The role of spindle microtubule assembly in the control of mitotic timing in Physarum: Induction of a novel type of tubular structure by griseofulvin treatment

Abstract

Synchronous macroplasmodia of the myxomycete Physarum polycephalum were exposed to the antimitotic drug griseofulvin (GF) for varying durations at different phases of the mitotic cycle. Continuous treatment with 50 μg/ml of GF induces mitotic delays dependent on phase, with mitotic delays of up to 6 h when treatment is begun in early G2, and little or no mitotic delays when treatment commences within 1 h of metaphase. All nuclei of GF-treated plasmodia enter an abnormal ring-chromosome metaphase, or stathmometaphase, which persists for about 2 h, and results in polyploid nuclei. Ultrastructural studies on stathmometaphase nuclei reveal total disruption of normal spindle microtubules with the concomitant appearance of enlarged tubular elements of short lengths, with typical diameters of 70–90 nm. These are hollow tubular structures with a wall thickness of 5–6 nm, composed of spherical subunits. No protofilament organization is present. We designate these structures “megatubules” to distinguish them from similar 40–60 nm macrotubules, often observed in Physarum plasmodia subjected to non-specific agents which disrupt spindle microtubules (e.g. heat and CO 2-induced anoxia). Pulses of GF (50 μg/ml) applied for 2 h throughout the mitotic cycle induce maximal mitotic delays at a critical phase (CP) at approx. 100 min before metaphase. This CP corresponds to the time of mitotic center duplication, and GF treatment then results in the suppression of microtubules of the developing microtubule-organizer region, TOR. It is inferred that the drug interferes with the normal process of spindle microtubule assembly, which is essential for signaling the onset of mitosis. Hence, TOR formation itself appears to be one event of the mitotic sequence which is part of the mechanism controlling the timing of the mitotic cycle.