Biomedical subjects

Michael Kühl

Publications and source records attributed to Michael Kühl.

31 records · Page 2Linked to original sources

PubMed2004

The WNT/calcium pathway: biochemical mediators, tools and future requirements.

Wnt proteins represent a family of secreted, lipid-modified glycoproteins that can activate different intracellular pathways. Upon binding to certain members of the Frizzled family of Wnt receptors some Wnts like Wnt-4, Wnt-5A or Wnt-11 are able to elicit an intracellular release of calcium ions. This calcium signaling acitivity is sufficient to activate calcium sensitive enzymes like protein kinase C (PKC), calcium-calmodulin dependent kinase II (CamKII) or calcineurin (CaCN). This so-called Wnt/calcium pathway plays important roles during dorso-ventral patterning of the embryo, regulating cell migration, as well as heart development, and might play a role during tumor suppression. The foci of this review are the biochemical aspects of Wnt/calcium signaling, the tools that are available to study Wnt/calcium signaling, and the open questions that need to be addressed in the future to validate this signaling pathway.

Animals↗

PubMed2004

Getting an embryo into shape.

Formation of a multicellular organism is a complex process involving differentiation and morphogenesis. During early vertebrate development, the radial symmetric organization of the egg is transferred into a bilateral symmetric organism with three distinct body axes: anteroposterior (AP), dorsoventral, and left-right. Due to cellular movements and proliferation, the body elongates along the AP axis. How are these processes coupled? Two recent publications now indicate that cell migration as well as orientated cell divisions contribute to axis elongation. The processes are coupled through the planar cell polarity pathway.1 At the same time, the AP axis is patterned independently of convergent extension. This process, however, is required for cell migration and represents a cue for polarized cell motility during gastrulation. Thus, it is AP polarity that instructs individual cells how to orientate with respect to the embryonic axis and provides positional information for the process of convergent extension.(2)

Animals↗

PubMed2003

Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos.

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Microbially mediated sulphide production in a thermal, acidic algal mat community in Yellowstone National Park.

Our objective in this study was to characterize prokaryotic sulphide production within the oxygenic, predominantly eukaryotic algal mat in an acidic stream, Nymph Creek, in Yellowstone National Park (YNP). We used microsensors to examine fluctuations in H2S and O2 concentrations over time through the vertical aspect of the approximately 3 mm mat in a 46-48 degrees C region of the creek. We also used analyses of PCR-amplified 16S rRNA gene sequences obtained from denaturing gradient gels, and PCR-amplified sequences of a functional gene associated with microbial sulphate respiration (dsrA) to characterize the bacterial community in the same region of the mat. During midday, photosynthesis rates were high within the first 500 micro m interval of the mat and high oxygen concentrations (600% air saturation) penetrated deeply (>1800 micro m) into the mat. During early evening and night, oxygen concentrations within the first 1100 micro m of the mat decreased over time from 60% air saturation (a.s) to 12% a.s. A precipitous decline in oxygen concentration occurred at a depth of 1100 micro m in all night measurements and anoxic conditions were present below 1200 micro m. Within this anoxic region, sulphide concentrations increased from nearly 0 micro M at 1200 micro m depth to 100 micro M at 2400 micro m depth. Enrichment cultures inoculated with Nymph Creek mat organisms also produced H2S. Sequence analyses of 16S rRNA and dsrA genes indicated the presence of at least five bacterial genera including species involved in dissimilative sulphate or sulphur reduction.

Eukaryota↗

PubMed2003

Cyanobacterial ecotypes in different optical microenvironments of a 68 degrees C hot spring mat community revealed by 16S-23S rRNA internal transcribed spacer region variation.

We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68 degrees C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O(2) and oxygenic photosynthesis demonstrated the existence of physiologically distinct Synechococcus populations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths.

Base Sequence↗

PubMed2002

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts.

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.

Animals↗

PubMed2002

The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.

Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.

Animals↗

PubMed2002

Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis.

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.

3T3 Cells↗

PubMed2002

Increasingly complex: new players enter the Wnt signaling network.

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.

Animals↗

PubMed2002

Cardiac specific expression of Xenopus Popeye-1.

Popeye genes code for putative transmembrane proteins that are predominantly expressed in heart and skeletal muscle. Here we report on the isolation and expression of a previously unknown Xenopus member of this family, Xenopus Popeye-1 (Xpop-1). Xpop-1 is 60-65% identical to other vertebrate Pop-1 genes at the protein level. Whole-mount in situ hybridization studies revealed a highly specific expression of Xpop-1 whose transcripts are restricted to the embryonic heart and become enriched in the forming ventricle. Interestingly, unlike other known vertebrate Popeye genes, Xpop-1 is exclusively expressed in cardiac tissue and absent from skeletal muscle.

Amino Acid Sequence↗

PubMed2002

Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation.

Wnt proteins form a family of secreted glycoproteins that are involved in different developmental processes such as differentiation, proliferation, cell migration and cell polarity. To exert its function, Wnt proteins activate different intracellular signaling cascades. Whereas the canonical, Wnt/beta-catenin pathway is well characterized, less is known about the function of non-canonical Wnt pathways in vertebrates. I here summarize recent findings implicating important roles for Wnt/Ca(2+) and Wnt/JNK signaling during different aspects of early Xenopus laevis development, namely axis formation and gastrulation movements.

Animals↗

PubMed2002

Conspicuous veils formed by vibrioid bacteria on sulfidic marine sediment.

We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10- micro m) cells that multiply by binary division and contain several spherical inclusions of poly-beta-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 micro M oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.

Bacteria↗

PubMed2002

Microscopic examination of distribution and phenotypic properties of phylogenetically diverse Chloroflexaceae-related bacteria in hot spring microbial mats.

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus Chloroflexus or with the type C cluster, a group of previously discovered Chloroflexaceae relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll a. Chloroflexus sp. filaments were rare at 60 degrees C but were more abundant at 70 degrees C, where they were confined to the upper millimeter of the mat. Both type C organisms and Chloroflexus spp. were observed to assimilate radiolabeled acetate under in situ conditions.

Autoradiography↗