Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.     

Pore formation is not associated with macroscopic redistribution of P2X7 receptors

The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.     

Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action

To determinethe mechanism of fatty acid modulation of rabbit pulmonary arterylarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, we studied effects of fatty acidsand other lipids on channel activity in excised patches withpatch-clamp techniques. The structural features of the fatty acidrequired to increase BK_Ca channel activity (or averagenumber of open channels, NP_o) were identified tobe the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which havea sufficiently long alkyl chain (C  8), produced a decrease inNP_o. Neutral and short-chain lipids did notalter NP_o. Screening of membrane surface chargewith high-ionic-strength bathing solutions (330 mM K⁺ or130 mM K⁺, 300 mM Na⁺) did not alter themodulation of the BK_Ca channel NP_oby fatty acids and other charged lipids, indicating that channelmodulation is unlikely to be due to an alteration of the membraneelectric field or the attraction of local counterions to the channel.Fatty acids and other negatively charged lipids were able to modulate BK_Ca channel activity in bathing solutions containing 0 mMCa²⁺, 20 mM EGTA, suggesting that calcium is not requiredfor this modulation. Together, these results indicate that modulationof BK_Ca channels by fatty acids and other charged lipidsmost likely occurs by their direct interaction with the channel proteinitself or with some other channel-associated component.

     

Differential effects of nutrient-limited primary production on primary, secondary or tertiary consumers

Nutritional imbalances between predator and prey are the rule rather than the exception at the lower end of food webs. We investigated the role of different grazers in the propagation of nutritionally imbalanced primary production by using the same primary producers in a three-trophic-level food chain and a four-trophic-level food chain experimental setup. The three-trophic-level food chain consisted of a classic single-cell primary producer (Rhodomonas salina), a metazoan grazer (the copepod Acartia tonsa) and a top predator (the jellyfish Gonionemus vertens), while we added a protozoan grazer (Oxyrrhis marina) as primary consumer to the food chain to establish the four-trophic-level food chain. This setup allowed us to investigate how nutrient-limitation effects change from one trophic level to another, and to investigate the performance of two components of our experimental food chains in different trophic positions. Stoichiometry and fatty acid profiles of the algae showed significant differences between the nutrient-depleted [no N and no P addition (?P), respectively] and the nutrient-replete (f/2) treatments. The differences in stoichiometry could be traced when O. marina was the first consumer. Copepods feeding on these flagellates were not affected by the nutritional imbalance of their prey in their stoichiometry, their respiration rates nor in their developmental rates. In contrast, when copepods were the primary consumer, those reared on the ?P algae showed significantly higher respiration rates along with significantly lower developmental rates. In neither of our two experimental food chains did the signals from the base of the food chains travel up to jelly fish, our top predator.