J. Cell Set. 75, 131-147 (1985) 131 Printed in Great Britain © The Company of Biologists Limited 1985 IMMUNOFLUORESCENCE MICROSCOPY OF MICROTUBULES IN INTACT CELL LINEAGES OF THE MOSS, PHYSCOMITRELLA PATENS I. NORMAL AND CIPC-TREATED TIP CELLS JOHN H. DOONAN 1 , DAVID J. COVE Department of Genetics, The University, Leeds, U.K. AND CLIVEW. LLOYD* Biosciences Division, Unilever Research Coltvorth Laboratory, Sharnbrook, Bedford, UK. SUMMARY Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde- derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that there was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC — a drug that is known to produce multiple microtubule- organizing centres — and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration - processes that are fundamental to the morphogenesis of filamentous organisms. INTRODUCTION Indirect immunofluorescence studies of the cytoskeleton have provided great insight into the cytoplasmic organization and behaviour of animal cells but there have been few studies on plants. This can be partly attributed to the technical difficulties associated with walled cells, which do not adhere and flatten to the substratum. Consequently, the short history of the application of indirect immunofluorescence to plants represents a series of technical advances. The first study employing anti- tubulin made use of wall-less Haemanthus endosperm to stain mitotic spindles • Presen t address: Department of Cell Biology, John Innes Institute, Colney Lane, Norwich, U.K. Key words: microtubules, tip growth, moss, cytoskeleton.