Using this tool, individual carbon nanotubes, gold nanorods, and melanoma cells with intrinsic melanin markers were identified in unstained (e.g. sentinel lymph nodes) and conventionally-stained tissues. In addition, we introduced a spectral burning technique for histology through selective laser bleaching areas with nondesired absorption background and nanobubble-based PT signal amplification. The obtained data demonstrated the promise of PT cytometry in the analysis of low-absorption samples and mapping of various individual nanoparticles' distribution that would be impossible with existing assays. Comparison of PT cytometry and photoacoustic (PA) cytometry previously developed by us, revealed that these methods supplement each other with a sensitivity advantage (up to 10-fold) of contactless PT technique in assessment of thin (≤100 μm) histological samples, while PA imaging provides characterization of thicker samples which, however, requires an acoustic contact with transducers. A potential of high-speed integrated PT-PA cytometry for express histology and immunohistochemistry of both intact and stained heterogeneous tissues with high sensitivity at the zepromolar concentration level is further highlighted.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.

Bleaching of red lake paints in encaustic mummy portraits

The present paper reports on the study of the development of whitish opacity in pink paints in encaustic mummy portraits. Non-invasive measurements carried out on two encaustic portraits belonging to the Ny Carlsberg Glyptotek, Copenhagen, by reflectance FTIR and UV–vis fluorescence have shown that the areas prone to the bleaching phenomenon had been painted with melted beeswax and an anthraquinone vegetal lake mixed with calcium sulphate hemihydrate and dihydrate. The hypothesis that the bleaching disease was neither related to a degradation of the dyes nor to an alteration of the wax but rather to a dehydration–hydration reaction of the CaSO4–H2O system, has been corroborated by the analyses of two microsamples from the bleached areas and ascertained by accelerated ageing experiments on encaustic models.

Effect of inorganic and organic nutrient addition on coral–algae assemblages from the Northern Red Sea

Previous studies in fringing reefs of the Northern Red Sea demonstrated that the in-situ competition of corals and algae in natural assemblages is highly variable between seasons displaying fast overgrowth of corals by benthic reef algae in fall that follows close to equilibrium between both groups of organisms in summer. This may be caused by up to 5-fold higher inorganic nutrient and 6-fold higher organic nutrient concentrations in fall and winter, thereby potentially promoting algae and cyanobacteria growth with concomitant phase shift. A long term mesocosm experiment (duration: 90 days) was conducted in order to study the effect of dissolved inorganic (ammonium, phosphate, nitrate, and mix of all three) and organic (glucose) nutrient addition onto the competitive process in the dominant coral–algae assemblages of the Northern Red Sea involving branching corals of the genus Acropora and a typical consortium of benthic turf algae. Nutrients were added in 3-fold higher concentrations compared to the annual averages, and the parameters algal growth, extension of bleached area on corals, tissue colour change and chlorophyll a concentrations were monitored at regular intervals over experimental duration. This revealed that elevated ammonium concentrations and elevated organic nutrient concentrations stimulate algal growth, while coral tissue pigmentation and chlorophyll a content were significantly decreased. But only in the elevated organic nutrient treatment all effects on corals were significantly pronounced when assembled with benthic turf algae. Supplementary logger measurements revealed that O 2 water concentrations were significantly lower in the elevated organic nutrient mesocosm compared to all other treatments, confirming side-effects on microbial activity. These findings indicate that organic nutrient input into coral reefs can affect physiology and metabolism of both corals and benthic turf algae. Reinforcing interaction between both groups of organisms along with involvement of microbes may facilitate phase shifts in coral reef ecosystems.

Photo-induced gratings in thin color center layers on lithium fluoride

We study the recording of permanent Bragg gratings on surface-colored lithium fluoride (LiF) crystals by using the interference pattern of a continuous-wave UV argon-ion laser operating at 244 nm. Gratings with spatial periodicity ranging from 400 to 1000 nm are written by using a phase-mask interferometer and are stable for several months after the writing process. Absorption and photoluminescence spectra show the bleaching of primary F and F -aggregate laser-active color centers as a result of the process. Confocal microscopy is used to determine the pitch and the profile of the fluorescent gratings. The UV laser-induced optical bleaching in highly colored LiF ultrathin layers is responsible for the periodic spatial modulation of absorption and photoemission properties that characterize the gratings. In the colored surface layer, a reduction of as much as 50% of the initial color-center-induced refractive-index increase has been estimated in the bleached areas.

Techniques for extraction of depth-resolved in vivo human retinal intrinsic optical signals with optical coherence tomography

To demonstrate acquisition and analysis methods for depth-resolved observation of slow retinal physiology induced changes in infrared backscatter in vivo. A dark-adapted human was briefly subjected to a localized photobleach. For 20 min before and 30 min after the stimulus, volumetric optical coherence tomograms were collected partially overlapping the bleached region. Tomograms were segmented into retinal layers by a newly described algorithm exploiting information in adjacent B-scans. En face fundus images extracted from major intraretinal layers were laterally registered manually. Time series summarizing the observed backscatter in selected layers for the bleached and unbleached areas are shown with a variety of corrections and normalizations applied: tomograms were corrected for inherent sensitivity roll-off, and the ratio between other layers and an assumed unchanging layer (retinal pigment epithelium), as well as the ratio of the stimulated area to the unstimulated area, were calculated. Adjacent B-scan information allows a simpler segmentation algorithm to be used. Sensitivity roll-off correction reduces signal variability due to eye motion. After normalizations, the signal correlated with the stimulus appears strongest at the photoreceptor inner-outer segment junction. Demonstrated methods manage data complexity and reduce uncorrelated signal variability. This single trial warrants further investigation of intrinsic optical signals to observe slow physiologic responses.

Dynamic Organization of Aminoacyl-tRNA Synthetase Complexes in the Cytoplasm of Human Cells

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.

Structural Memory in the Contractile Ring Makes the Duration of Cytokinesis Independent of Cell Size

Cytokinesis is accomplished by constriction of a cortical contractile ring. We show that during the early embryonic divisions in C. elegans, ring constriction occurs in two phases--an initial phase at a constant rate followed by a second phase during which the constriction rate decreases in proportion to ring perimeter. Cytokinesis completes in the same amount of time, despite the reduction in cell size during successive divisions, due to a strict proportionality between initial ring size and the constant constriction rate. During closure, the myosin motor in the ring decreases in proportion to perimeter without turning over. We propose a "contractile unit" model to explain how the ring retains a structural memory of its initial size as it disassembles. The scalability of constriction may facilitate coordination of mitotic events and cytokinesis when cell size, and hence the distance traversed by the ring, varies during embryogenesis and in other contexts.

The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD KZ), originating from Pseudomonas aeruginosa bacteriophage ϕKZ, has been examined using a fusion protein of PBD KZ and green fluorescent protein (PBD KZ-GFP). A fluorescence recovery after photobleaching analysis of bound PBD KZ-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 × 10 7 M −1 for the PBD KZ-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD KZ-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

PML3 Orchestrates the Nuclear Dynamics and Function of TIP60

The promyelocytic leukemia (PML) protein is a major component to govern the PML nuclear body (NB) assembly and function. Although it is well defined that PML NB is a site recruiting sumoylated proteins, the mechanism by which PML protein regulates the process remains unclear. Here we show that PML3, a specific PML isoform, interacts with and recruits TIP60 to PML NBs. Our biochemical characterization demonstrates that PML3 physically interacts with TIP60 via its N-terminal 364 amino acids. Importantly, this portion of TIP60 is sufficient to target to the PML NBs, suggesting that PML3-TIP60 interaction is sufficient for targeting TIP60 to the NBs. The PML3-TIP60 interaction is specific, since the region of TIP60 binding is not conserved in other PML isoforms. The physical interaction between PML3 and TIP60 protects TIP60 from Mdm2-mediated degradation, suggesting that PML3 competes with MDM2 for binding to TIP60. Fluorescence recovery after photobleaching analysis indicates that the PML3-TIP60 interaction modulates the nuclear body distribution and mobility of TIP60. Conversely, the distribution and mobility of TIP60 are perturbed in PML3-deficient cells, accompanied by aberrations in DNA damage-repairing response. Thus, PML3 orchestrates the distribution, dynamics, and function of TIP60. Our findings suggest a novel regulatory mechanism by which the PML3 and TIP60 tumor suppressors cooperate to ensure genomic stability.

CXXC5 Is a Novel BMP4-regulated Modulator of Wnt Signaling in Neural Stem Cells

Bone morphogenetic proteins such as BMP4 are essential for proper development of telencephalic forebrain structures and induce differentiation of telencephalic neural stem cells into a variety of cellular fates, including astrocytic, neuronal, and mesenchymal cells. Little is yet understood regarding the mechanisms that underlie the spatiotemporal differences in progenitor response to BMP4. In a screen designed to identify novel targets of BMP4 signaling in telencephalic neural stem cells, we found the mRNA levels of the previously uncharacterized factor CXXC5 reproducibly up-regulated upon BMP4 stimulation. In vivo, CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon, including the developing choroid plexus. CXXC5 showed partial homology with Idax, a related protein previously shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl co-localized in the cytoplasm and interacted in co-immunoprecipitation experiments. Moreover, fluorescence resonance energy transfer (FRET) experiments verified that CXXC5 and Dvl2 were located in close spatial proximity in neural stem cells. Studies of the functional role of CXXC5 revealed that overexpression of CXXC5 or exposure to BMP4 repressed the levels of the canonical Wnt signaling target Axin2, and CXXC5 attenuated Wnt3a-mediated increase in TOPflash reporter activity. Accordingly, RNA interference of CXXC5 attenuated the BMP4-mediated decrease in Axin2 levels and facilitated the response to Wnt3a in neural stem cells. We propose that CXXC5 is acting as a BMP4-induced inhibitor of Wnt signaling in neural stem cells.

A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ∼1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.