Spatial distribution and functional significance of activated vinculin in living cells

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin β by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics

Role of Dlg5/lp-dlg, a Membrane-Associated Guanylate Kinase Family Protein, in Epithelial-Mesenchymal Transition in LLc-PK1 Renal Epithelial Cells

Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT

Report and preliminary results of R/V SONNE Cruise SO251 - Extreme events Archived in the GEologial Record of JAPAN's subduction margins (EAGER-JAPAN)

Leg A SO251-1, Yokohama - Yokohama, 04.10.2016 - 15.10.2016, Leg B SO251-2, Yokohama - Yokohama, 18.10.2016 - 02.11.201

Impact of mediastinal, liver and lung 123I-metaiodobenzylguanidine (123I-MIBG) washout on calculated 123I-MIBG myocardial washout

PURPOSE: In planar (123)I-metaiodobenzylguanidine ((123)I-MIBG) myocardial imaging mediastinum (M) activity is often used as a background correction in calculating "washout" (WO). However, the most likely sources for counts that might produce errors in estimating myocardial (Myo) activity are lung (Lu) and liver (Li), which typically have higher counts/pixel (cpp) than M. The present study investigated the relationship between changes in Lu, Li and Myo activity between early and late planar (123)I-MIBG images, with comparison to M as the best estimator of non-specific background activity. METHODS: Studies on 98 subjects with both early (e) and late (l) planar (123)I-MIBG images were analysed. There were 68 subjects with chronic heart failure (CHF), 14 with hypertension (HTN) but no known heart disease and 16 controls (C). For each image, regions of interest (ROIs) were drawn: an irregular whole Myo, Lu, upper M and Li. For each ROI, WO was calculated as [(cpp(e)-cpp(l:decay corrected))/cpp(e)]x100%. RESULTS: Multivariable forward stepwise regression analysis showed that overall a significant proportion of the variation in Myo WO could be explained by a model containing M WO and Lu WO (37%, p < 0.001). Only in controls was M WO the sole variable explaining a significant proportion of the variation in Myo WO (27%, p = 0.023). CONCLUSION: Although increased Myo WO in CHF subjects reflects disease severity, part of the count differences measured on planar (123)I-MIBG myocardial images likely reflects changes in the adjacent and surrounding Lu tissue. The results for the controls suggest that this is the only group where a mediastinum correction alone may be appropriate for cardiac WO calculation

Correction of non-intrusive drill core physical properties data for variability in recovered sediment volume

Non-intrusive track-based physical properties measurements of sediment cores recovered during ocean drilling are often biased by imperfect recovery within sediment core liners, particularly in heterogeneous and/or partially lithified sediments. These biases result in misrepresentation in measurements of true sediment physical properties, and can complicate integration of the composite site records assembled from recovered cores with borehole logs of the stratigraphic section. Here we develop a strategy utilizing gamma ray attenuation (GRA) density to generate mass-specific magnetic susceptibility (MS) and natural gamma radiation (NGR) data. Shipboard GRA density is collected in all cores that comprise a site at equivalent or higher resolution than the corresponding MS and NGR data. All instruments are calibrated assuming a volume of sediment in their detector windows equivalent to that present in a perfectly full core liner; changes in sediment bulk density related to compaction, and/or imperfect sediment recovery resulting in a partially filled core liner thus influence all three measurements proportional to their detector sensitivities. In principle it may be possible to correct MS or NGR data for variable sediment volume by normalizing them to GRA measured at equivalent depth on a sensing track, assuming that the volumetric bias is comparable in all three datasets. Because GRA is measured in much greater detail, it must be smoothed by the known measurement windows of the other parameters for the assumption of comparable analytical sediment volume to be true. Normalizing MS or NGR by the equivalently smoothed GRA in down-hole records should thus remove the bias associated with variable sediment volume in the detector windows, allowing for robust mass-specific determination of these volume-based sediment physical properties.© The Authors 2015. Published by Oxford University Press on behalf of The Royal Astronomical Society. The published article can be found at: http://gji.oxfordjournals.org/content/202/2/1317Keywords: Downhole methods, Ocean drillingKeywords: Downhole methods, Ocean drillin

Mid-Pleistocene climate transition drives net mass loss from rapidly uplifting St. Elias Mountains, Alaska

Erosion, sediment production and routing on a tectonically active continental margin reflect both tectonic and climatic processes; partitioning the relative importance of these processes remains controversial. Gulf of Alaska contains a preserved sedimentary record of Yakutat Terrane collision with North America. Because tectonic convergence in the coastal St. Elias orogen has been roughly constant for 6 Myr, variations in its eroded sediments preserved in the offshore Surveyor Fan constrain a budget of tectonic material influx, erosion, and sediment output. Seismically imaged sediment volumes calibrated with chronologies derived from Integrated Ocean Drilling Program boreholes shows that erosion accelerated in response to Northern Hemisphere glacial intensification (~2.7 Ma) and that the 900-km long Surveyor Channel inception appears to correlate with this event. However, tectonic influx exceeded integrated sediment efflux over the interval 2.8-1.2 Ma. Volumetric erosion accelerated following the onset of quasi-periodic (~100-kyr) glacial cycles in the mid-Pleistocene climate transition (1.2-0.7 Ma). Since then erosion and transport of material out of the orogen has outpaced tectonic influx by 50-80%. Such a rapid net mass loss explains apparent increases in exhumation rates inferred onshore from exposure dates and mapped out-of-sequence fault patterns. The 1.2 Myr mass budget imbalance must relax back toward equilibrium in balance with tectonic influx over the time scale of orogenic wedge response (Myrs). The St. Elias Range provides a key example of how active orogenic systems respond to transient mass fluxes, and the possible influence of climate driven erosive processes that diverge from equilibrium on the million-year scale

Expression of Kruppel-Like Factor KLF4 in Mouse Hair Follicle Stem Cells Contributes to Cutaneous Wound Healing

Kruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated.Quantitative real time polymerase chain reaction (RT-PCR) analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP) transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/CreER™) transgenic mice with KLF4(flox) mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreER™ and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreER™ and KLF4(flox) double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised cell migration.KLF4 expression in mouse hair bulge stem cells plays an important role in cutaneous wound healing. These findings may enable future development of KLF4-based therapeutic strategies aimed at accelerating cutaneous wound closure

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts

Autosomal-dominant hypotrichosis with woolly hair : novel gene locus on chromosome 4q35.1-q35.2

Hypotrichosis simplex (HS) with and without woolly hair (WH) comprises a group of rare, monogenic disorders of hair loss. Patients present with a diffuse loss of scalp and/or body hair, which usually begins in early childhood and progresses into adulthood. Some of the patients also show hair that is tightly curled. Approximately 10 genes for autosomal recessive and autosomal dominant forms of HS have been identified in the last decade, among them five genes for the dominant form. We collected blood and buccal samples from 17 individuals of a large British family with HS and WH. After having sequenced all known dominant genes for HS in this family without the identification of any disease causing mutation, we performed a genome-wide scan, using the HumanLinkage-24 BeadChip, followed by a classical linkage analysis; and whole exome-sequencing (WES). Evidence for linkage was found for a region on chromosome 4q35.1-q35.2 with a maximum LOD score of 3.61. WES led to the identification of a mutation in the gene SORBS2, encoding sorbin and SH3 domain containing 2. Unfortunately, we could not find an additional mutation in any other patient/family with HS; and in cell culture, we could not observe any difference between cloned wildtype and mutant SORBS2 using western blotting and immunofluorescence analyses. Therefore, at present, SORBS2 cannot be considered a definite disease gene for this phenotype. However, the locus on chromosome 4q is a robust and novel finding for hypotrichosis with woolly hair. Further fine mapping and sequencing efforts are therefore warranted in order to confirm SORBS2 as a plausible HS disease gene