NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation.

Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.

Role of PDZK1 protein in apical membrane expression of renal sodium-coupled phosphate transporters.

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.

Rapid Inflammasome Activation Is Attenuated in Post-Myocardial Infarction Monocytes.

Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation that has not been well defined in the context of acute myocardial infarction (AMI). Rapid transcription-independent inflammasome activation induced by simultaneous TLR priming and triggering stimulus was measured by caspase-1 (CASP1) activity and interleukin release. Both classical and intermediate monocytes from healthy donors exhibited robust CASP1 activation, but only classical monocytes produced high mature interleukin-18 (IL18) release. We also recruited a limited number of coronary artery disease (CAD, n=31) and AMI (n=29) patients to evaluate their inflammasome function and expression profiles. Surprisingly, monocyte subpopulations isolated from blood collected during percutaneous coronary intervention (PCI) from AMI patients presented diminished CASP1 activity and abrogated IL18 release despite increased NLRP3 gene expression. This unexpected attenuated rapid inflammasome activation was accompanied by a significant increase of TNFAIP3 and IRAKM expression. Moreover, TNFAIP3 protein levels of circulating monocytes showed positive correlation with high sensitive troponin T (hsTnT), implying an association between TNFAIP3 upregulation and the severity of tissue injury. We suggest this monocyte attenuation to be a protective phenotype aftermath following a very early inflammatory wave in the ischemic area. Damage-associated molecular patterns (DAMPs) or other signals trigger a transitory negative feedback loop within newly recruited circulating monocytes as a mechanism to reduce post-injury tissue damage.

Differential modulation of the molecular dynamics of the type IIa and IIc sodium phosphate cotransporters by parathyroid hormone.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c.

Hypophosphatemia in vitamin D receptor null mice: effect of rescue diet on the developmental changes in renal Na+ -dependent phosphate cotransporters.

We analyzed vitamin D receptor (VDR) (-/-) mice fed either a normal diet or a rescue diet. Weanling VDR (-/-) mice had hypophosphatemia and hyperphosphaturia. Renal Na(+)-dependent inorganic phosphate (Pi) cotransport activity was significantly decreased in weanling VDR (-/-) mice. In VDR (+/+) mice, renal Npt2a/Npt2c/PiT-2 protein levels were significantly increased at 21 and 28 days of age compared with that at 1 day of age. Npt2c and PiT-2 protein levels were maximally expressed at 28 days of age. Npt2a protein levels were significantly decreased in mice at 28 days of age compared with 21 and 60 days of age. In VDR (-/-) mice, Npt2a/Npt2c/PiT-2 protein levels were considerably lower than those in age-matched VDR (+/+) mice at 21 and 28 days of age. The reduced Npt2a/Npt2c/PiT-2 protein recovered completely in VDR-null mice fed the rescue diet. Although Pi transport activity and Npt2b were reduced in the proximal intestine in VDR (-/-) mice, Npt2b protein levels were not reduced in the distal intestine in VDR (-/-) mice. The rescue diet did not affect intestinal Npt2b protein levels in VDR (-/-) mice. Thus, reduced intestinal Pi absorption in VDR (-/-) mice does not seem to be the only factor that causes hypophosphatemia; reduced Npt2a, Npt2c, or PiT-2 protein levels during development might also cause hypophosphatemia and rickets in VDR (-/-) mice. Furthermore, dietary intervention completely normalized the expression of the renal phosphate transporters (Npt2a/Npt2c/PiT-2) in VDR (-/-) mice, suggesting that the lack of VDR activity is not the cause of impaired renal phosphate reabsorption.

Liver X receptor-activating ligands modulate renal and intestinal sodium-phosphate transporters.

Cholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium-phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels.

Shank2 redistributes with NaPilla during regulated endocytosis.

Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells.

Impact of the Gut Microbiota on Atorvastatin Mediated Effects on Blood Lipids.

BACKGROUND AND AIMS: The mechanisms of interindividual variation of lipid regulation by statins, such as the low-density lipoprotein cholesterol (LDL) lowering effects, are not fully understood yet. Here, we used a gut microbiota depleted mouse model to investigate the relation between the gut microbiota and the regulatory property of atorvastatin on blood lipids. METHODS: Mice (C57BL/6) with intact gut microbiota or antibiotic induced abiotic mice (ABS) were put on standard chow diet (SCD) or high fat diet (HFD) for six weeks. Atorvastatin (10 mg/kg body weight/day) or a control vehicle were applied per gavage for the last four weeks of dietary treatment. Blood lipids including total cholesterol, very low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and sphingolipids were measured to probe microbiota-dependent effects of atorvastatin. The expression of genes involved in hepatic and intestinal cholesterol metabolism was analyzed with qRT-PCR. The alteration of the microbiota profile was examined using 16S rRNA qPCR in mice with intact gut microbiota. RESULTS: HFD feeding significantly increased total blood cholesterol and LDL levels, as compared to SCD in both mice with intact and depleted gut microbiota. The cholesterol lowering effect of atorvastatin was significantly attenuated in mice with depleted gut microbiota. Moreover, we observed a global shift in the abundance of several sphingolipids upon atorvastatin treatment which was absent in gut microbiota depleted mice. The regulatory effect of atorvastatin on the expression of distinct hepatic and intestinal cholesterol-regulating genes, including Ldlr, Srebp2 and Npc1l1 was altered upon depletion of gut microbiota. In response to HFD feeding, the relative abundance of the bacterial phyla Bacteroidetes decreased, while the abundance of Firmicutes increased. The altered ratio between Firmicutes to Bacteroidetes was partly reversed in HFD fed mice treated with atorvastatin. CONCLUSIONS: Our findings support a regulatory impact of atorvastatin on the gut microbial profile and, in turn, demonstrate a crucial role of the gut microbiome for atorvastatin-related effects on blood lipids. These results provide novel insights into potential microbiota-dependent mechanisms of lipid regulation by statins, which may account for variable response to statin treatment.

PTH-induced internalization of apical membrane NaPi2a: role of actin and myosin VI.

Parathyroid hormone (PTH) plays a critical role in the regulation of renal phosphorous homeostasis by altering the levels of the sodium-phosphate cotransporter NaPi2a in the brush border membrane (BBM) of renal proximal tubular cells. While details of the molecular events of PTH-induced internalization of NaPi2a are emerging, the precise events governing NaPi2a removal from brush border microvilli in response to PTH remain to be fully determined. Here we use a novel application of total internal reflection fluorescence microscopy to examine how PTH induces movement of NaPi2a out of brush border microvilli in living cells in real time. We show that a dynamic actin cytoskeleton is required for NaPi2a removal from the BBM in response to PTH. In addition, we demonstrate that a myosin motor that has previously been shown to be coregulated with NaPi2a, myosin VI, is necessary for PTH-induced removal of NaPi2a from BBM microvilli.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate.

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Into the Wild: GWAS Exploration of Non-coding RNAs.

Genome-wide association studies (GWAS) have proven a fundamental tool to identify common variants associated to complex traits, thus contributing to unveil the genetic components of human disease. Besides, the advent of GWAS contributed to expose unexpected findings that urged to redefine the framework of population genetics. First, loci identified by GWAS had small effect sizes and could only explain a fraction of the predicted heritability of the traits under study. Second, the majority of GWAS hits mapped within non-coding regions (such as intergenic or intronic regions) where new functional RNA species (such as lncRNAs or circRNAs) have started to emerge. Bigger cohorts, meta-analysis and technical improvements in genotyping allowed identification of an increased number of genetic variants associated to coronary artery disease (CAD) and cardiometabolic traits. The challenge remains to infer causal mechanisms by which these variants influence cardiovascular disease development. A tendency to assign potential causal variants preferentially to coding genes close to lead variants contributed to disregard the role of non-coding elements. In recent years, in parallel to an increased knowledge of the non-coding genome, new studies started to characterize disease-associated variants located within non-coding RNA regions. The upcoming of databases integrating single-nucleotide polymorphisms (SNPs) and non-coding RNAs together with novel technologies will hopefully facilitate the discovery of causal non-coding variants associated to disease. This review attempts to summarize the current knowledge of genetic variation within non-coding regions with a focus on long non-coding RNAs that have widespread impact in cardiometabolic diseases.

Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells.

Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.

Cytotoxicity of peroxisome proliferator-activated receptor alpha and gamma agonists in renal proximal tubular cell lines.

Fibrates and thiazolidinediones are agonists of peroxisome proliferator-activated receptors (PPAR) alpha and gamma, pharmacologically designed to control dyslipidemia and insulin resistance, respectively. Several works have reported the toxicity of some agonists in a number of tissues. In this work we have analyzed the toxicity of two PPARalpha (WY14643 and clofibrate) and two PPARgamma (pioglitazone and ciglitazone) agonists, using three different renal proximal tubular cell lines: Opossum OK, pig LLC-PK1, and murine MCT. Cell death was determined by the activity of intracellular lactate dehydrogenase. WY14643 and ciglitazone increased cell death with LC50 values of 92-124 microM and 8.6-14.8 microM, respectively, depending on the cell line. Clofibrate and pioglitazone were, however, non-cytotoxic even at concentrations of 10 and 100 higher than the corresponding EC50, which suggests that cell death is independent of PPAR activation. Discrimination between apoptosis or necrosis was analyzed by light microscopy and stress fiber morphology, double staining with acridine orange and ethidium bromide, binding of annexin V, caspase-3 activity, and DNA laddering. With these methods, no signs of apoptosis were observed, which suggests a direct necrosis of the compounds on these renal proximal tubular cell lines.

Anacetrapib, but not evacetrapib, impairs endothelial function in CETP-transgenic mice in spite of marked HDL-C increase.

BACKGROUND AND AIMS: High-density lipoprotein cholesterol (HDL-C) is inversely related to cardiovascular risk. HDL-C raising ester transfer protein (CETP) inhibitors, are novel therapeutics. We studied the effects of CETP inhibitors anacetrapib and evacetrapib on triglycerides, cholesterol and lipoproteins, cholesterol efflux, paraoxonase activity (PON-1), reactive oxygen species (ROS), and endothelial function in E3L and E3L.CETP mice. METHODS: Triglycerides and cholesterol were measured at weeks 5, 14 and 21 in E3L.CETP mice on high cholesterol diet and treated with anacetrapib (3 mg/kg/day), evacetrapib (3 mg/kg/day) or placebo. Cholesterol efflux was assessed ex-vivo in mice treated with CETP inhibitors for 3 weeks on a normal chow diet. Endothelial function was analyzed at week 21 in isolated aortic rings, and serum lipoproteins assessed by fast-performance liquid chromatography. RESULTS: Anacetrapib and evacetrapib increased HDL-C levels (5- and 3.4-fold, resp.) and reduced triglycerides (-39% vs. placebo, p = 0.0174). Total cholesterol levels were reduced only in anacetrapib-treated mice (-32%, p = 0.0386). Cholesterol efflux and PON-1 activity (+45% and +35% vs. control, p < 0.005, resp.) were increased, while aortic ROS production was reduced with evacetrapib (-49% vs. control, p = 0.020). Anacetrapib, but not evacetrapib, impaired endothelium dependent vasorelaxation (p < 0.05). In contrast, no such effects were observed in E3L mice for all parameters tested. CONCLUSIONS: Notwithstanding a marked rise in HDL-C, evacetrapib did not improve endothelial function, while anacetrapib impaired it, suggesting that CETP inhibition does not provide vascular protection. Anacetrapib exerts unfavorable endothelial effects beyond CETP inhibition, which may explain the neutral results of large clinical trials in spite of increased HDL-C.

MicroRNAs in lipid metabolism and atherosclerosis.

Numerous studies have examined the role of microRNAs (miRNAs) in cell homeostasis and cardiovascular disease and have markedly improved our understanding of RNA biology in general and the potential role of miRNAs in atherosclerosis. In atherosclerosis, several miRNAs, such as miR-33a,b, miR-92a, miR-126 and others, have been identified that are relevant mediators of pathological processes, including regulation of cholesterol and lipid biosynthesis, lipoprotein metabolism and cholesterol efflux, but also immune responses, endothelial cell biology and vascular function. Further understanding of the specific roles of miRNAs in the distinct cell types involved in atherosclerosis initiation, progression and resolution may reveal new intervention strategies for the prevention and treatment of atherosclerotic cardiovascular disease.

High-density lipoproteins as modulators of endothelial cell functions: alterations in patients with coronary artery disease.

Alteration of endothelial cell functions, including reduced endothelial nitric oxide (NO) availability, increased endothelial cell apoptosis, adhesion molecule/chemokine expression and pro-thrombotic activation are thought to contribute to the pathophysiology of atherosclerosis and coronary-artery-disease (CAD) with its clinical complications, such as acute coronary syndromes. High-density lipoproteins (HDL) from healthy subjects or reconstituted HDL have been observed to exert potential direct anti-atherogenic effects by modulating these endothelial cell functions. Importantly, endothelial effects of HDL have now been reported to be highly heterogeneous, and are modulated as part of immune responses. More recently, this has also been observed for HDL of patients with CAD, where HDL becomes potentially pro-inflammatory and endothelial-protective properties are markedly altered. Several mechanisms may lead to these altered endothelial effects of HDL in patients with CAD, including oxidative modification of HDL-associated lipids and proteins, such as apoA-I and paraoxonase-1, and alterations of HDL-proteome. These findings have to be considered with respect to interpretation of recent clinical studies failing to demonstrate reduced cardiovascular events by HDL-cholesterol raising strategies in patients with CAD. Both clinical and genetic studies suggest that HDL-cholesterol levels alone are not a sufficient therapeutic target in patients with CAD. The focus of this review is to summarize the role of HDL onto endothelial homeostasis and to describe recently characterized molecular pathways involved. We highlight how structural and functional modifications of HDL particles in patients with CAD may perturb the physiological homeostasis and lead to a loss of endothelial-protective properties of HDL in patients with CAD.

Nuclear receptor LXR: a new partner for sodium-dependent phosphate cotransporters.

New pharmaceutical research approaches are focusing on trying to alleviate the perturbed phosphate (Pi) homeostasis associated with the onset of chronic kidney disease; this includes activation of some of the nuclear receptors. We have recently reported the down regulation of the intestinal and renal sodium-phosphate (NaPi) cotransporters by the liver X receptor (LXR) agonists, and the consequent decrease of the serum Pi levels. In this review, we describe our current knowledge of the different proteins involved in the renal and intestinal actions of LXR.

Myosin VI is required for maintenance of brush border structure, composition, and membrane trafficking functions in the intestinal epithelial cell.

Characterization of the intestinal epithelium of the Snell's waltzer (sv/sv) mouse revealed that myosin VI (Myo6) is required for proper brush border (BB) ultrastructure, composition and membrane traffic. The defects observed were distinct from that observed in the myosin Ia KO, even though Myo6 is lost from the BB in this KO. Myo6 is expressed throughout the length of the small and large intestine; it is localized to the subapical inter-microvillar (MV) domain and basolateral membrane. Defects in the BB include apparent lifting of the plasma membrane off of the actin cytoskeleton in the inter-MV region, fusion of MV, and disorganized morphology of the terminal web. The molecular composition of the sv/sv BB is altered. This includes increased expression of myosin Va, myosin Ie and the MV actin binding proteins espin and phosphorylated-ezrin; myosin Id is reduced. Changes in endocytic components include reduced clathrin and adaptin ß, and increased disabled-2. Endocytic uptake of lumenal lactoferrin is inhibited in adult, but not neonatal intestinal epithelial cells. There is increased BB membrane-associated expression of both the Na(+)/H(+) exchanger, NHE3 and the Na(+)/phosphate transporter, NaPi2b. These results suggest that Myo6 is involved in the regulated trafficking of NHE3 and NaPi2b between the BB membrane and endosome.

Nanometer-scale imaging by the modulation tracking method.

We developed an optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample. The feedback approach produces nanometer-resolved 3D images of very small and moving features in live cells in seconds. We show images of microvilli in live cultured opossum kidney cells expressing NaPi co-transporter proteins with different GFP constructs and images of cell protrusions in a collagen matrix with a resolution of about 20 nm. We found that in the microvilli the NaPi proteins can be found clustered. Along cell protrusions in 3D we identified cellular adhesions to the extracellular matrix. Our approach to super-resolution and to 3D nanoimaging is different than other proposed methods that break the diffraction limit using non-linear effects or are based on single molecule localization.