Faculty Bibliography

AIMS: It is well-known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant "Cx43D378stop") led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: a) the morphology of clusters formed by the microtubule plus-end tracking protein "end binding 1" (EB1), b) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin rich sites, c) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. METHODS AND RESULTS: Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa and microtubules. CONCLUSIONS: NaV1.5 and EB1 localization at cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.

BACKGROUND: The cardiac intercalated disc (ID) has been extensively studied by conventional transmission electron microscopy (EM). Yet, novel methods for tissue preservation (high-pressure freezing), image (3D tomographic EM) and analysis (image segmentation) that greatly improve image quality/resolution, have not been applied to the ID. Recent studies show that, at the ID, the gap junction protein Connexin43 is part of an interactome (a "connexome"). Here, we provide a structural characterization of the connexome. METHODS: Adult mouse ventricular tissue was prepared by high-pressure freezing and freeze substitution and embedded in resin. 200 nm thick sections were imaged with a 200kV electron microscope (FEI TF20). Images were collected at a set magnification of 9.6k on a 4kx4k CCD camera set to 2x binning, giving an effective pixel size of 1.76 nm. Dual-axis tilt series (1 masculine steps, +/-70 masculine per axis) were acquired using SerialEM. Protomo software was used for aligning projection images and reconstructing tomograms. Visualization/segmentation of objects of interest was performed in Amira. RESULTS: In addition to classic ID structures, we observed (a) close proximity between gap junctions and mitochondria of opposing cells; (b) a complex network of tubular structures running perpendicular to the long cell axis; these structures showed a hollow interior, with an estimated inner diameter of ~40 nm and were often adjacent to gap junctions or desmosomes; (c) triads formed by lateral edges of gap junctions and desmosomes, with a rough budding vesicle separating the two structures; (d) budding vesicles of approximately 50 nm interrupting the continuity of one side of the gap junction plaque; (e) vesicular bodies of approx. 65 nm in diameter in the intercellular space, and in proximity to gap junction-containing regions. CONCLUSIONS: We describe the nanometric landscape that surrounds gap junctions. We speculate that the connexome includes a physical association with molecules of the mitochondria, desmosome and microtubular network, and propose that microsomes may pass from one cell to another at the ID. Functional characterization of these structures may lead to novel clues as to the mechanisms of inherited or acquired arrhythmias that involve disruption of the ID.

The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the presence of the proteasomal inhibitor MG132. The present data complete the remarkable insight that the structural integrity of three closely juxtaposed cytoplasmic organelles - Golgi apparatus, endoplasmic reticulum and mitochondria - is dependent on nitric oxide.

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila beta-catenin) expression and activity by directly targeting the 3'-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.

AIMS: The shRNA-mediated loss of expression of the desmosomal protein plakophilin-2 leads to sodium current (I(Na)) dysfunction. Whether pkp2 gene haploinsufficiency leads to I(Na) deficit in vivo remains undefined. Mutations in pkp2 are detected in arrhythmogenic right ventricular cardiomyopathy (ARVC). Ventricular fibrillation and sudden death often occur in the 'concealed phase' of the disease, prior to overt structural damage. The mechanisms responsible for these arrhythmias remain poorly understood. We sought to characterize the morphology, histology, and ultrastructural features of PKP2-heterozygous-null (PKP2-Hz) murine hearts and explore the relation between PKP2 abundance, I(Na) function, and cardiac electrical synchrony. METHODS AND RESULTS: Hearts of PKP2-Hz mice were characterized by multiple methods. We observed ultrastructural but not histological or gross anatomical differences in PKP2-Hz hearts compared with wild-type (WT) littermates. Yet, in myocytes, decreased amplitude and a shift in gating and kinetics of I(Na) were observed. To further unmask I(Na) deficiency, we exposed myocytes, Langendorff-perfused hearts, and anaesthetized animals to a pharmacological challenge (flecainide). In PKP2-Hz hearts, the extent of flecainide-induced I(Na) block, impaired ventricular conduction, and altered electrocardiographic parameters were larger than controls. Flecainide provoked ventricular arrhythmias and death in PKP2-Hz animals, but not in the WT. CONCLUSIONS: PKP2 haploinsufficiency leads to I(Na) deficit in murine hearts. Our data support the notion of a cross-talk between desmosome and sodium channel complex. They also suggest that I(Na) dysfunction may contribute to generation and/or maintenance of arrhythmias in PKP2-deficient hearts. Whether pharmacological challenges could help unveil arrhythmia risk in patients with mutations or variants in PKP2 remains undefined.

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high beta-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid beta (sAbeta) peptide is converted into highly toxic oligomeric Abeta and fibrillar Abeta that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAbeta and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Abeta or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Abeta in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Abeta40/42 levels, as well as reduced Abeta oligomer levels. This type of immunomodulation has the potential to be a universal beta-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases

Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis