Roles of phospholipase D isoforms and phosphatidylinositol kinases in Golgi structure and function

Date

2004

Authors

Freyberg, Zachary Zisha

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid (PA), a molecule known to have multiple physiological roles including involvement in release of nascent secretory vesicles from the trans -Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. However, due to its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. I have used highly sensitive antibodies to PLD1 and PLD2 together with immunofluorescence, immunogold electron microscopy as well as cell fractionation to identify the intracellular localizations of the endogenous PLD isoforms in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched in the Golgi apparatus and also present in cell nuclei. Similarly, a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and dense cytoplasmic puncta; a fraction of which co-localized with caveolin-1 and the plasma membrane. Most significantly, cryo-immunogold electron microscopy demonstrated that in pituitary GH3 cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively, consistent with a model where PLD2 plays a role in Golgi vesicular transport.;The phospholipid phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5)P 2] not only stimulates PLD activity, but mediates rearrangement of the cortical actin cytoskeleton and membrane ruffling to drive cell motility. Following phorbol ester stimulation, both PLD1 and PLD2 translocated into plasma membrane ruffles. Importantly, the phosphoinositide kinases, phosphatidylinositol 4-kinase type IIIalpha and -beta (PI4KIIIalpha/beta) as well as phosphatidylinositol(4)P 5-kinase Ialpha and -beta (PIP5KIalpha/beta) co-localized with both PLD isoforms and PtdIns(4,5)P2 at sites of ruffling.;Microinjection of an anti-PI4KIIIalpha inhibitory antibody resulted in significant Golgi fragmentation, while inhibition of PI4KIIIbeta led to only subtle dilation of Golgi cisternae. Surprisingly, PI4KIIIalpha inhibition caused a dramatic dilation of the endoplasmic reticulum (ER), an organelle not previously implicated in PI4K function. Biochemical assays of PI4K activity confirmed an ER and Golgi localization of the PI4Ks suggesting a novel role for the PI4Ks in ER-Golgi traffic.

Description

Keywords

Cellular biology., Molecular biology.

Citation

Source: Dissertation Abstracts International, Volume: 64-09, Section: B, page: 4150.;Advisors: Dennis Shields.