Christoph Ruediger Bauer

Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells.

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.

SLP-2 is required for stress-induced mitochondrial hyperfusion

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin‐related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress‐induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L‐OPA1, MFN1, and the mitochondrial inner membrane protein SLP‐2. In the absence of SLP‐2, L‐OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro‐survival response against stress.

Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP‐tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312‐aa domain of Sir4 (Sir4PAD) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4PAD‐mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku‐ and Esc1‐dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

Apoptotic Cells Provide an Unexpected Source of Wnt3 Signaling to Drive Hydra Head Regeneration

Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.

Circadian gene expression is resilient to large fluctuations in overall transcription rates

Mammalian circadian oscillators are considered to rely on transcription/translation feedback loops in clock gene expression. The major and essential loop involves the autorepression of cryptochrome (Cry1, Cry2) and period (Per1, Per2) genes. The rhythm‐generating circuitry is functional in most cell types, including cultured fibroblasts. Using this system, we show that significant reduction in RNA polymerase II‐dependent transcription did not abolish circadian oscillations, but surprisingly accelerated them. A similar period shortening was observed at reduced incubation temperatures in wild‐type mouse fibroblasts, but not in cells lacking Per1. Our data suggest that mammalian circadian oscillators are resilient to large fluctuations in general transcription rates and temperature, and that PER1 has an important function in transcription and temperature compensation.

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4α, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

PLEKHA7 Is an Adherens Junction Protein with a Tissue Distribution and Subcellular Localization Distinct from ZO-1 and E-Cadherin

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.

The Circadian Clock Starts Ticking at a Developmentally early Stage

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.

The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion

BackgroundCell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology.ResultsWe show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation.ConclusionCollectively, our manuscripts and the works on Necl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types.

Opening a Gateway for Chemiluminescence Cell Imaging: Distinctive Methodology for Design of Bright Chemiluminescent Dioxetane Probes

Chemiluminescence probes are considered to be among the most sensitive diagnostic tools that provide high signal-to-noise ratio for various applications such as DNA detection and immunoassays. We have developed a new molecular methodology to design and foresee light-emission properties of turn-ON chemiluminescence dioxetane probes suitable for use under physiological conditions. The methodology is based on incorporation of a substituent on the benzoate species obtained during the chemiexcitation pathway of Schaap’s adamantylidene–dioxetane probe. The substituent effect was initially evaluated on the fluorescence emission generated by the benzoate species and then on the chemiluminescence of the dioxetane luminophores. A striking substituent effect on the chemiluminescence efficiency of the probes was obtained when acrylate and acrylonitrile electron-withdrawing groups were installed. The chemiluminescence quantum yield of the best probe was more than 3 orders of magnitude higher than that of a standard, commercially available adamantylidene–dioxetane probe. These are the most powerful chemiluminescence dioxetane probes synthesized to date that are suitable for use under aqueous conditions. One of our probes was capable of providing high-quality chemiluminescence cell images based on endogenous activity of β-galactosidase. This is the first demonstration of cell imaging achieved by a non-luciferin small-molecule probe with direct chemiluminescence mode of emission. We anticipate that the strategy presented here will lead to development of efficient chemiluminescence probes for various applications in the field of sensing and imaging.