Article

Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton

Department of Physiology, University of Alberta, Edmonton, Canada.
FEBS Journal (Impact Factor: 3.99). 09/2008; 275(19):4675-83. DOI: 10.1111/j.1742-4658.2008.06616.x
Source: PubMed

ABSTRACT TRPP2, also called polycystin-2, the gene product of PKD2, is a membrane protein defective in 10-15% of cases of autosomal dominant polycystic kidney disease. Mutations in PKD2 are also associated with extrarenal disorders, such as hepatic cystogenesis and cardiovascular abnormalities. TRPP2 is a Ca-permeable nonselective cation channel present in the endoplasmic reticulum and plasma membrane, as well as in cilia of renal epithelial and embryonic nodal cells, in which it likely forms part of a flow sensor. Recent studies have identified a number of TRPP2-interacting proteins, of which many are cytoskeletal components. Work from our and other laboratories indicates that cytoskeletal partner proteins seem to play important, albeit highly complex, roles in the regulation of TRPP2 expression, localization and channel function. This minireview covers current knowledge about cytoskeletal interactions with TRPP2, and suggests that mutations in proteins of the TRPP2-cytoskeleton complex may be implicated in the pathogenesis of autosomal dominant polycystic kidney disease.

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    ABSTRACT: Mutations in the PKD2 gene cause autosomal dominant polycystic kidney disease (ADPKD), a common, inherited disease that frequently leads to end-stage renal disease (ESRD). Swine show substantial similarity to humans physiologically and anatomically, and are therefore a good model system in which to decipher the structure and function of the PKD2 gene and to identify potential therapeutic targets. Here we report the cloning and characterization of the porcine PKD2 cDNA showing that the full-length gene (3370 bases) is highly expressed in kidney, with minimal expression in the liver. RNA interference (RNAi) is a promising tool to enable identification of the essential components necessary for exploitation of the pathway involved in cellular processes. We therefore designed four shRNAs and nine siRNAs targeting the region of the porcine PKD2 gene from exons 3 to 9, which is supposed to be a critical region contributing to the severity of ADPKD. The results from HeLa cells with the dual-luciferase reporter system and porcine kidney cells (LLC-PK1) showed that sh12 could efficiently knock down the PKD2 gene with an efficiency of 51% and P1 and P2 were the most effective siRNAs inhibiting 85% and 77% respectively of PKD2 expression compared with untreated controls. A subsequent functional study of the transient receptor potential polycystic (TRPP) 2 channel protein indicated that the decreased expression of TRPP2 induced by siRNA P1 and P2 could release the arrest of the cell cycle from G0/G1 promoting progression to S and G2 phases. Our data, therefore, provides evidence of potential knock-down target sites in the PKD2 gene and paves the way for the future generation of transgenic ADPKD knock-down animal models.
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    ABSTRACT: Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.
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    ABSTRACT: The endoplasmic reticulum (ER) and the Golgi apparatus are organelles that produce, modify and transport proteins and lipids and regulate Ca2+ environment within cells. Structurally they are composed of sheets and tubules. Sheets may take various forms: intact, fenestrated, single or stacked. The ER, including the nuclear envelope, is a single continuous network, while the Golgi shows only some level of connectivity. It is often unclear, how different morphologies correspond to particular functions. Previous studies indicate that the structures of the ER and Golgi are dynamic and regulated by fusion and fission events, cytoskeleton, rate of protein synthesis and secretion, and specific structural proteins. For example, many structural proteins shaping tubular ER have been identified, but sheet formation is much more unclear. In this study, we used light and electron microscopy to study morphological changes of the ER and Golgi in mammalian cells. The proportion, type, location and dynamics of ER sheets and tubules were found to vary in a cell type or cell cycle stage dependent manner. During interphase, ER and Golgi structures were demonstrated to be regulated by p37, a cofactor of the fusion factor p97, and microtubules, which also affected the localization of the organelles. Like previously shown for the Golgi, the ER displayed a tendency for fenestration and tubulation during mitosis. However, this shape change did not result in ER fragmentation as happens to Golgi, but a continuous network was retained. The activity of p97/p37 was found to be important for the reassembly of both organelles after mitosis. In EM images, ER sheet membranes appear rough, since they contain attached ribosomes, whereas tubular membranes appear smooth. Our studies revealed that structural changes of the ER towards fenestrated and tubular direction correlate with loss of ER-bound ribosomes and vice versa. High and low curvature ER membranes have a low and high density of ribosomes, respectively. To conclude, both ER and Golgi architecture depend on fusion activity of p97/p37. ER morphogenesis, particularly of the sheet shape, is intimately linked to the density of membrane bound ribosomes. Endoplasmakalvosto (ER) ja Golgi ovat organelleja, jotka tuottavat, muokkaavat ja kuljettavat proteiineja ja lipidejä sekä säätelevät kalsiumin oikeaa pitoisuutta solun sisällä. Ne koostuvat putkimaisista ja laattarakenteista, jotka voivat olla ehjiä, reikiintyneitä, yksittäisiä tai pinoutuneita. ER, johon kuuluu myös tumakalvo, on yksi yhtenäinen verkosto, kun taas Golgissa on erillisiä alaosastoja ja siis vähemmän verkostoitumista. Erilaisten rakenteiden ajatellaan tukevan erityisiä toimintoja. Useimpien rakenteiden kohdalla on kuitenkin epäselvää, mitkä toiminnot niissä sijaitsevat tai miten rakenne palvelee ko. toimintoa. Aiemmissa tutkimuksissa on todettu, että ER ja Golgi ovat dynaamisia ja niiden rakennetta muovaavat erilaiset kalvoston fuusio- ja fissiotapahtumat, solun tukiranka, proteiinituotanto ja eritys sekä rakenneproteiinit. Esimerkiksi monta putkia muodostavaa ER:n proteiinia on jo tunnistettu, mutta laattarakenteiden syntymekanismi on paljon epäselvempi. Tässä väitöskirjassa on käytetty valo- ja elektronimikroskopiaa ER:n ja Golgin rakenteiden tutkimiseen nisäkässoluissa. Eri rakenteiden määrän, laadun, paikan ja dynamiikan huomattiin vaihtelevan solutyypin mukaan. Löysimme myös näihin rakenteisiin vaikuttavan tekijän, p37:n, joka säätelee tunnettua fuusiotekijää, p97:ää. Solunjakautumisen aikana Golgi hajoaa reikiintymisen ja putkiverkoston muodostumisen kautta pieniksi rakkuloiksi ja putkiksi. Meidän tuloksemme osoittavat, että ER:ssä tapahtuu samantapaisia muodonmuutoksia, mutta ER ei hajoa palasiksi. Myös näiden muutosten laajuus vaihtelee solutyypin mukaan. P97/p37 osallistuu molempien organellien rakenteen palauttamiseen solunjakautumisen jälkeen. Elektronimikroskooppikuvissa ER:n laattarakenteet näyttävät yleensä karkealta, koska niihin on kiinnittynyt ribosomeja, jotka valmistavat proteiineja. Tutkimalla useita eri soluja, solusyklin vaiheita ja ER:n rakenteita, me havaitsimme, että ribosomien määrän väheneminen korreloi ER:n reikiintymisen tai putkiston muodostuksen tai yleensä kalvon kaarevuuden lisääntymisen kanssa. Tästä voidaan päätellä, että laattamainen ER on erikoistunut ribosomien toimintaan, josta sen muoto myös on riippuvainen.

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