Cells interact with their environment at the plasma membrane (PM)*
and respond to stimuli by rearranging their surfaces through modification of cell junctions, cell polarity, and the organization of the cortical actin cytoskeleton. Although signaling pathways and actin dynamics have well-documented roles in these responses, the contribution of membrane traffic to these processes is less clear. Nonetheless, evidence is accumulating that the insertion and uptake of membrane at the PM is important in regulating PM morphology (Mellman, 2000
). Requirements for membrane traffic in neutrophil migration (Lawson and Maxfield, 1995
) and phagocytosis (Bajno et al., 2000
) have been demonstrated. Similarly, GTPases involved in the regulation of membrane traffic, such as Rab5 (Spaargaren and Bos, 1999
), Rab8 (Peranen et al., 1996
), and ADP-ribosylation factor (Arf) 6 (Radhakrishna et al., 1996
) have been shown to influence cortical actin structure at the PM, lending support to the concept that membrane trafficking can influence the composition and structure of the plasma membrane.
We have been studying the Arf6 GTPase in cells to elucidate the relationship between Arf6-regulated membrane traffic and cell morphology at the PM. In HeLa and many other cells, Arf6 regulates the movement of membrane between the PM and a nonclathrin-derived endosomal compartment (Radhakrishna and Donaldson, 1997
). PM proteins and lipids that are internalized by this endocytic pathway can be recycled back to the PM. This membrane recycling requires activation of Arf6 and the presence of actin filaments (Radhakrishna and Donaldson, 1997
). Activation of Arf6 has also been implicated in regulated exocytosis (Galas et al., 1997
), suggesting that recycling of plasma membrane and regulated exocytosis may be related or share some properties.
Several investigations have shown that Arf6-dependent regulation of membrane recycling influences the actin cytoskeleton along the PM. Expression of a dominant negative mutant of Arf6, T27N, that inhibits membrane recycling inhibits cell spreading (Song et al., 1998
), Rac-mediated ruffling (Radhakrishna et al., 1999
), and Fc-mediated phagocytosis (Zhang et al., 1998
). Acute activation of Arf6 by either treatment of cells with aluminum fluoride (AlF) (Radhakrishna et al., 1996
) or coexpression of guanine nucleotide exchange factors (GEFs) for Arf6, ARNO (Frank et al., 1998
), or EFA6 (Franco et al., 1999
) results in ruffling protrusive structures along the PM. Although expression of the constitutively active Arf6 mutant, Q67L, also generates protrusive structures and alterations in cortical actin (Radhakrishna et al., 1996
), a variety of changes in cell morphology is observed, and the phenotype has not been explored thoroughly. Some studies investigating the morphology of cells expressing Arf6 Q67L report extensive invaginations of the PM, often tortuous and complex (Peters et al., 1995
; D'Souza-Schorey et al., 1998
). In another recent report, constitutively active Arf6 was shown to decrease stress fiber formation, suggesting antagonism of Rho function (Boshans et al., 2000
). The variable morphology of cells expressing Arf6 Q67L can be attributed to the consequence of persistent activation of Arf6 effectors over extended periods during transient transfection. Understanding how activated Arf6 causes these morphological changes would clarify the normal functions of Arf6 in cells.
An important advance in understanding Arf function was the recent demonstration that members of the Arf family can activate type I phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that catalyses the formation of phosphatidylinositol 4,5-bisphosphate (PIP2
) from phosphatidylinositol 4-phosphate (Honda et al., 1999
; Godi et al., 1999
; Jones et al., 2000
). Synthesis and turnover of PIP2
have been implicated in a variety of cellular events including membrane trafficking (Martin, 1997
), control of actin polymerization (Janmey, 1994
; Yamamoto et al., 2001
), regulation of ion channel transporters (Kobrinsky et al., 2000
), and signal transduction where it serves as a substrate for enzymes such as PLC and phosphatidylinositol 3-kinase (Czech, 2000
). This pleiotropic role of PIP2
illustrates the importance of understanding where in the cell PIP2
is synthesized and how this process is regulated spatially. Although all Arfs have been shown to stimulate PIP 5-kinase activity in vitro, in cells it is Arf6 that colocalizes with PIP 5-kinase at the PM (Honda et al., 1999
). Protrusions and ruffles generated by activation of Arf6 result in the recruitment of PIP 5-kinase and PLD2 into the PM structures (Honda et al., 1999
), suggesting that Arf6 might function in cells through activation of PIP 5-kinase. However, no data has been published showing the distribution of these proteins and their lipid product, PIP2
, in this membrane trafficking pathway.
Here, we have investigated the effects of Arf6 activation and PIP 5-kinase on the distribution and local formation of PIP2 in order to better understand the role of this lipid in the regulation of membrane traffic through the Arf6 pathway. We find that the Arf6 PM-endosomal recycling pathway can serve as a mechanism to regulate the availability and activity of PIP 5-kinase and therefore PIP2 levels at the PM. We show that activation of Arf6 by expression of its exchange factor, EFA6, allows for uptake and recycling of membrane through this pathway, whereas expression of constitutively active Arf6Q67L or PIP 5-kinase alters the trafficking of membrane such that PM proteins enter the cell and become trapped in PIP2-positive actin-coated vacuolar structures. These results establish that Arf6-dependent regulation of PIP 5-kinase and PIP2 levels and their localization is crucial for proper regulation of trafficking through the Arf6 PM-endosomal recycling pathway.