Re-establishment of the interphase microtubule array in vacuolated plant cells, studied by confocal microscopy and 3-D imaging

There are two conflicting ideas about the site of reassembly of cortical microtubules following cytokinesis. Some observations indicate that microtubules (MTs) radiate from the surface of the postcytokinetic nuclear envelope, before becoming organized at the cortex. On the other hand, results of regrowth experiments, following MT depolymerization by drugs, suggest that the array may assemble directly upon the cortex. In this study, we have taken advantage of the significant separation between nucleus and cortex, in large, vacuolated epidermal cells, to determine which of these two potential sites supports the earliest stages of regrowth in the undrugged, native state. MTs in stem epidermis of Datura stramonium L. were stained by indirect immunofluorescence. This was performed on hand-cut sections of tissue in which the cells were not separated by enzymes or distorted by air- drying. Epidermal cells with these sheets were optically sectioned by confocal laser scanning microscopy and three-dimensional images reconstructed, rotated and viewed stereoscopically using computer methods. During metaphase, no MTs can be detected at the cortex but MTs begin to re-colonize the cell surface during early cytokinesis. Thick cables of MTs radiate from the nucleus parallel to the cell plate as well as in other directions, along transvacuolar strands, out to the cortex. Microtubules grow out over the cortex where the thick bundles make contact, as well as from the edges of the fully developed phragmoplast. These early cortical MTs do not form regular transverse arrays: they either appear to be random or to grow in branching V- shaped patterns. The cortical array is therefore not organized immediately but at a later stage. It is concluded that MT bundles, radiating from the nucleus, are involved in the earliest stages of cortical array formation.