Telomere protection by mammalian Pot1 requires interaction with Tpp1

The shelterin complex at mammalian telomeres contains the single-stranded DNA-binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA-binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.     

Structure and function insights garnered from in silico modeling of the thrombospondin type-1 domain-containing 7A antigen

The thrombospondin type-1 domain containing 7A (THSD7A) protein is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD7A antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this protein contains 21 extracellular domains and with the exception of the first two domains, has a regular repeating pattern of TSP-1-like followed by F-spondin-like domains. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-1-like domains. Of the 21 domains, 18 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like domains have insertions in the canonical TSP fold, most notably the coiled coil region in domain 4, which may be utilized in protein-protein binding interactions, suggesting that this protein functions as a heparan sulfate binding site.     

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (>400Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the S1-casein mammary gland-specific promoter operatively linked to 37Kb of the human 1(I) procollagen structural gene and 3 flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(1)₃] type I procollagen were detected (up to 8mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in mil; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.     

A role for p21-activated kinase in endothelial cell migration

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and increased stress fibers and focal adhesions. The DN effect mapped to the most NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.     

Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters

The dopamine transporter plays an essential role in the modulation of dopaminergic neurotransmission by mediating the reuptake of dopamine into presynaptic neurons. In cells expressing the dopamine transporter, activation of protein kinase C by phorbol esters results in a significant reduction in dopamine uptake. This phorbol ester-mediated inhibition of dopamine transport is associated with a decrease in V(max), although the apparent affinity of the transporter for dopamine remains unchanged. Using a green fluorescent protein-tagged dopamine transporter stably expressed in Madin-Darby canine kidney cells, we show in live cells that the decrease in transporter activity is caused by the rapid internalization of carriers from the plasma membrane. This redistribution of the transporter is specific to phorbol ester activation and is unaffected by the presence of either substrates or inhibitors of the carrier. Upon the addition of phorbol esters, transporters at the cell surface are rapidly endocytosed through a clathrin-mediated and dynamin-dependent mechanism into early endosomes, where they colocalize with transferrin. The internalized carrier is targeted to the endosomal/lysosomal pathway and is completely degraded within 2 h of protein kinase C activation. Phorbol ester-mediated alterations in the trafficking of the dopamine transporter may serve as a mechanism for controlling extracellular dopamine levels in the central nervous system.     

Feline viruses in wildcats from Scotland

Few data are available on the prevalence of feline viruses in European wildcats (Felis silvestris). Previous surveys have indicated that wildcats may be infected with the common viruses of domestic cats, apart from feline immunodeficiency virus (FIV). In the present study, 50 wildcats trapped throughout Scotland (UK) between August 1992 and January 1997 were tested for evidence of viral infection. All were negative for FIV by several serological or virological methods. By contrast, 10% of the cats were positive for feline leukemia virus (FeLV) antigen and infectious virus was isolated from 13% of a smaller subset. Of the wildcats tested for respiratory viruses, 25% yielded feline calicivirus (FCV) and although no feline herpesvirus was isolated, 16% of the samples had neutralizing antibodies to this virus. Antibodies to feline coronavirus (FCoV) were found in 6% of samples. Feline foamy virus (FFV) was an incidental finding in 33% of samples tested. This study confirms that wildcats in Scotland are commonly infected with the major viruses of the domestic cat, except for FIV.     

Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation.