Cytochalasin B II: Selective Inhibition of Cytokinesis in Xenopus laevis Eggs

Abstract

Carter (1) has described inhibition of cytoplasmic division (cytokinesis) with unimpaired nuclear division (karyokinesis) in mammalian cells treated with cytochalasin B (CB), a metabolite from the mold, Helminthosporium dematioideum. We report a similar effect of CB on the initial cleavages of Xenopus Zaevis embryos. Our data on both the duration and magnitude of drug effect define temporal limits for the act of cytokinesis and suggest that a pool of material which will be used for subsequent cleavages is available prior to the first cleavage. The demonstration that nuclear division can be separated from cytoplasmic division has been made before with both chemical and physical agents (2-7). However, because CB seems to have minimal effects on other ceIl processes (8), we felt that it would be an ideal tool for the definition of those events surrounding cytoplasmic division.Methods. Batches of 10-1 5 eggs, fertilized within approximately 2 min of each other, were collected from X . Zaezris adults hormonally induced to mate ( 9 ) . The outer jelly coats were removed using watchmakers forceps and the eggs were exposed to 1-10 pg/mlpared by diluting, with distilled water, stock solutions of 1 mg/ml of CB in dimethyl sulfoxide (DMSO) . Although CB is only sparingly soluble in pure water, it is readily soluble in dilute solutions of DMSQ. ControIs were exposed to 1% DMSO in distilled water, the maximum concentration of DMSO used in the experimental solutions. I n our initial experiments we found that solutions of 10 pg/ml or more of CB caused pigment clumping and rotation of the eggs, both signifying death, while controls in 1% DMSO were unaffected. The remainder of our experiments were run with doses of 1-5 pglml.Further details of procedure, ie., concentration of CB, time and duration of exposure to CB, are given in the text below and in the figure legend. Results. We have focused our attention on the first two cleavages and the beginning of the third. CB produced consistent alterations in the external features of the cleavage furrow as well as in the internal structure seen in histological sections. The following external changes occurred when the eggs were placed in 5 pg/ml of CB immediately after removal of the outer jelly coat: (a) the first two cleavage furrows began at the usual time and position and with a normal appearance; the third cleavage furrow began a t the usual time but was disoriented, i.e., it was nearly meridional rather than equatorial ; (b) the progression of the first two furrows around the egg appeared normal; (c) however, about the time each furrow was completed at the vegetal pole, "pigment separation" was observed in the depths of the furrow a t the animal pole; ( d ) pigment separation became more pronounced and continued along the furrow which became shallower and finally disappeared leaving a white band of poorly pigmented cortex as the only surface indication of the cleavage plane. Events (c) and (d) are referred to below as "rever~al" of the furrow.Certain alterations in internal struct...

Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos

“…Treatment with critical doses of such agents led to cleavage arrest but did not completely inhibit the nuclear cycle. Although similar effects on cleavage and the nuclear cycle were described with cytochalasin B (Hammer et al, 1971), different results were obtained with anti-microtubule agents in other laboratories. For example, nuclear division was arrested, along with cleavage, following treatment with colchicine (Takeichi et al, 1985).…”

On the molecular basis of action of cytochalasin B

“…This is best illustrated by the studies on inhibition of cytokinesis by cytochalasin B. This is the first system where the effects of the drug were observed (32), and it has been the subject of detailed studies conducted by several investigators (12,13,33,34). There is strong evidence unrelated to cytochalasin B that the contractile ring plays an important role in the cleavage of eukaryotic cells during cell division (12,35).…”

Actin in Dividing Cells: Contractile Ring Filaments Bind Heavy Meromyosin

“…In studies involving newt eggs Perry and coworkers (7) have recently demonstrated that the CR complexes with HMM, and they therefore concluded that it contains an actin-like component. Unfortunately their conclusion is not automatically applicable to contractile rings in other cells because CR microfilaments in amphibian eggs seem to be unique on two grounds: (i) they are thicker (8-10 nm) (8,9) than CR microfilaments elsewhere (4-6 nm) and (ii) whereas cytochalasin B brings about disruption of CR microfilaments in HeLa cells and sea urchin eggs (4,5), the drug elicits very little response in cleaving amphibian eggs, either in terms of inhibiting contraction or disrupting the CR (8,10,11). In this last connection, it might be reasoned that since cytochalasin B has little or no effect on native actomyosin from muscle (12)(13)(14) (Fig.…”