Latrunculin A Accelerates Actin Filament Depolymerization in Addition to Sequestering Actin Monomers

Ikuko Fujiwara, Mark E. Zweifel, Naomi Courtemanche, Thomas D. Pollard

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

Latrunculin A (LatA), a toxin from the red sea sponge Latrunculia magnifica, is the most widely used reagent to depolymerize actin filaments in experiments on live cells. LatA binds actin monomers and sequesters them from polymerization [1, 2]. Low concentrations of LatA result in rapid (tens of seconds) disassembly of actin filaments in animal [3] and yeast cells [2]. Depolymerization is usually assumed to result from sequestration of actin monomers. Our observations of single-muscle actin filaments by TIRF microscopy showed that LatA bound ATP-actin monomers with a higher affinity (Kd = 0.1 μM) than ADP-Pi-actin (Kd = 0.4 μM) or ADP-actin (Kd = 4.7 μM). LatA also slowly severed filaments and increased the depolymerization rate at both ends of filaments freshly assembled from ATP-actin to the rates of ADP-actin. This rate plateaued at LatA concentrations >60 μM. LatA did not change the depolymerization rates of ADP- actin filaments or ADP-Pi-actin filaments generated with 160 mM phosphate in the buffer. LatA did not increase the rate of phosphate release from bulk samples of filaments assembled from ATP-actin. Thermodynamic analysis showed that LatA binds weakly to actin filaments with a Kd >100 μM. We propose that concentrations of LatA much lower than this Kd promote phosphate dissociation only from both ends of filaments, resulting in depolymerization limited by the rate of ADP-actin dissociation. Thus, one must consider both rapid actin depolymerization and severing in addition to sequestering actin monomers when interpreting the effects of LatA on cells. Fujiwara et al. address how Latrunculin A (LatA) depolymerizes actin filaments. Observations of single filaments revealed that LatA not only sequesters actin monomers but also promotes subunit dissociation from the ends of filaments assembled from ATP-actin monomers by rapid “aging” due to fast phosphate dissociation from terminal ADP-Pi-subunits.

Original languageEnglish (US)
Pages (from-to)3183-3192.e2
JournalCurrent Biology
Volume28
Issue number19
DOIs
StatePublished - Oct 8 2018

Bibliographical note

Funding Information:
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01GM026338 to T.D.P. and R01GM122787 to N.C. and JSPS KAKENHI grant numbers 15K21106 and 17K07373 to I.F. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Dimitrios Vavylonis, Shuichi Takeda, and Yuichiro Maéda for their advice on the interpretation of the experiments and Jeffrey Kuhn for generously supplying a length analysis software.

Keywords

  • ADP
  • ATP
  • actin
  • capping protein
  • depolymerization
  • formin
  • latrunculin A
  • phosphate
  • polymerization

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