The role of nerve- versus muscle-derived factors in mammalian neuromuscular junction formation.

Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin.

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin.

As the only barrier between blood and bile compartments hepatocellular tight junctions play a crucial role in cholestasis-induced increase of biliary permeability. The molecular basis of this reversible defect is not known. We, therefore, examined expression, phosphorylation, distribution and colocalization of the junctional proteins occludin, claudin-1-3, ZO-1 and ZO-2 in rats after bile duct ligation and release of ligation. In control rats, claudin-1 and ZO-2 displayed a lobular gradient with highest expression levels in periportal cells, whereas claudin-2 showed a reciprocal distribution. Other proteins were evenly expressed in the liver lobule. Ligation resulted in upregulation of ZO-2 (2.7-fold), ZO-1 (1.4-fold) and occludin (1.2-fold) but not of claudins. Only ZO-2 showed increased phosphorylation. Distribution patterns were unchanged except for a strong accumulation of ZO-2 in perivenous hepatocytes. Colocalization analysis demonstrated that perivenous ZO-2 was the only protein examined revealing strongly increased overlap with occludin and ZO-1, whereas claudins and other proteins displayed a decrease. All changes were partially reversed by release of ligation. We conclude that differential expression of claudin-1-2 and ZO-2 has functional implications for bile formation. The moderately increased ZO-1 and occludin levels account for the known elongation of tight junction strands. The highly increased expression and changed distribution of ZO-2 suggests that ZO-1 is partly substituted by ZO-2, an alteration possibly causing impaired barrier function.

The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells.

The transmembrane receptor CD44 conveys important signals from the extracellular microenvironment to the cytoplasm, a phenomena known as "outside-in" signaling. CD44 exists as several isoforms that result from alternative splicing, which differ only in the extracellular domain but yet exhibit different activities. CD44 is a binding partner for the membrane-cytoskeleton cross-linker protein ezrin. In this study, we demonstrate that only CD44 standard (CD44s) colocalizes and interacts with the actin cross-linkers ezrin and moesin using well-characterized cell lines engineered to express different CD44 isoforms. Importantly, we also show that the association CD44s-ezrin-actin is an important modulator of Fas-mediated apoptosis. The results highlight a mechanism by which signals from the extracellular milieu regulate intracellular signaling activities involved in programmed cell death.

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes.

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.

Heat stress enhances recovery of hepatocyte bile acid and organic anion transporters in endotoxemic rats by multiple mechanisms.

Heat stress (HS) reduces the many sequelae of lipopolysaccharide (LPS)-induced endotoxemia. Without HS, endotoxins have been shown to induce a transcriptional down-regulation of hepatocyte transport proteins for bile acids and organic anions. We performed experiments in isolated perfused rat livers at various times after LPS administration with and without HS pretreatment to determine whether HS would correct deficient transport of bromosulfophthalein (BSP). Possible mechanisms involved were investigated in livers from intact animals. In isolated perfused livers, LPS injection reduced BSP excretion to 48% compared with saline-injected controls (P < 0.01). When HS was applied 2 hours prior to LPS, BSP excretion increased to 74% of controls (P < 0.05 vs LPS and controls). Expression of the basolateral (Oatp1a1) and canalicular (Mrp2) organic anion transporter involved in the transport of BSP recovered more rapidly when HS preceded LPS application. Recovery of mRNA levels of these transporters occurred also earlier. Coimmunoprecipitation experiments and immunoelectron microscopy using a double immunogold labeling of heat shock protein 70 (HSP70) and various hepatocyte transporters suggested colocalization with HSP70 for the canalicular bile acid transporter (Bsep) in the subcanalicular space. In contrast, no colocalization was shown for Ntcp and anion transporters. In conclusion, we could show that HS enhances recovery of organic anion transporters and bile acid transporters following endotoxemia. Faster recovery of mRNA seems to be a key mechanism for anion transporters, whereas physical interaction with HSP70 plays a role in preservation of bile acid transporters. This interaction of HSP70 and canalicular transporters occurs only in pericanalicular vesicles but not when the protein is integrated into the plasma membrane.

A versatile in vitro assay for investigating angiogenesis of the heart.

Neovascularization in the heart is usually investigated with models of angiogenesis in vivo. Here we present a simple model that allows investigating heart angiogenesis in mice and rats in vitro. Small pieces of left ventricular myocardium were cultured in three-dimensional fibrin gels for 10 days. A single mouse heart allowed assessing 24 conditions, each tested in octuplicates. Rat recombinant VEGF164, human recombinant bFGF, and human recombinant PDGF-BB were used under normoxia (21% O2) and hypoxia (3% O2), and outgrowth of endothelial sprouts from heart pieces was quantified. In 4-week-old OF1 mice, endothelial sprouts formed spontaneously. In contrast, in 12-week-old adult mice, virtually no sprouts formed under normoxia. Under hypoxia, sprout formation increased substantially. Different growth factors induced formation of distinct patterns of sprouts and unorganized single cells. Sprouts were composed of endothelial cells with smooth muscle cells or pericytes interacting with them, as assessed by immunohistochemistry. Taken together, our model is suited for investigation of angiogenesis of the heart in vitro. It may allow performing extensive series of experiments in vitro including rapid screening of pharmacological compounds and assessment of mechanisms of heart angiogenesis in transgenic animals in an easy straightforward manner.

Colocalization analysis yields superior results after image restoration.

Colocalization analysis is a powerful tool for the demonstration of spatial and temporal overlap in the distribution patterns of fluorescent probes. In unprocessed images, background affects image quality by impairing resolution and obscuring image detail in the low-intensity range. Because confocal images suffer from background levels up to 30% maximum intensity, colocalization analysis, which is a typical segmentation process, is limited to high-intensity signal. In addition, noise-induced, false-positive events ("dust") may skew the results. Therefore, suppression of background is crucial for this type of image analysis. Analysis of synthetic and biological objects demonstrates that median filtering is able to eliminate noise-induced colocalization events successfully. Its disadvantages include the occasional generation of false-positive and false-negative results as well as the inherent impairment of resolution. In contrast, image restoration by deconvolution suppresses background to very low levels (<10% maximum intensity), which makes additional objects in the low-intensity but high-frequency range available for analysis. The improved resolution makes this technique extremely suitable for examination of objects of near resolution size as demonstrated by correlation coefficients. Deconvolution is, however, sensitive to overestimation of the background level. Conclusions for practical application are: (1) In raw images, colocalization analysis is limited to the intensity range above the background level. This means the higher the RS/N the better. Unfortunately, images of most biological specimens have a low RS/N. (2) Filtering improves the result substantially. The reduction of background levels and the concomitant increase of the RS/N are generated at the expense of resolution. This is a quick and simple method in cases where resolution is not a major concern. (3) If colocalization in the low-intensity range and/or maximum resolution play a role, deconvolution should be used.

Heat stress prevents impairment of bile acid transport in endotoxemic rats by a posttranscriptional mechanism.

BACKGROUND & AIMS: Endotoxemia leads to reduction of bile acid transporters in the hepatocyte membrane and impaired bile acid transport. Because heat stress ameliorates other sequelae of endotoxemia, studies were performed to determine whether heat stress would correct deficient bile acid transport caused by endotoxin. METHODS: Body temperature of rats was elevated to 42 degrees C for 10 minutes. Lipopolysaccharide was injected after different time intervals, and maximal transport for cholyltaurine was measured in perfused rat livers. Sodium-dependent and -independent uptake was studied in isolated hepatocytes. Protein expression, messenger RNA levels, and tissue distribution of the bile acid transporters sodium taurocholate cotransporting protein (ntcp) and bile salt export pump (bsep) were also analyzed. RESULTS: In the perfused liver, cholyltaurine transport was reduced by 59% by endotoxin, but transport was not reduced when heat stress was applied 2 hours before injection of lipopolysaccharide. The protective effect coincided with maximal expression of heat shock proteins 70 and 25. Sodium-dependent and -independent transport was preserved by heat stress. Expression of bile acid transporters in plasma membrane fractions was reduced after injection of lipopolysaccharide but not if lipopolysaccharide was preceded by heat stress. In contrast, messenger RNA levels of bile acid transporters were not preserved by heat stress. CONCLUSIONS: Heat stress preserves bile acid transporters during endotoxemia by a posttranscriptional mechanism.