An unexplained sequestration of latrunculin A is required in neutrophils for inhibition of actin polymerization

Abstract

Latrunculin A (LatA) is a toxic natural product that causes disruption of the actin cytoskeleton in many eukaryotic cells at submicromolar concentrations. LatA has been found to bind G-actin with a dissociation constant of 0.2 microM, and more recently to bind profilin-G-actin and, weakly, thymosin beta4-G-actin. A number of investigators have used LatA as a G-actin sequestering agent. Thus, we studied neutrophil chemotaxis and its requisite conversion of G-actin to F-actin, supported by an extensive pool of G-actin, mainly bound to thymosin beta4. Calculations suggest that the affinity of LatA is insufficient to cause significant sequestration of this pool, and the pool's buffering action should protect neutrophils from depletion of productive G-actin species by submicromolar LatA. Nonetheless, we found that both chemoattractant stimulated migration and F-actin polymerization in neutrophils were inhibited by LatA at these concentrations. The latter effect was accompanied by sequestration of LatA and showed a cell density dependence that was consistent with G-actin sequestration. The apparent contradiction between the calculations and the experimental observations could be reconciled by assuming the presence of an accessory species, of unknown normal function, which forms a high affinity ternary complex with LatA and G-actin, thus causing the cells to concentrate LatA. Other models that could not be ruled out also invoke new actions of LatA, suggesting caution in the interpretation of its effects on cells.

G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers

“…This observation is consistent with a previous report estimating a negligible change in the free G-actin concentration in neutrophils treated with 100 nM LatA (Pring et al, 2002). In this report, the , either free or bound to thymosin-β4 (T), profilin (P) and LatB (L).…”

“…Scale bars: 2 μm. also been estimated based on the different amounts of F-actin in stimulated and unstimulated leukocytes (Pring et al, 2002). Hence, we and Pring et al consistently predict several-fold free G-actin increases associated with the increased G-actin:F-actin ratio.…”

“…LatB competes with thymosin-β4 binding to G-actin (Yarmola et al, 2000). A previous study has estimated that free G-actin does not change upon treatment with 100 nM latrunculin A (LatA) despite accumulation of this drug in the neutrophil cytoplasm (Pring et al, 2002). The authors concluded that an unknown mechanism might be required to explain the impaired migration of neutrophils treated with 100 nM LatA.…”

“…4C). Using the same approach as the previous study (Pring et al, 2002), which calculates the equilibrium state between G-actin and its binding molecules, we estimated the concentration of total Gactin and free G-actin from the observed F-actin decrease. This calculation based on the different observation again predicts increases in free G-actin to 2.0 and 3.5 μM at 1 and 2 minutes, respectively (Fig.…”

Hem-1 Complexes Are Essential for Rac Activation, Actin Polymerization, and Myosin Regulation during Neutrophil Chemotaxis

“…The importance of F-actin in this loop [9] and the profound defects in F-actin distribution induced by Hem-1 depletion (Figure 4) raise the possibility that decreased Rac activation and Akt/PKB phosphorylation simply reflect the requirement of Hem-1–containing complexes for actin polymerization. To test the idea that Hem-1–containing leading edge complexes support the feedback loop by mechanisms independent of WAVE2-dependent actin polymerization, we treated control or Hem-1–depleted cells with latrunculin B, which sequesters monomeric actin [59,60] and blocks chemoattractant-induced actin polymerization in neutrophils [9,61,62]. Hem-1 depletion proved to inhibit fMLP-stimulated PAK phosphorylation (Figure 5D), Rac activation (Figure 5E), and Akt/PKB phosphorylation (Figure 5F) in latrunculin-treated cells to an even greater degree than in untreated cells.…”