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      Role of Isoprenoids in Cytoskeleton Integrity and Albumin Endocytosis by Opossum Kidney Cells

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          Background: The actin cytoskeleton has been increasingly implicated in endocytic events that are involved in the reabsorption of filtered protein by the proximal tubule. Isoprenylated small G proteins have emerged as key regulators of the actin cytoskeleton. This study examines the role of isoprenoid intermediates in organization of the cytoskeleton, and the effect of modification of the cytoskeleton on albumin endocytosis. Methods: The effect of lovastatin on cytoskeleton morphology of opossum kidney cells (OK cells) was determined by staining with fluorescent isothiocyanate (FITC)-phalloidin followed by examination using confocal microscopy. Quantitative effects on albumin binding and uptake were determined using a well-established method. Results: Inhibition of isoprenoid synthesis by lovastatin led to morphological disruption of the cytoskeleton with a concentration-dependent decrease in albumin uptake into OK cells. Addition of mevalonate, but not cholesterol, ameliorated the effects of lovastatin on the cytoskeleton and albumin uptake. Selective inhibition of isoprenoid intermediates with a farnesyltransferase inhibitor suggests that geranylgeranylated proteins mediate cytoskeleton organization. Conclusion: These results confirm the importance of cytoskeleton integrity in albumin endocytosis in renal proximal tubules. While the present data suggest that synthesis of isoprenoids via the mevalonate pathway is a critical step in maintenance of the cytoskeleton, the role of individual small G proteins in control of the cytoskeleton and other endocytic events is yet to be defined.

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          Most cited references 9

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          Protein prenyltransferases.

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            Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

            Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.
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              Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells.

              Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. Elimination of Cdk inhibitor protein p27Kip1 during the G1 to S phase is required for the activation process. An inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase prevents its elimination and leads to G1 arrest. Mevalonate and its metabolite, geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, restore the inhibitory effect of pravastatin on the degradation of p27 and allow Cdk2 activation. By the addition of geranylgeranyl pyrophosphate, Rho small GTPase(s) are geranylgeranylated and translocated to membranes during G1/S progression. The restoring effect of geranylgeranyl pyrophosphate is abolished with botulinum C3 exoenzyme, which specifically inactivates Rho. These results indicate (i) among mevalonate metabolites, geranylgeranyl pyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation; (ii) geranylgeranylated Rho small GTPase(s) promote the degradation of p27 during G1/S transition in FRTL-5 cells.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                July 2004
                17 November 2004
                : 97
                : 3
                : e77-e85
                aVictorian Paediatric Renal Service, Royal Children’s Hospital, and bDepartment of Physiology, University of Melbourne, Parkville, Vic., Australia
                78641 Nephron Exp Nephrol 2004;97:e77–e85
                © 2004 S. Karger AG, Basel

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                Page count
                Figures: 7, Tables: 1, References: 30, Pages: 1
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                Original Paper

                Cardiovascular Medicine, Nephrology

                Isoprenoids, Endocytosis, Proteinuria, Cytoskeleton


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