Intracellular Step see Table

Table 1. Representative Studies Reflecting the Use of Clostridium botulinum C2 Toxin as a Research Tool

Cell Line or Tissue

Research Problem


3T3 Cells

Autoregulation of actin polymerization

Bershadsky et al.,


Adrenal Y-l Cells

Role of microfilaments in steroid release Considine et al.,



Autoregulation of actin synthesis

Reuner et al., 1991

HIT-T15 Cells

Role of actin filaments in insulin

Li et al., 1994



G/F-actin transition in smooth muscle

Mauss et al., 1989



Receptor-mediated cell activation

Melamed et ai,



Proliferation of Epstein-Barr Virus

Melamed et al.,



Role of cytoskeleton in nerve growth

Melamed et al.,

factor signaling



Mitogen-activated protein kinases and

Melamed et al.,

signal transduction



Role of microfilaments in motility of

Verschueren et ai,


lymphoid cells


derived cell line


Acetylcholine release

Simpson, 1982


Mast Cells

Histamine release

Böttinger et al., 1987


Cholesterol esterification

Tabas et al., 1994


Activation of oxidase

Al-Mohanna et al.,



Ligand-evoked lipid mediator

Grimminger et al.,




Ligand-evoked signal transduction and Grimminger et al.,




Nitric oxide stimulation of ADP-

Clancy et al., 1995


PC-12 Cells

Release of norepinephrine

Matter etal., 1989

Xenopus Oocytes

Potassium channel clustering by the

Honore etal., 1992


The first area of investigation in which C2 toxin was used as a research tool was in the analysis of storage and release of chemical mediators (Considine and Simpson, 1991). This was a natural outgrowth of the fact that other clostridial toxins, and particularly botulinum neurotoxin and tetanus toxin, have profound effects on transmitter release (Simpson, 1989a; Montecucco and Schiavo, 1994). It therefore seemed logical to determine whether clostridial binary toxins had similar effects. In addition, at the time that most of the original work on binary toxins and chemical mediators was done, there was a prevailing belief that the cytoskeleton played an integral role in governing the location and mobility of vesicles. Thus, it was reasonable to assume that any toxin that disrupted the cytoskeleton would have an impact on storage or release of mediators.

The first report in this area compared the actions of botulinum neurotoxin and botulinum binary toxin on transmission in the phrenic nerve-hemidiaphragm preparation (Simpson, 1982). There was the expected finding that neurotoxin blocked transmission by blocking acetylcholine release from nerve terminals, but the binary toxin had no effect. Apart from showing that C2 toxin did not block exocytosis, this study showed that the toxin did not act on the diaphragm to block muscle twitch. This observation is in keeping with the fact that the predominant form of actin in striated muscle is not that which is ADP-ribosylated by C2 toxin.

In related studies, norepinephrine action was studied on isolated strips of thoracic aorta (Simpson, 1982). It was found that the toxin neither altered agonist-induced responses nor the high affinity reuptake system.

release of mediators The effect of C2 toxin on release of granular mediators such as norepinephrine (Matter etal., 1989) and histamine (Bottinger etal., 1987) has been examined, but there is no clear picture that emerges from this work. Studies published to date indicate that the toxin does not have any effect on basal release. However, there may be effects on stimulated release, but the nature and even the direction of effect hinges on experimental conditions. In contrast to granular mediators, the basal release of at least one non-granular mediator is affected by toxin. The constitutive release of steroids in Y-l adrenal cells was markedly stimulated by C2 toxin (Considine et a/., 1992).

To the extent that these studies are revealing, they demonstrate that there is no simple and universal scheme that links integrity of the cytoskeleton with storage and release of mediators. This may not be a surprising finding. Even if the cytoskeleton did play a simple and direct role in mediator release, the indirect consequences of producing cellular collapse would probably cause many other outcomes that would complicate interpretation of results. This intuitively obvious matter may lead to what are the most important conclusions that can be drawn from studies with C2 toxin:

1. If an action of C2 toxin can be documented (i.e., ADP-ribosylation of actin; disaggregation of the cytoskeleton), but there is no change in the cellular response under study, then this particular response is not immediately dependent on integrity of the cytoskeleton.

2. If an action of C2 toxin can be documented, and if there is a change in the cellular response in question, then this response may be governed by the cytoskeleton. However, before a direct link between actin structure and cell response can be accepted, work must be done to demonstrate that the loss of response is not an indirect (and mundane) result of causing cell collapse. In relation to the second point, it is worth noting that most of the literature on C2 toxin as a research tool is flawed by the fact that few investigators have established whether changes in cell response are the direct or indirect consequence of modifying the cytoskeleton. There are, however, two areas of investigation for which this is not a serious problem. C2 toxin has been used as a tool to analyze cell regulation of actin synthesis, and in separate studies it has been used to examine cell motility. It is safe to assume that changes in the actin based cystoskeleton would affect directly both of these phenomena.

The relationship between cytosolic levels of monomeric actin and regulation of actin the levels of mRNA associated with synthesis of actin has been ex- synthesis amined by Reuner et a/. (1991) and by Bershadsky et al. (1995). Although the two groups used different cell lines (hepatocytes and 3T3 cells), their experimental approaches and observations were similar. C2 toxin was used to promote depolymerization and thus an increase in cytosolic levels of monomeric actin. This treatment produced both a reduction in the rate of synthesis of actin and a decrease in the levels of mRNA. The latter finding was apparently due to a shortened half-life of mRNA (Bershadsky et al., 1995). The down regulation in synthesis of actin was a specific outcome, because synthesis of other proteins was largely unaffected. The down regulation could also be linked to increased levels of free actin rather than changes in the cytoskeleton, because the result was obtained both in monoculture and in cell suspension. It was interesting that procedures that promote polymerization had the opposite effect, causing both the levels of mRNA as well as the rates of actin synthesis to increase. This work demonstrates that there is autoregulation of actin synthesis.

The relationship between actin polymerization and cell motility was effect on cell motility examined by Verschueren et al. (1995). Using a lymphoma-derived cell line, they showed that C2 toxin treatment greatly diminished pseu-dopodal protrusion as well as the ability to invade a monolayer of fibroblast-like cells. This is a predictable finding, and one that must be due at least in part to loss of actin filaments.

In contrast to the studies reviewed above, in which a particular type of phenomenon was examined in various cell types (e.g., secretion, autoregulation of actin synthesis), there is other work in which several types of phenomena have been examined in a single cell type

(see Table 1). Thus, Melamed and colleagues have analyzed the relationship between actin and various cell responses in lymphocytes, ranging from receptor-mediated cell activation to proliferation of Epstein-Barr virus (Melamed et a/., 1991; Melamed et a/., 1994; Melamed et a/., 1995a, b). Other groups have investigated various phenomena in neutrophils (Al-Mohanna et a/., 1987; Grimminger et a/., 1991a, b; Clancy eta/., 1995). As discussed above, it is sometimes difficult to know whether an observed change in cell response is direct and can be related to specific changes in cytosolic actin or actin filaments, or indirect and merely due to a collapse in cell structure.

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