Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus, Gastropoda)

Abstract

The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3-1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5-50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100-200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs - as in untreated eggs - microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.

Cell lineage-specific inhibition of cytokinesis by concanavalin A in a molluscan embryo (Nassarius reticulatus, Gastropoda)

“…Previous studies have d e m o n s t r a t e d that the vegetal m e m b r a n e o f the Nassarius egg is e n d o w e d with special properties, such as increased lateral m o b i l i t y o f m e m b r a n e lipids, as well as a higher density o f i n t r a m e m b r a n e particles (Speksnijder et al 1985a, b). In addition, in our accomp a n y i n g p a p e r (Speksnijder et al 1991), we concluded that the p a r t i c u l a r effects o f C o n A on p o l a r lobe formation are p r o b a b l y due to binding o f C o n A to sites at the vegetal pole. Thus, the cell lineage-specific effects of Con A on cleavage could be explained by inheritance of this vegetal membrane by the CD-blastomere.…”

“…It seems, therefore, that the egg cortex is not capable of responding fully to this furrowing signal and that the assembly of a contractile ring, or its contraction, is inhibited by the action of Con A. Our accompanying paper indeed shows that Con A-binding greatly affects the organization of filamentous (F)-actin in the egg cortex (Speksnijder et al 1991), which we suggest may be caused by altered levels of intracellular calcium ions.…”

“…The mechanism by which binding of Con A to sites at the vegetal membrane affects cleavage in the CDblastomere probably involves a transmembrane signal triggered by cross-linking of Con A-binding sites (see Speksnijder et al 1991). This signal may induce the production of intracellular messengers, which in turn could affect cytokinesis by inhibiting the stimulus for furrowing or, alternatively, the responsiveness of the cortex to the furrowing signal.…”

“…Incubation with high doses of Con A at this trefoil stage results in the regression of the cleavage furrow and irreversible inhibition of further cleavage. In addition, the constriction of the polar lobe is shifted toward the animal pole (Speksnijder et al 1991). When low doses, below 1 ~tg/ ml, are used during the same period, first cleavage will proceed normally in most cases although the resorption of the polar lobe into the CD-blastomere may not be entirely complete.…”

“…1 a, b), which becomes shifted toward the animal pole. The effects of Con A on polar lobe formation have been investigated in detail in our accompanying paper (Speksnijder et al 1991).…”