Phosphatidylinositol 5-Kinase Stimulates Apical Biosynthetic Delivery via an Arp2/3-dependent Mechanism

Abstract

The mechanisms by which polarized epithelial cells target distinct carriers enriched in newly synthesized proteins to the apical or basolateral membrane remain largely unknown. Here we investigated the effect of phosphatidylinositol metabolism and modulation of the actin cytoskeleton, two regulatory mechanisms that have individually been suggested to function in biosynthetic traffic, on polarized traffic in Madin-Darby canine kidney cells. Overexpression of phosphatidylinositol 5-kinase (PI5K) increased actin comet frequency in Madin-Darby canine kidney cells and concomitantly stimulated trans-Golgi network (TGN) to apical membrane delivery of the raft-associated protein influenza hemagglutinin (HA), but did not affect delivery of a non-raft-associated apical protein or a basolateral marker. Modulation of actin comet formation by pharmacologic means, by overexpression of the TGN-localized inositol polyphosphate 5-phosphatase Ocrl, or by blockade of Arp2/3 function had parallel effects on the rate of apical delivery of HA. Moreover, HA released from a TGN block was colocalized in transport carriers in association with PI5K and actin comets. Inhibition of Arp2/3 function in combination with microtubule depolymerization led to a virtual block in HA delivery, suggesting synergistic coordination of these cytoskeletal assemblies in membrane transport. Our results suggest a previously unidentified role for actin comet-mediated propulsion in the biosynthetic delivery of a subset of apical proteins.

Phosphoinositides in Cell Architecture

“…It might therefore be more accurate to think of PTEN function as simply preventing PtdIns(3,4,5)P 3 accumulation at the apical surface than as a major source of PtdIns(4,5)P 2 . Consistent with this idea is the observation that phosphatidylinositol 5-kinase (PI(5)-kinase), a kinase known to stimulate PtdIns(4,5)P 2 production, localized to the apical region of MDCK cells and increased biosynthetic delivery of apical proteins (Guerriero et al 2006). There are likely to be additional mechanisms to generate and amplify apical PtdIns(4,5)P 2 that await discovery.…”

Apical trafficking in epithelial cells: signals, clusters and motors

“…that stimulate formation of N-WASP-Arp2/3-dependent actin comets and slowed by inhibitors of comet formation (Guerriero et al, 2006) (Fig. 1, route 1).…”

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…Studies in polarized epithelial cells (MDCK) have shown that the Rho GTPase cdc42, an actin cytoskeleton organizer found at the Golgi (Erickson et al, 1996;Kroschewski et al, 1999;Musch et al, 2001), actindepolymerizing drugs (Lazaro-Dieguez et al, 2007), and Arp 2/3 (Rozelle et al, 2000;Guerriero et al, 2006) affect differentially the exit of apical and basolateral proteins from the TGN. In none of these cases, however, the specific roles of This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -08 -0891) on November 5, 2008. the actin cytoskeleton in cargo protein exit have been documented.…”