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1.  Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells 
Molecular Biology of the Cell  2009;20(8):2337-2350.
The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.
PMCID: PMC2669039  PMID: 19244346
2.  HPV16 and BPV1 Infection Can Be Blocked by the Dynamin Inhibitor Dynasore 
American journal of therapeutics  2008;15(4):304-311.
The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin’s contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin’s function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.
PMCID: PMC2519006  PMID: 18645330
dynasore; papillomavirus endocytosis; dynamin inhibitor; viral infection; papillomavirus antiviral; dynamin-mediated endocytosis
3.  Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells 
Molecular Biology of the Cell  2007;18(10):3978-3992.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
PMCID: PMC1995710  PMID: 17686995
4.  Endocytic Trafficking of CFTR in Health and Disease 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the debilitating genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only into the patho-physiology of cystic fibrosis, but also provides potential drug targets to help cure this debilitating disease.
PMCID: PMC1964799  PMID: 17098482

Results 1-5 (5)