Marine toxins

In 1980 the marine toxin Latrunculin was isolated from the red sea sponge Latmnculia magnifica

(Kashman et al. 1980). Subsequent investigations have found latrunculin to be mediating its toxic effects by disrupting the actin cytoskeleton (Spector et al. 1983). Since then, several bioactive compounds from sea sponges have been identified as actin depolymerising agents. These

compounds have been grouped into three classes based on their structure (Refer to fig 1.1). The first group is represented by latrunculin, which consists of a 2-thiazolidinone macrolide with no long side chains. The second class of compounds has a macrolide structure with a single side chain. The side chains of this class are very similar, while the macrolide rings vary in structure. This class includes - Tolytoxin, mycalolide B, kabiramide D and aplyronin A. The third class is composed of toxins, which have symmetric structures contisting of a macrolide ring with two diametrically opposed side chains. Examples of this class include swinholide A and misakinolide A (also named bistheonellide A). The compounds in these three groups all inhibit actin polymerisation, although they exhibit some differences in their F-actin dirupting activity. These agents are up to 100x times more potent than the cytochalasins at disrupting the stress fibres of cultured cells (Yahara et al. 1982). Their apparent specificity in targeting actin alone (excluding mycalolide B) and their ability to pass across the plasma membrane has made them a very attractive tool for a wide range of actin-related cellular research.

1.5.3.1 Class I: Latrunculin

Purification of this toxin showed that latrunculin consist of two isoforms, Latrunculin A and B (Kashman et al. 1980). Investigations into their biological activity in both neuroblastoma and fibroblast mouse cells found submicromolar concentrations of both isoforms disrupted the microfilament structures of cultured cells, while leaving microtuble structures unaltered (Spector et al. 1983).

Rapidly cycling microfilamentous structures such as stress fibres were found to be particularly susceptible. These effects of Latrunculin were found to be fully reversible. Comparative studies have shown that both isoforms appear to induce similar cellular effects, although latrunculin A is slightly more potent than latrunculin B (Spector et al. 1989). The more widely used alpha isoform has been used at concentrations ranging from -0.1 to 400 |iM (Lappalainen and Drubin, 1997). Pyrene actin assays revealed the stochiometry of latrunculin binding to G-actin was 1:1, with a kd of between 0.18 and 0.22|iM (Çoué et al. 1987). Subsequent kinetic studies show that latrunculin slowly depletes F-actin filaments (Ayscough et al. 1997). It thus appears that latrunculin induces actin deploymerisation by sequestering monorrler, released from the filaments, preventing their its re-incorporation into filaments (Ayscough et al. 1997). Thus over a period of time cellular F-actin

becomes depleted by latrunculin. The rate of latrunculin-mediated filament depletion should therefore reflect the rate of actin depolymerisation in vivo (Belmont et al. 1999). This accounts for the susceptibility of stress fibres - which rapidly turnover, to the effects of latrunculin. Its binding site on G-actin was defined using a set of congenic charged-to-alanine mutated yeast actins, - used to locate phalloidin’s binding site on actin. Four of these yeast mutants were completely resistant to the effects latrunculin. As these resistant strains exhibited no defects in their general uptake mechanisms, the interaction between latrunculin and actin appears to be highly specific. The residues mutated in these resistant alleles were either directly associated, through salt bridges or hydrogen bonds, with the nucleotide itself, or formed salt bridges to position other residues that associated with the nucleotide. The close proximity of the latrunculin binding-site to the ATP-binding pocket results in latrunculin inhibiting nucleotide exchange (Ayscough et al.

1997). Thus actin monomers associated with latrunculin will be predominenty in their ADP bound form. With this apparent specificity, latrunculin has provided and excellent tool to investigate such actin dependent process as endocytosis (Lamaze et al. 1997), cell polarity (Ayscough et al. 1997) and the activity of the actin-binding protein cofilin (Lappalainen and Drubin, 1997).

1.5.3.2 Class II: mycalolide B and kabiramide D

Mycalolide B is isolated from the Mycale sp sea sponge. Mycalolide B (5 |im) and kabiramide D (5 ixm) like latrunculin both induce depolymerisation of filamentous actin (refer to Fig 1.1). However in 3Y1 cells mycalolide B also binds covalently to proteins other than actin. This non­ specific binding is thought to occur via its a-p-unsaturated ketone moiety, which is highly reactive with the thiol groups in cysteine residues (Wada et al. 1998a). kabiramide D - an analogue of mycalolide B, but lacking the ability to react with thiol groups, binds non-covalently and specifically to actin. Preliminary findings suggest that kabiramide D in addition to sequestering actin monomers, also severs filaments. It appears that marine actin-depolymerising toxins with long side chains, including mycalolide B, kabiramide D and misakinolide A bind to a common site on actin, which differs from that of latrunculin and cytochalasin D (Wada et al. 1998a). These findings favour the use of kabiramide D, rather than mycalolide B, as a tool to manipulate the cytoskeleton.

1.5.3.3 Class I I I : swinholide A, misakinolide A

Swinholide A and misakinolide A were isolated from the sea sponges Theonella sp and Theonella swinhoei respectively (refer to figure 1.1). These are extremely potent agents with 10-50 nM inducing substantial F-actin depletion after only 60min. The structures of these two compounds are very similar, differing only in the macrolide ring, where Swinholide A has two additional double-bonded carbons (Terry et al. 1997). In spite of this similarity in structure, the two compounds have surprisingly different effects on actin filament dynamics in vitro. Both compounds bind with high affinity to two actin monomers in a configuration, which prevents the actin monomers from participating in either filament elongation, or nucléation reactions. However swinholide A severs actin filaments while misakinolide A caps the barbed end of filaments. Swinholide A binds to two actin subunits (Ka = 9 x 1 0^^ ^ -2) jp the presence of 2 mM MgCl2 (Bubb et al. 1995). It then rapidly severs F-actin in a highly co-operative manner; for efficient severing swinholide A needs to be used at a concentration that approaches the cellular actin concentration. Misakinolide A prevents elongation of actin filaments consistent with misakinolide- actin complexes capping the barbed end of filaments (Kd 50 nM), but as mentioned it does not exhibit any detectable severing activity (Terry et al. 1997). Surprisingly misakinolide A does not appear to prevent actin disassembly. This is unexpected as it binds to the barbed end with a higher affinity than actin itself. To explain this phenomenon it has been suggested that dissociation of misakinolide A from the barbed end is accompanied by the loss of more than one actin subunit.

The differences in activity between Swinholide A and misakinolide A are reflected in their effects in living cells, with swinholide A being significantly more potent in depleting F-actin. However swinholide A is cytotoxic except when the exposure is very brief. The effects of misakinolide A on the other hand are fully reversible and thus provide a more useful tool in studies of a longer duration.

1.5.3.4 Summary of “actin depleting” marine toxins

The marine toxins kabiramide D, swinholide A (short applications), misakinolide A and latrunculin therefore provide powerful and inexpensive tools for the quantitative and specific removal of the actin cytoskeleton of living cells.