PLANEACIÓN

Bundling N etw orks Severing nnd ca pping Nucleallon Monomer b inding Capping Elongation

(Taken from Way and Weeds, 1990).

1.7e. Inhibitors of Actin.

Many cytoskeletal toxins function by mimicking the effects of ABPs or regulators of actin.

(i) Cvtochalasins.

Reversible cytotoxins, eg. cytochalasins, are useful

tools for assessing actin-based motility, cytoskeletal rearrangements and acting as models for ABPs. Cytochalasins

are fungal metabolites (Carter, 1972) that primarily

growing filaments with high affinity (reviewed by Bray,

1979; Cooper, 1987). However, the final viscosity is

markedly reduced implying a shortened filament length

(Bonder and Mooseker, 1987). Experiments in vitro show

that treatment with substochiometric concentrations of cytochalasins initially accelerate F-actin assembly (Brown and Spudich, 1979a; Brenner and Korn, 1980). A similar induction of F-actin polymerization was observed in human

leukocytes (Rao et al., 1992). In vivo, cytochalasin action

may be modified by the interaction of ABPs (Rao et al., 1992) acting either synergistically or in competition. It appears that, as a result of an increase in Mg^""-dependent

ATP hydrolysis by actin monomers, the critical

concentration required for monomer addition at both ends is slightly increased in the presence of cytochalasin (Bonder and Mooseker, 1987) thus, in the long term, the rate of polymerization is reduced, reflecting ADP-monomer addition.

The apparent disparity that an inhibitor can initially

stimulate assembly may be the result of a second, less

well characterised action of cytochalasins. Indeed, it has been proposed that cytochalasins could interfere with dimer

formation (Goddette and Frieden, 1986); nucléation of

filament assembly (Bonder and Mooseker, 1987); prevent annealing; or, alternatively, cleave existing filaments

(Rao et al., 1992) . The contradictory effects seen when

cells are treated with a low dose of cytochalasin as compared with a high dose can be interpreted as the result of dual functions of the reagent eg. capping versus

severing (Brown and Spudich, 1979a). In the presence of

ATP, F-actin is more sensitive to cytochalasins implying an interference of ATP with cytochalasin treatment (Brown and

Spudich, 1979a) . It has also been suggested that CD may

preferentially cap ADP-actin ends thus its bimodal effect may reflect the proportion of ADP ends in a population

(review. Cooper, 1987) . Cytochalasins vary in their

effectiveness on actin ATPase activity (CD>CB), (Brenner

At the cellular level, cytochalasins gross morphological changes, such as rounding up of cultured mouse fibroblasts through contraction of actin cables, and induction of arborisation (Yahara et al., 1982). In these cells CB and CD were also found to induce actin rodlet formation in the nucleus.

(ii) Phalloidin.

Phalloidin is a toxin from the green death cap

mushroom. Amanita phalloides which binds specifically to F-

actin (Faulstich et al., 1988). Fluorescent derivatives of phalloidin have proven to be excellent probes for F-actin localisation in fixed cells. Phalloidin is limited in its utility because its action on F-actin is irreversible,

(ill) Botulinum C2-toxin.

A third type of toxin which has also provided insight into the mechanisms of polymerization, is botulinum C2-

toxin, (Aktories and Wegner, 1989) which functions by

modifying actin subunits. In this case, residue arg-177 is ADP-ribosylated, causing a physical alteration in the monomer which prevents assembly thus disrupting the G- to F-actin equilibrium (Reuner et al., 1987).

1.7f. Actin and the Nucleus.

Many workers have postulated a mechanical contribution

by actin in chromosome separation (Gawadi, 1971) . However,

it is now generally accepted that localization of actin to the mitotic apparatus is probably a result of cytoplasmic contamination after nuclear envelope breakdown (Sanger,

1975) . The absence of actin in the nucleus during mitosis

in species where the nuclear envelope remains intact

supports this notion (Butt and Heath, 1988) . A minor actin

isoform, slightly more acidic (0.05-0.1 pH unit

difference), constituting 1.5% of total cell actin, has

been isolated from nuclei of Acanthamoeha castellan! (Kumar

et al., 1984) . However, its precise in vivo location is

uncertain and it may reside on the nuclear envelope rather than the nucleoplasm. The recent discovery that a mammalian

homolog of yeast act2 , centractin, resides in the microtubule organising centre which juxtaposes the nucleus,

suggests a potential association of this second actin population with the nuclear embedded SPBs of yeast.

In Neozygite, a population of actin, distinct from the

peripheral and apical plaques seen at areas of cell wall growth, is found around the nucleus during interphase and disperses at the onset of septum formation (Butt and Heath, 1988) . This peri-nuclear actin "shell" may be a storage depot (Butt and Heath, 1988) or, alternatively, a means of maintaining the correct position of the nucleus as is seen

in S.cerevisiae (Palmer et al., 1992).

Various stresses such as 10% DMSO, heat shock (Hirono

et al., 1987), or treatment with cytochalasin B and

cytochalasin D (Yahara et al., 1982) can artificially induce intranuclear incorporation of F-actin bundles or rodlets.

1.7g. Actin in Saccharomvces cerevisiae.

In S .cerevisiae, actin is distributed asymmetrically

and changes in a defined way through the cell cycle (Kilmartin and Adams, 1984; Adams and Pringle, 1984). The site of bud emergence is anticipated by a ring of punctate patches composed of F-actin which disperses as buds enlarge. Cells with large buds have actin cables aligned along their longitudinal axes. Cortical patches of actin localize to areas of cell surface growth. At the mother-bud neck a single or double F-actin ring, distinct from the bud emergence ring, develops prior to cytokinesis, at which time there are no F-actin cables in the mother cell.

Cortical patches at areas of cell growth may be

transmembrane linkages that rivet the cytoskeleton to the cell membrane in a similar way to fibroblast adhesion plaques sticking to a surface, or, alternatively they may be acting to strengthen the tip during growth (Adams et a l ., 1991).

Botstein, 1985) are osmotically sensitive and tend to grow uniformly rather than in a polarised manner. Yeast actin behaves like other actins except that its polymerization appears to be unusually sensitive to calcium (Greer and

Schekman, 1982b) , although this may have been due to a

contaminating protein.

Originally it was thought that actin in S .cerevisiae

was encoded by a single gene, ACTl (Ng and Abelson, 1980). Recently, however, a second actin, encoded by ACT2 and 47%

identical to ACTl was found in S .cerevisiae, (Schwob and

Martin, 1992) . ACT2 disruptants block with large buds

implying a role for ACT2 late in the cell cycle, possibly

in cytokinesis. Moreover, the consensus motif, TPLK,

involved in phosphorylation by p3 4 =^0 2^ is found in this actin-like protein and suggests that direct regulation of actin through phosphorylation is feasible.

1.7h. Actin in Schizosaccharomvces vombe.

As in S .cerevisiae, filamentous actin (F-actin)

predicts sites of cell wall deposition in S.pombe, thus it

is intimately involved in maintaining polarity of growth and septum formation. The changes in distribution of F- actin during the cell cycle have been investigated using rhodamine conjugated phalloidin (Marks and Hyams, 1985) and follows the scheme shown below: