|Distribution and function of polycystin-2 in mouse retinal ganglion cells.|
S Kaja,O A Mafe,R A Parikh,P Kandula,C A Reddy,E V Gregg,H Xin,P Mitchell,M A Grillo,P Koulen
The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.
|The centrosomal protein pericentrin identified at the basal body complex of the connecting cilium in mouse photoreceptors.|
Mühlhans, J; Brandstätter, JH; Giessl, A
Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known.We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium.Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene.
|Polycystin-2 expression and function in adult mouse lacrimal acinar cells.|
Kaja, S; Hilgenberg, JD; Rybalchenko, V; Medina-Ortiz, WE; Gregg, EV; Koulen, P
Investigative ophthalmology & visual science
Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca(2+) homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca(2+) concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca(2+) release channels, transient receptor potential polycystin family (TRPP) channels.Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca(2+) channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system.All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues.The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology.
|The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.|
Tran U, Zakin L, Schweickert A, Agrawal R, Döger R, Blum M, De Robertis EM, Wessely O
The RNA-binding protein Bicaudal C is an important regulator of embryonic development in C. elegans, Drosophila and Xenopus. In mouse, bicaudal C (Bicc1) mutants are characterized by the formation of fluid-filled cysts in the kidney and by expansion of epithelial ducts in liver and pancreas. This phenotype is reminiscent of human forms of polycystic kidney disease (PKD). Here, we now provide data that Bicc1 functions by modulating the expression of polycystin 2 (Pkd2), a member of the transient receptor potential (TRP) superfamily. Molecular analyses demonstrate that Bicc1 acts as a post-transcriptional regulator upstream of Pkd2. It regulates the stability of Pkd2 mRNA and its translation efficiency. Bicc1 antagonized the repressive activity of the miR-17 microRNA family on the 3\'UTR of Pkd2 mRNA. This was substantiated in Xenopus, in which the pronephric defects of bicc1 knockdowns were rescued by reducing miR-17 activity. At the cellular level, Bicc1 protein is localized to cytoplasmic foci that are positive for the P-body markers GW182 and HEDLs. Based on these data, we propose that the kidney phenotype in Bicc1(-/-) mutant mice is caused by dysregulation of a microRNA-based translational control mechanism.Full Text Article