Minisymposium 6: Cytoskeleton

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Abs # M0601: LIM Domain Proteins : A Novel Family of Plant Actin Binding Proteins

Presenter:	Steinmetz, Andre A       Contact Presenter
Authors	Steinmetz, Andre A (A)   Hoffmann, Celine  (A)   Dieterle, Monika  (A)   Gatti, Sabrina  (A)   Friederich, Evelyne  (B)   Shen, Wen-Hui  (C)   Thomas, Clement  (A)
Affiliations:	(A): Plant Molecular Biology Laboratory, CRP-Sante, Luxembourg (B): Laboratory of Molecular Biology, Genetic Analysis and Modeling, CRP-Sante, Luxembourg (C): Institute of Plant Molecular Biology, CNRS, Strasbourg

Plant LIM domain proteins were first identified by their coding sequences following differential screening of a floral cDNA library from sunflower. Very abundant in mature pollen grains of several plant species (PLIMs), mRNAs encoding LIM proteins were also detected later at low levels in other cell types (WLIMs). We have focused our studies on the two tobacco proteins NtWLIM1 and NtWLIM2. When expressed as GFP fusions in BY2 cells following stable Agrobacterium-mediated transformation, they were both detected in the cytoplasm as well as in the nucleus. Interestingly however, their cytoplasmic distribution pattern was markedly different: while the cytoplasmic fraction of NtWLIM1 was all concentrated along sharply defined filaments, only a small cytoplasmic fraction of NtWLIM2 accumulated along filamentous structures in a rather diffuse manner, with a considerable amount of the protein remaining free in the cytoplasm. Similar observations were made when the two proteins were expressed in Arabidopsis leaves following agroinfiltration. Using cytoskeleton-depolymerizing drugs we were able to identify the filamentous structures as actin filaments. High and low-speed co-sedimentation assays have shown that the binding of the NtWLIM1 protein to F-actin is direct, and induces the formation of actin bundles as well as their stabilization against latrunculin B action. Bundling of actin filaments in the presence of this protein was also directly observed by fluorescence light microscopy. When expressed in human fibroblasts and simian kidney cells, the NtWLIM1- and NtWLIM2-GFP fusion proteins bind to stress fibers and accumulate in focal adhesions, similarly to the animal Cystein-Rich Proteins (CRPs) with whom they share their structural organization. Typical structural features of this class of proteins are their small size (about 20 kDa), and the presence of two conserved double zinc finger motifs (LIM domains) separated by a 40 to 50 residue spacer. Flowering plants express up to 6 LIM proteins which can differ significantly in their amino acid sequence and are therefore expected to have different, but also overlapping functions. Our aim is to identify the functional differences of these proteins and their respective roles in the architecture and stability/dynamics of the plant actin cytoskeleton. This work is supported by the Luxembourg Ministry of Culture, Higher Education and Research.