Arabidopsis Projects


Regulation of Actin Filament Ends
Supported by the Dept. of Energy–Energy Biosciences Division (DE-FG02-04ER15526)
Staff: Dr. Xia Wang

Summary:
The cytoskeleton is a dynamic network of actin filaments and microtubules that coordinates plant growth and morphogenesis. The actin cytoskeleton, in particular, powers the movements of cytoplasmic streaming and drives the motility and positioning of various organelles, including photosynthetic plastids. Growing evidence implicates actin dynamics in the transport of vesicles between different compartments of the endomembrane system. Specific examples include secretory traffic that delivers non-cellulosic polysaccharides to the cell wall, remodeling of wall composition by endocytosis, and transport of storage proteins from the trans-Golgi network to vacuoles. These intracellular movements are certain to require actin polymerization and construction of higher-order structures. Changes in cytoskeletal architecture are choreographed by more than 70 different actin-binding proteins in eukaryotic cells, many of which are present in plants. A growing list of recent findings, however, reveals that these plant proteins can have surprisingly different activities or forms of regulation. To understand the actin dynamics that underpin intracellular motility and actin-based processes, a critical need is to learn how cells modulate the activity at filament ends; i.e., how ends are created or prevented from growing and shrinking. We have identified and characterized two classes of protein that interact with the barbed or fast growing end of actin filaments, villin/gelsolin and capping protein. The heterodimeric capping protein from Arabidopsis, AtCP, binds to filament ends with high affinity (Kd 12¬–24 nM) and prevents addition of actin monomers or the profilin-actin complex. Once bound, AtCP dissociates from filament ends extremely slowly. This suggests that regulation of uncapping will be one fundamental mechanism used by cells to allow actin filament elongation. We find that AtCP is regulated by phosphatidic acid (PA), a structural and signaling lipid with widely recognized importance in various stress responses. PA inhibits capping protein activity and stimulates uncapping. This provides a novel link between specific organelles or fluxes in PA levels and control of actin dynamics. Further study of AtCP forms the basis for much of the current proposal. Our working hypothesis is that AtCP is responsible for maintaining a large pool of unpolymerized actin subunits, by preventing the assembly of profilin–actin complexes, and for keeping the amount of polymeric actin remarkably low in plant cells. We also propose that filament uncapping by phospholipids, and yet to be identified protein partners, will modulate the activity of AtCP in plant cells. This provides a unique mechanism for driving motility of specific organelles/vesicles or for locally stimulating actin polymerization. Finally, we propose that there will be cross-talk between PA signaling and actin dynamics that impacts the ability of plants to respond to biotic and abiotic stresses. To test these hypotheses we will embark on a multifaceted approach that combines reverse-genetics with cell biology and state-of-the-art biochemical/biophysical analyses of a key actin-binding protein and its partners.

The specific aims of this proposal include:

  1. detailed phenotypic analysis of cp homozygous mutant plants and subcellular localization of CP,
  2. identification and characterization of protein binding partners and multimeric complexes that regulate the activities of CP,
  3. dissecting the cross-talk between PA and actin dynamics by mutagenesis of AtCP protein and study of plants with altered PA levels or signaling networks.

 

Through characterization of a key actin-binding protein that is involved in cytoskeletal organization, dynamics and function, we will further elucidate roles for the actin cytoskeleton in plant growth and development. Specifically, we will understand how CP senses and cross-talks with phospholipid (PA) signaling and how it regulates fundamental processes like germination and cell expansion. Through the identification of protein binding partners, we will gain insight about CP regulation in the cell and generate data on the assembly of cytoskeletal networks. The results obtained from these studies have obvious relevance for all actin-based functions, including intracellular transport and organelle positioning, morphogenesis, cell wall deposition and turnover, endocytosis, and response to biotic and abiotic stress. Realizing these goals will offer the potential to manipulate delivery of materials within cells, biopolymer formation, and accumulation of storage proteins during seed development.

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