Biomedical subjects

Sophia Y Breusegem

Publications and source records attributed to Sophia Y Breusegem.

6 recordsLinked to original sources

PubMed2006

Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH(2) end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

Animals↗

PubMed2006

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy.

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Forster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment.

Animals↗

PubMed2005

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells.

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells.

We previously showed an inverse correlation between membrane cholesterol content and Na-P(i) cotransport activity during the aging process and adaptation to alterations in dietary P(i) in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P(i) cotransport activity and apical membrane Na-P(i) protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with beta-methyl cyclodextrin (beta-MCD) resulted in a significant increase in Na-P(i) cotransport activity accompanied by a moderate increase in apical membrane Na-P(i) protein abundance and no alteration of total cellular Na-P(i) protein abundance. Conversely, acute cholesterol enrichment achieved with beta-MCD/cholesterol resulted in a significant decrease in Na-P(i) cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P(i) protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-P(i) cotransport activity by diverse mechanisms that also include significant interactions of Na-P(i) protein with lipid microdomains.

Animals↗

PubMed2004

Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

Animals↗

PubMed2002

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.

AT Rich Sequence↗