Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ('lipid rafts') were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3 - mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs

Down regulation of small intestinal ion transport in PDZK1- (CAP70/NHERF3) deficient mice

The PDZ-binding protein PDZK1 (CAP70/PDZ-dc-1/NHERF3) in vitro binds to cystic fibrosis transmembrane conductance regulator (CFTR), the anion exchangers SLC26A3 and SLC26A6 and the Na(+)/H(+) exchanger NHE3, all of which are major transport proteins for intestinal anion secretion and salt absorption. This study was undertaken to search for a role of PDZK1 in regulating electrolyte transport in native murine small intestine. Short circuit current (I (SC)) and HCO-(3) secretory rate (J(HCO-)(3)) were measured to assess electrogenic anion secretion; (22)Na(+) fluxes to assess sodium absorption in isolated small intestine. NHE3, CFTR, as well as NHERF1, NHERF2, and PDZK1 messenger RNA (mRNA) expression levels, and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative polymerase chain reaction (PCR) and Western analysis. NHE3 localization was performed by immunohistochemistry. In pdzk1 -/- jejunal mucosa, basal net Na(+) absorption as well as the inhibition of Na(+) absorption by forskolin was significantly reduced. In pdzk1 -/- duodenal mucosa, identical basal I (SC) and (J(HCO-)(3)) but a significant, yet mild, reduction of forskolin-stimulated Delta(J(HCO-)(3)) and DeltaI (SC) was observed compared to +/+ tissue. Tissue conductance, morphological features, and the DeltaI (SC) and increase in (22)Na(+) absorption in response to luminal glucose was identical in pdzk1 +/+ and -/- small intestine, ruling out a general absorptive defect. While CFTR mRNA expression levels were unchanged, NHE3 mRNA expression levels were significantly increased in small intestinal mucosa of pdzk1 -/- mice. Total enterocyte and BBM abundance was not significantly different, suggesting an increased NHE3 turnover, possibly due to reduced NHE3 membrane retention time. Lack of the PDZ-adapter protein PDZK1 in murine small intestine causes a mild reduction in maximal CFTR activation, but a severe defect in electroneutral Na(+) absorption

Effects of spatial pattern and relatedness in an experimental plant community

Many plant species show limited dispersal resulting in spatial and genetic substructures within populations. Consequently, neighbours are often related between each other, resulting in sibling competition. Using seed families of the annuals Capsella bursa-pastoris and Stachys annua we investigated effects of spatial pattern (i.e. random versus aggregated) on total and individual performance at the level of species and seed families under field conditions. At the level of species, we expected that inferior competitors increase, while superior competitors decrease their performance within neighbourhoods of conspecifics. Thus, we expected a species by spatial pattern interaction. Sibling competition, however, might reduce the performance of competitors, when genetically related, rather than non-related individuals are competing. Therefore, aggregations at the level of seed families could decrease the performance of competitors. Alternatively, if the opposite outcome would be observed, kin selection might be hypothesized to have occurred in the past. Because heavy seeds are expected to disperse less than light seeds, we further hypothesized that kin selection might be more likely to occur in superior competitors with heavy, locally dispersed seeds (e.g. Stachys) compared to inferior competitors with light, more distantly dispersed seeds (e.g. Capsella). We found a significant species by spatial pattern interaction. Indeed, the inferior competitor, Capsella, showed increased reproductive biomass production in aggregated compared to random patterns. Whereas, the performance of the superior competitor, Stachys, was to some extent decreased by intraspecific aggregation. Although statistically not significant, effects of intrafamily aggregations tended to be rather negative in Capsella but positive in Stachys. Our results confirmed that spatial patterns affect growth and reproduction of plant species promoting coexistence in plant communities. Although, we could not provide strong evidence for sibling competition or kin selection, our results suggested that competition among relatives was more severe for Capsella (lighter seeds) compared to Stachys (heavier seeds)