Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

Abstract

The dynamics of microtubule (MT) disassembly and reassembly were studied in the green alga Ernodesmis verticillata, using indirect immunofluorescent localization of tubulin. This alga possesses two distinct MT arrays: highly-ordered, longitudinally-oriented cortical MTs, and shorter perinuclear MTs radiating from nuclear surfaces. Perinuclear MTs are very labile, completely disassembling in the cold (cells on ice) within 5-10 min or in 25 μM amiprophos-methyl (APM) within 15-30 min. Although cortical MTs are generally absent after 3 h in APM, it takes 45-60 min before any cold-induced depolymerization is apparent, and some cortical MTs persist after 6 h of cold treatment. The extent of immunofluorescence of cytoplasmic (depolymerized?) tubulin is inversely proportional to the abundance of cortical MTs. Recovery of MT arrays upon warming or upon removal of APM occurs within 30-60 min for the perinuclear MTs, but the cortical arrays take much longer to regain their normal patterns. The cortical MTs initially reappear in a random distribution with respect to the cell axis, but within 3-4 d of warming (or 24-36 h of removing APM) they are nearly parallel to each other and to the cell's longitudinal axis. Thus, although the timing differs, the actual patterns of depolymerization and recovery are similar, irrespective of whether physical or chemical agents are used. Longer-term treatments in 1 μM APM indicate that despite the rapid disappearance of perinuclear MTs, a loss of the uniform nuclear spacing occurs gradually over 1-6 d. Similar disorganization of nuclei is obtained with long-term treatment with 1 μM taxol, where a gradual loss of perinuclear MTs is accompanied by an increased abundance of mitotic spindles. This implies that perinuclear MTs can disassemble in vivo in the presence of taxol, and that they are not the sole components involved in maintaining nuclear spacing in these coenocytes. The results indicate that both nuclear and cortical sites of MT nucleation may exist in this organism, and that MT reassembly and re-organization are temporally distinct events in cells that have highly-ordered arrays of long MTs.