Phalloidin

Structure of Phalloidin (MW = 788).

 

Source                       

green toadstool Amanites phalloides

Use

Phalloidin stabilises actin filaments irreversibly, followed by bundling

Mode of action         

They bind tightly to F-actin, but not to G-actin. They lower the critical concentration for assembly by 10-30-fold (decrease the rate constant of actin dissociation, association rate is equal at the pointed end, slightly decreased at the barbed end). Stochiometry: 1 phalloidin per 1-2 actin protomers nM. Binding is covalent to amino acids Glu-117, Met-119, and Met-355 (near the nucleotide binding site).

Properties                 

Kd of binding is 20-80 nM. MW 788.9. Stock solution 6.6 or 66 µM in ethanol, can be stored at -20°C

  • Very specific, however cells die after phalloidin treatment (in contrast to cytochalasin treatment).
  • Therefore other side targets cannot be excluded.
  • Fluorescence-labelled phalloidin at saturating concentrations is used to determine the amount of F-actin (fluorometrically after methanol extraction).
  • Plant cells and protoplasts: BY-2 cells 1 µM rescue division synchrony in ADF2ox (Durst et al. 2013). Protoplast regeneration also 1 µM (Zaban et al. 2013). 30 min 1 µM alters volume regulation in grapevine protoplasts (Liu et al. 2013), 2 h 1 µM alter defence gene expression in grapevine cells (Qiao et al. 2010), 30 min 1 µM stabilise membrane (Berghöfer et al. 2009)
  • Rice coleoptiles: 1 h with 1-10 µM alters auxin transport (Nick et al. 2009)

Publications              

Mode of action

Cooper JA (1987) Effects of Cytochalasin and Phalloidin on Actin. J Cell Biol 105, 1473-1478

Estes JE, Selden LA, Gershman LC (1981) Mechanisms of action of phalloidin on the polymerization of muscle actin. Biochemistry 20, 708-712

Publications from our group using this inhibitor:

75. Nick P, Han M, An G (2009) Auxin stimulates its own transport by actin reorganization. Plant Physiology 151, 155-167

81. Qiao F, Chang X, Nick P (2010) The cytoskeleton enhances gene expression in the response to the Harpin elicitor in grapevine. J Exp Bot.61, 4021-4031

93. Zaban B, Maisch J, Nick P (2013) Dynamic actin controls polarity induction de novo in protoplasts, J Int Plant Biol 55, 142–159

97. Durst S, Nick P, Maisch J (2013) Actin-Depolymerizing Factor 2 is Involved in Auxin Dependent Patterning, J Plant Physiol 170, 1057-1066

98. Liu Q, Qiao F, Ismail A, Chang X, Nick P (2013) The Plant Cytoskeleton Controls Regulatory Volume Increase. BBA Membranes 1828, 2111–2120