Moniliformin sodium salt Until now the molecular interaction

Until now, the molecular interactions of Tmod or Lmod with Moniliformin sodium salt had been assessed in pyrene-actin polymerization assays [13], [23], [27], [28], [44], [45] or directly measured using non-denaturing polyacrylamide gel-electrophoresis (for Tmod isoforms only [23]), ITC (for Tmod1 and Lmod2) [43], [46], and for Lmod2 only, bioLayer interferometry [44] and NMR spectroscopy [12]. Although the use of these techniques has given valuable insight, there may be challenges in their interpretation. The latter three methods needed high concentrations and used Lmod2 fragments. Crystal structures of complexes between Lmod2 or Tmod1 and actin also were obtained using fragments [43], [44], [46]. The use of fragments is convenient for understanding the function of multidomain proteins, as well as for estimating the binding kinetics between Tmod or Lmod and their binding partners. However, they may not reflect the binding expected between full-length proteins. Additionally, the tendency of actin to polymerize rapidly under physiological conditions complicates the measurement of its binding constants with Tmod or Lmod, as well as prevents crystallization [44]. In order to overcome this problem, previous efforts for measuring the interactions of Tmod or Lmod with actin involved the use of latrunculin B [43] or mutated actin [44] to stabilize the monomeric state of actin and prevent polymerization. Atomic force microscopy (AFM) with its ability to measure interactions between single molecules allows quantification of protein-protein interactions with high accuracy under native conditions and using full-length proteins [47]. This technique is an exciting complement to previous studies of the structure/function relationship between Tmods and actin. In this study, we utilized AFM to characterize the unbinding forces between G-actin and proteins of the tropomodulin family. By creating mutants or fragments of Tmod2 and Lmod2, we assigned specific unbinding forces to their individual actin-binding sites. Our findings demonstrated that the N-terminal domain of Lmod2 interacts with actin and confirmed the existence of the N-terminal actin-binding site.
Materials and methods
Results and discussion
Conclusions The unbinding forces between G-actin and proteins from the tropomodulin family were quantified and characterized using single molecule force spectroscopy. By creating mutants or fragments of Tmod2 and Lmod2, we assigned specific unbinding forces to their individual actin-binding sites. Our results demonstrate how the differences between the number and the strength of the actin-binding sites translate to the actin-sequestering and -nucleating abilities of Lmod and Tmod. Finally, our results confirm that Lmod2 binds at least three molecules of actin. In agreement with our recent report [12] and data published by Chen and co-authors [44], we conclude that the N-terminal domain of Lmod2 contains an actin-binding site. AFM provides a valuable approach to quantify interactions between biomolecules [52]. AFM's ability to quantify biomolecular forces with pico-newton resolution in liquid environments [47], [57] allowed researchers to investigate protein-ligand [58], protein-DNA [59], protein-protein [60], and protein-biomolecule [61] interactions at the nanoscale. The ability to work with full-length proteins, the use of relatively low protein concentrations (∼0.5–3 μM in this study) and stability of actin in a monomeric form make AFM a promising technique for measuring forces of interaction of G-actin with other actin-nucleating and actin-sequestering proteins.
Acknowledgements The authors thank Christopher Keller for assistance in Tmod2[L73D] preparation. This work was supported by the National Institutes of Health grants GM081688 and GM120137 to ASK. We thank Dr. Carol Gregorio for providing WT-Lmod2, Lmod21-514 and Lmod21-201.
Introduction In plants, the actin cytoskeleton plays an important role in cell development, cell morphogenesis, and the establishment and maintenance of cell polarity. Myosin is a motor protein that has a conserved motor domain with ATPase and actin-binding activities. Using the motor domain, myosin converts chemical energy via adenosine triphosphate (ATP) hydrolysis into physical movement along the actin filaments [[1], [2], [3]]. Phylogenetic analyses of 2269 myosin motor domains from 328 organisms have revealed 35 myosin classes in eukaryotes [4]. Among the myosin species, plant myosins are classified into class VIII and XI. The actin–myosin XI cytoskeleton is predominantly involved in vesicle trafficking, organelle movement and positioning in plant cells [5]. Particularly, cytoplasmic streaming, which refers to general intracellular transport, occurs widely in plants ranging from algae to angiosperms [6]. Cytoplasmic streaming is inhibited by treatment with an actin polymerization inhibitor (cytochalasin B), indicating that actin is involved in the generation of motive force [6]. Actin filaments are observed in a variety of plants [7], reflecting the broad occurrence of cytoplasmic streaming. Although many results have confirmed that the actin–myosin XI cytoskeleton generates organelle movement [[8], [9], [10]] and cytoplasmic streaming [6,11,12], the mechanisms underlying these intracellular transport systems have not been clearly understood.