Reassembly of microtubules in Nitella tasmanica: assembly of cortical microtubules in branching clusters and its relevance to steady-state microtubule assembly

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Abstract
Giant internodal cells of Nitella tasmanica have cortical microtubules beneath the plasma membrane and endoplasmic microtubules associated with sub-cortical actin bundles and nuclei. We depolymerized the microtubules with oryzalin and followed their reassembly by immunofluorescence. At 18°C (the standard temperature of culture), microtubules were lost from young cells within 10 min and the first microtubules were detected in the cortex within 20 min of washing out the herbicide. Microtubules of older cells disassembled and re-formed more slowly. Continued cortical microtubule assembly was at acute angles to the first-formed microtubules, building branching clusters of microtubules. At 25°C, cortical microtubule assembly generated less extensively branched clusters and was completed more rapidly. Larger clusters but shorter MTs were generated in older cells. Reassembly of microtubules in the endoplasm only began 50 min after the removal of oryzalin. We therefore conclude that assembly proceeded independently in the cortex and endoplasm. Cortical assembly involves scattered assembly events initiating microtubules from which, as the latter elongate, further microtubules assemble as branches. We suggest that similar processes operate in steady-state cells and we explain with a simple model why branched clusters of microtubules are unusually large after microtubule depolymerization. By proposing that these processes show differential changes in activity with temperature and during cell ageing, we can account in qualitative terms for the age- and temperature-dependent differences in microtubule reassembly patterns.