We have used the ability to reconstitute Listeria motility in Xenopus egg extracts to test directly the role of the gelsolin and ADF/cofilin proteins in the promotion of actin filament depolymerization. We have raised specific antibodies to gelsolin and to XAC, the major ADF/cofilin protein known to be present in Xenopus egg extracts. Using immunodepletions and adding back purified proteins, our studies demonstrate that ADF/cofilin is the major factor responsible for the rapid turnover of actin filaments in Listeria tails. Removal of 75% of the ADF/cofilin from extracts considerably lengthened Listeria tails and greatly increased the total actin polymer mass present in the tails. Both tail length and actin polymer mass in tails were restored to control levels by adding back pure XAC at physiological concentrations to XAC-depleted extracts. In addition, adding excess XAC shortens Listeria tails and reduces the polymer mass in the tail, and AMPPNP actin is resistant to depolymerization by both XAC and whole extracts. Taken together, these data strongly implicate XAC as a central component of the machinery responsible for rapid actin filament turnover in extracts. Although we have no direct evidence, we predict that ADF/cofilin proteins are required for the dynamic organization of populations of actin filaments within living cells that turn over rapidly, such as the filaments within lamellipodia. Three arguments support our prediction: Xenopus egg extracts can support the actin polymerization and depolymerization in Listeria tails at rates comparable to those in intact cells and suggest they reflect the actin dynamics within cell cytoplasm. XAC is required to maintain the turnover of actin filaments in these tails in extracts. Because ADF/cofilin proteins are essential in every species in which they have been found, we may infer that they are required for essential processes like rapid actin turnover in the cell. Finally, since XAC is concentrated in Listeria tails and the leading edge of cells, we suspect it is also required for rapid filament turnover in vivo.
In contrast to XAC, gelsolin depletions had no significant effect on Listeria tail length or tail polymer mass, leaving the functional role of gelsolin in the regulation of actin dynamics an open question. Since gelsolin does not affect the depolymerization of Listeria tails, its concentration in these tails is curious. Perhaps gelsolin is concentrated in these tails strictly because of its actin binding activity, or by another of its activities such as actin capping. Although the Xenopus egg extracts used in our assays contain 5 mM EGTA, it is difficult to analyze any local concentrations of Ca2+ that may be due to vesicle release. Since Ca2+ is required for gelsolin activity, it is possible that the conditions in our cell-free egg extracts are not able to support the activity of gelsolin. Therefore, we cannot rule out a role for gelsolin in tail dynamics in vivo. However, the presence of gelsolin in Listeria tails does not greatly affect actin assembly or disassembly in Listeria tails in Xenopus extracts.
In our addition of varying amounts of excess XAC to extracts until all the actin was driven into abnormal rod structures, Listeria
tails shrank to ~5 μm but no further. The persistence of a resistant tail segment in up to sixfold the normal concentrations of XAC suggests that an additional factor may control the extent of actin depolymerization by XAC. Several investigators have suggested that the energy of ATP hydrolysis could be used to regulate the lifetime of a filament (Pollard, 1986
; Carlier, 1988
; Maciver et al., 1991
; Moon and Drubin, 1995
). In vitro studies of actin filament assembly have shown that upon polymerization of ATP-actin, the bound ATP is hydrolyzed and the terminal phosphate is slowly released (Carlier, 1987
). This slow rate of phosphate loss relative to the polymerization rate should then produce filaments that contain a segment of ADP + inorganic phosphate (ADP.Pi). Since ATP- and ADP.Pi-bound actin are known to make stronger intersubunit bonds than ADP-actin (Carlier, 1991
; Carlier et al., 1985
), phosphate release could play a role in regulating actin filament stability. However, since in vitro filaments depolymerize very slowly, phosphate release alone is not sufficient to direct rapid disassembly of actin filaments in the cell. In vivo, perhaps the role of slow phosphate release in actin is to regulate either the binding or activity of actin-severing proteins. Severing proteins such as ADF and cofilin may preferentially sever the ADP subunits of actin filaments while the ATP and ADP.Pi subunits are resistant to severing (Fig. ). In support of this, the depolymerizing activity of actophorin, an Acanthamoeba
member of the ADF/cofilin family, can be inhibited by addition of 25 mM phosphate, which presumably mimics ADP.Pi filaments (Maciver et al., 1991
). In addition, actophorin has been shown to bind tightly to ADPcontaining G-actin and weakly to ATP-actin (Maciver and Weeds, 1994
). However, other studies show that chick ADF has a higher affinity for ATP-actin than for ADP-actin (Hayden et al., 1993
) and have left the role of the bound nucleotide in regulating depolymerization in other species an open question. Our results showing the resistance of AMPPNP-containing actin filaments to the depolymerizing activities of both XAC and concentrated Xenopus
egg extracts lend support to the idea that the nucleotide content of an actin filament controls filament lifetime by regulating the activity of ADF/cofilin family members.
Figure 7 Model for recycling of actin by ADF/cofilin proteins. At areas of high filament turnover in the cell, ATP-bound actin is induced to polymerize by a complex of proteins. The ATP within the filament is hydrolyzed and the terminal phosphate is slowly (more ...)
What is the mechanism of actin depolymerization by XAC? XAC could depolymerize by end-wise removal of subunits, by severing, or by both mechanisms. We interpret our results with AMPPNP actin as favoring the endwise mechanism. We reason that a severing protein would not be greatly affected by an actin filament with only 12% of its subunits substituted with AMPPNP. By contrast, an end-wise depolymerizing protein would be blocked whenever AMPPNP subunits were present at the filament ends. Thus, the large inhibition of depolymerization by AMPPNP filaments may be accounted for if XAC primarily depolymerized using an end-wise mechanism. The most direct way of distinguishing severing and end-wise mechanisms will come from imaging depolymerization by ADF/ cofilin proteins.
On the basis of our findings, we can postulate a model for how XAC recycles actin subunits in the Listeria
tail (Fig. ). A complex of proteins at the back of Listeria
(Welch et al., 1997
) induces polymerization of ATP-bound actin. Once actin polymerizes, the bound ATP is hydrolyzed and the terminal phosphate is slowly released. The resulting ADP-containing actin subunits interact more weakly within the filament than the ATP subunits and allow binding of XAC. XAC either depolymerizes single subunits or short fragments of filament. XAC is then released from the depolymerized actin subunit and recycled for another round of depolymerization. The ADP in the depolymerized actin is then exchanged for ATP by bulk mass or by catalysis from profilin, and this subunit is now available for another round of polymerization or remains unpolymerized by binding thymosin β4
or other sequestering proteins.
This model provides a framework for understanding how an actin subunit is recycled from one round of polymerization to the next in regions of the cell that rapidly turn over actin filaments. Clearly, many questions remain regarding the details of this model. Future work will need to address whether XAC works primarily by severing or end-wise mechanisms in the cell. We are currently examining how XAC is recycled for another round of depolymerization after it is bound to the actin subunit and the role that XAC phosphorylation may play in its recycling. Other studies will need to focus on how the actin subunit is recycled for repolymerization. Analysis of the nucleotide content of actin has revealed that ~90% of unpolymerized actin in the cell is bound to ATP (Rosenblatt et al., 1995
), suggesting that the actin nucleotide is exchanged early in the pathway. Information about whether the ATP- or ADP-bound actin is used for polymerization is still lacking. The 10% of actin that is ADP bound could be bound to XAC. This population of actin could undergo nucleotide exchange and be used directly for another round of polymerization, leaving the remaining 90% of actin sequestered from polymerization. However, the lack of an effect that XAC depletion has on the rate of polymerization at early time points would suggest that this is not the case. Future studies will need to address what population of actin is used for polymerization.