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Role of Phospholipase D in Actin Cytoskeletal Reorganization of Epithelium Cells
Author(s) -
Pastore Mayra,
Frohman Michael,
Du Guangwei
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1373-d
Subject(s) - pld2 , phospholipase d , microbiology and biotechnology , cytoskeleton , small hairpin rna , actin cytoskeleton , biology , actin , downregulation and upregulation , transfection , phosphatidic acid , cell , signal transduction , chemistry , cell culture , biochemistry , gene knockdown , phospholipid , genetics , membrane , gene
Phospholipase D (PLD) is involved in many intra/extra‐cellular signaling transduction processes. The signaling relay is achieved through the production phosphatidic acid (PA) and choline from phosphodiester hydrolysis of phosphatidylcholine. The PLD isoforms, PLD1 and PLD2, has been reported to be involved in epithelium cell tumorogenesis through cytoskeletal rearrangement, but the mechanism remains unclear. Our project investigates the potential role and mechanism that PLD has over actin cytoskeletal rearrangement during tumor progression. The function of PLD in NMuMG epithelium cells was explored through overexpression and downregulation of the individual isoforms. First, we examined the subcellular localization using EGFP‐tagged‐PLD expression constructs, then assayed changes in cell‐cell tight junctions due to overexpression. To downregulate PLD expression, we adapted short hairpin RNA (shRNA) expressed as a fusion with the human microRNA‐30 (mir‐30) precursors driven by a CMV promoter. To date, several constructs expressing PLD1 or PLD2 shRNA have been assayed with Western Blotting after transient cell transfection. Our results revealed that PLD1 localizes to the perinuclear region and PLD2 to the plasma membrane, in par with previous reports. Analyzing Western Blotting, we concluded that shRNAs successfully downregulated the protein's overexpression. However, there is not enough evidence to conclude PLD overexpression alters actin cytoskeleton, although, redistribution of actin units from plasma membrane to the nuclear region was observed Funded by the NSF SUNY AGEP Project, Award No. 35583