Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

Use of wetlands at the mouth of the Yangtze River by shorebirds during spring and fall migration

ABSTRACT.   The mouth of the Yangtze River is an important stopover site for migratory shorebirds using the East Asian-Australasian Flyway. From 1984 to 2004, we censused and banded shorebirds and monitored hunting activities at the mouth of the Yangtze River to understand how shorebirds used the study area. Counts and banding data revealed greater numbers of shorebirds at the mouth of the Yangtze River during northward migration (spring) than during southward migration (fall), with ratios varying from 1.5:1 to 7.2:1 at different sites from 1984 to 2005. The most common species observed during spring (northward) migration were Great Knots ( Calidris tenuirostris ), Red Knots ( Calidris canutus ), Bar-tailed Godwits ( Limosa lapponica ), Sharp-tailed Sandpipers ( Calidris acuminata ), and Red-necked Stints ( Calidris ruficollis ). During spring 2003–2004, 96.98% of the shorebirds observed were adults (ASY or older) and 3% were after hatching-year and second-year birds (AHY or SY). In contrast, almost all (94.73%) birds counted during the fall were hatching-year (HY) birds. These results indicate that adult shorebirds either use a different migration route during fall migration or use the same route, but do not stop at the mouth of the Yangtze River. HY birds, however, may depend on the coastal stopover sites for feeding during their first southward passage.     

Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory

The neuronal ubiquitin/proteasomal pathway has been implicated in the pathogenesis of Alzheimer's disease (AD). We now show that a component of the pathway, ubiquitin C-terminal hydrolase L1 (Uch-L1), is required for normal synaptic and cognitive function. Transduction of Uch-L1 protein fused to the transduction domain of HIV-transactivator protein (TAT) restores normal enzymatic activity and synaptic function both in hippocampal slices treated with oligomeric Abeta and in the APP/PS1 mouse model of AD. Moreover, intraperitoneal injections with the fusion protein improve the retention of contextual learning in APP/PS1 mice over time. The beneficial effect of the Uch-L1 fusion protein is associated with restoration of normal levels of the PKA-regulatory subunit IIalpha, PKA activity, and CREB phosphorylation.     

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.     

Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.     

The biology of Ku and its potential oncogenic role in cancer

Ku is a heterodimeric protein made up of two subunits, Ku70 and Ku80. It was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. It is a highly versatile regulatory protein that has been implicated in multiple nuclear processes, e.g., DNA repair, telomere maintenance and apoptosis. Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival. Recent reports suggest that there is a positive relationship between Ku and the development of cancer, making Ku an important candidate target for anticancer drug development. Specifically, prior studies suggest that a delicate balance exists in Ku expression, as overexpression of Ku proteins promotes oncogenic phenotypes, including hyperproliferation and resistance to apoptosis; whereas deficient or low expression of Ku leads to genomic instability and tumorigenesis. Such observations through various experimental models indicate that Ku may act as either a tumor suppressor or an oncoprotein. Hence, understanding the link between the various functions of Ku and the development of cancer in different cell systems may help in the development of novel anticancer therapeutic agents that target Ku. These studies may also increase our understanding of how Ku autoantibodies are generated in autoimmune diseases.