Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid inArabidopsis   

Abstract

Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics.

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

“…S2); this observation is consistent with the proteomic detection of actin at isolated vacuoles (16,17). Interference with actin affects the formation of transvacuolar strands (18,19), raising the question of whether the actin network is mechanistically linked to vacuolar morphogenesis required for auxin-reliant growth repression. To assess the role of actin in the vacuolar morphology of epidermal root cells, we first interfered with actin dynamics pharmacologically.…”

“…Auxin treatment seemingly leads to the formation of multiple small luminal vacuoles (15), and several studies previously have addressed the mechanisms of vacuolar fragmentation (16,18,(23)(24)(25). Accordingly, actin and its motor protein myosin could contribute the force required for auxin-dependent vacuolar fission and fusion events.…”

Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

“…The creation and turnover of actin filaments, as well as their assembly into higherorder structures, are tightly regulated at spatial and temporal levels by a plethora of actin-binding proteins, which control nucleation, polymerization, capping, severing and crosslinking (dos Remedios et al, 2003;Higaki et al, 2007;Staiger and Blanchoin, 2006;Thomas et al, 2009;Winder and Ayscough, 2005). Recent live cell studies combining reliable fluorescent actin markers with novel high-resolution imaging techniques such as spinning-disc confocal microscopy and variable-angle epifluorescence microscopy (VAEM) have provided key insights into actin cytoskeleton dynamics at the cell cortex of plant cells (Augustine et al, 2011;Era et al, 2009;Henty et al, 2011;Henty-Ridilla et al, 2013;Khurana et al, 2010;Konopka and Bednarek, 2008;Li et al, 2012;Smertenko et al, 2010;Staiger et al, 2009;Tóth et al, 2012). In striking contrast to in vitro actin treadmilling (Bugyi and Carlier, 2010;Selve and Wegner, 1986), in vivo remodeling of plant cortical actin arrays was found to follow a socalled 'stochastic dynamics' process that is dominated by fast elongation and prolific severing of single filaments Okreglak and Drubin, 2010;Staiger et al, 2009).…”

“…Actin-bundling proteins exhibit a modular organization and combine one or more actin-binding domains (ABDs), with regulatory domains conferring sensitivity to specific stimuli such as phospholipids or variations in pH or calcium concentration (Bartles, 2000;Furukawa and Fechheimer, 1997;Huang et al, 2006;Li et al, 2012;Papuga et al, 2010;Puius et al, 1998;Wang et al, 2008). Despite the frequent pairwise arrangement of their ABDs, some actin-bundling proteins undergo dimerization to crosslink actin filaments (Mimura and Asano, 1986;Thomas, 2012;Thomas et al, 2009).…”

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

“…To quantify F-actin bundling at the shank and tip of pollen tubes, an average skewness value and F-actin density were measured using ImageJ as described previously (Higaki et al, 2010;Li et al, 2012).…”