Heterodimeric Capping Protein fromArabidopsisIs Regulated by Phosphatidic Acid

Abstract

The cytoskeleton is a key regulator of morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. Changes in the cellular architecture are often assumed to require actin-binding proteins as stimulus-response modulators, because many of these proteins are regulated directly by binding to intracellular second messengers or signaling phospholipids. Phosphatidic acid (PA) is gaining widespread acceptance as a major, abundant phospholipid in plants that is required for pollen tube tip growth and mediates responses to osmotic stress, wounding, and phytohormones; however, the number of identified effectors of PA is rather limited. Here we demonstrate that exogenous PA application leads to significant increases in filamentous actin levels in Arabidopsis suspension cells and poppy pollen grains. To investigate further these lipid-induced changes in polymer levels, we analyzed the properties of a key regulator of actin filament polymerization, the heterodimeric capping protein from Arabidopsis thaliana (AtCP). AtCP binds to PA with a K d value of 17 M and stoichiometry of ϳ1:2. It also binds well to PtdIns(4,5)P 2 , but not to several other phosphoinositide or acidic phospholipids. The interaction with PA inhibited the actin-binding activity of CP. In the presence of PA, CP is unable to block the barbed or rapidly growing and shrinking end of actin filaments. Precapped filament barbed ends can also be uncapped by addition of PA, allowing rapid filament assembly from an actin monomer pool that is buffered with profilin. The findings support a model in which the inhibition of CP activity in cells by elevated PA results in the stimulation of actin polymerization from a large pool of profilin-actin. Such regulation may be important for the response of plant cells to extracellular stimuli as well as for the normal process of pollen tube tip growth. INTRODUCTIONMembrane phospholipids are key regulators of cellular signaling responses and reorganization of the cytoplasmic architecture in all eukaryotic cells. Although much early work in this arena focused on the role of polyphosphoinositide (PPI) turnover, there is growing evidence for the function of other acidic phospholipids as signaling intermediates. In particular, phosphatidic acid (PA) and lysophospholipids are implicated in diverse signaling cascades (Meijer and Munnik, 2003;Wang, 2004;Testerink and Munnik, 2005). PA is generated through two major pathways: hydrolysis of the structural lipid phosphatidylcholine by phospholipase D (PLD) enzymes, and phosphorylation of the lipid second messenger diacylglycerol (DAG) by DAG kinase (Meijer and Munnik, 2003;Wang, 2004;Testerink and Munnik, 2005). In animal cells, actin cytoskeleton polymerization is stimulated by PA (Ha and Exton, 1993;Ha et al., 1994;Zhou et al., 1995;Siddiqui and English, 1997;Shin et al., 1999), and both PA and PLD activity have been implicated in multiple stress signaling responses of plant cells (Meijer and Munnik, 2003;Wang, 2004).Transient increases in cellular PA in response ...

Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro

“…Therefore, elevated levels of PA expectedly increase the amount of actin in the cells. Indeed, exogenous PA is reported to enhance the amount of filamentous F-actin in Arabidopsis and tobacco suspension cultures and Arabidopsis epidermal cells (Huang et al 2006;Pleskot et al 2010;. As recently discovered, phosphatidic acid has a regulating role in the case of microtubules as well.…”

“…It is proven that activity of phospholipase D increased after cold exposure (Ruelland et al 2002), leading to the elevation of intracellular PA levels. In Arabidopsis, phosphatidic acid negatively affected the actin-binding ability of a heterodimeric capping protein (CP) that binds actin filaments at the barbed ends (Huang et al 2006;Pleskot et al 2012). This regulatory protein decreases filament length and annealing frequency through the lowering of dynamic activity at filament ends.…”

Phospholipids

“…And do the products of PA, diacylglycerol (DAG) and diacylglycerol pyrophosphate (DGPP), also regulate the cytoskeleton? Moreover, PtdIns(4,5)P 2 also bind to AtCP and inhibit its capping activity except PA. 25,27 What are the molecular effect of PtdIns(4,5)P 2 on microfilament organization to enhance tolerance under abiotic stresses? Therefore, in future work, it is necessary to further clarify more phospholipids, including PA, DAG, PtdIns(4,5)P 2 and InsP 3 , and their binding or interacting cytoskeletal proteins upon stresses.…”

“…24 Interestingly, PLD and its derived product-PA have been proved to interact with MFs cytoskeleton and regulate MFs dynamics. 25 It was depicted PLDβ1-PA controlled MFs dynamics in tobacco pollen tube. 26 Most of pollen tube MFs depolymerized in the presence of n-butanol, which is a specific inhibitor of PA generation, and the depolymerized MFs could be recovered by addition of PA. NtPLDβ1 preferentially bound to MFs and G-actins, and its activity was enhanced by MFs, but was inhibited by G-actins, thus forming a positive feedback loop.…”

Role of phosphatidic acid in plant galactolipid synthesis

“…PDK1 [68]). Some others are related with the cytoskeleton such as the actin cap protein AtCP [69]. PA binding can induce a cascade of changes not only on membrane proteins but also in the surrounding domain inside the membrane [70].…”