The role of F-actin in determining the division plane of carrot suspension cells. Drug studies

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Abstract
Following the report that a network of F-actin is associated with the nucleus throughout the division cycle, we have examined the involvement of F-actin in determining the division plane of carrot suspension cells. This was achieved by treating cells with drugs and then staining the unfixed cells with rhodaminyl lysine phallotoxin in detergent extraction buffer. In interphase, actin cables radiate from the nucleus but at the cortex become more or less transversely arranged in the pattern already known for cortical microtubules. Concentration of the cortical F-actin into a band at preprophase draws most of the nucleus-associated actin into a transvacuolar disc, thereby forming the phragmosome within which mitosis and cytokinesis occur. In addition to this transversely aligned structure, F-actin is also associated with the spindle poles during mitosis but these filaments tend to align at right angles to the phragmosomal actin. F-actin therefore defines transverse and longitudinal vectors as division approaches. Depolymerization of F-actin with cyto-chalasin D can cause the spindle axis to reorientate such that the pole-pole axis comes to lie, abnormally, parallel with the phragmosome. The cytokinetic apparatus (the phragmoplast) develops centrifugally within the phragmosome. There has been considerable speculation on the nature of the elements that guide the phragmoplast to the cortical site previously occupied by the preprophase band of microtubules. This study demonstrates that F-actin bridges the leading margin of the outgrowing phragmoplast to the opposing cortex. Radial actin strands therefore provide a ‘memory’ of the predetermined division plane whose perimeter had been marked at preprophase by a band composed of microtubules and F-actin. This relationship was perturbed with the herbicide, chloroisopropylphenyl carbamate. Preprophase bands of actin appear to form normally in herbicidetreated cells. However, cytokinesis does not occur within this predicted plane since the drug perturbs the mitotic spindle, forming three nuclei which become separated by Y-shaped, actin-containing phragmoplasts. Cytoplasmic actin strands connect the edges of the phragmoplast to the cortex. It is suggested that the irregular distribution of F-actin, which radiates from the herbicide-altered mitotic apparatus, provides alternative paths for outgrowth of the abnormal phragmoplasts. Caffeine is known to cause failure of cell plate formation. But apart from inducing cytoplasmic ‘starbursts’ of F-actin in interphase cells it does not appear to have any effect on F-actin-containing division structures. It is concluded that the formation of a transvacuolar phragmosome, spindle alignment and the ‘correct’ outgrowth of a planar cytokinetic apparatus to the predetermined boundary of the division site all involve F-actin.