Comparative immunocytochemistry of the cytoskeleton in filamentous fungi with dikaryotic and multinucleate hyphae
Authors: Salo Vanamo , Niini Sara S. , Virtanen Ismo , Raudaskoski Marjatta 
Published: April 2021

ABSTRACT
Indirect immunofluorescence (HF) microscopy of microtubules of five filamentous fungi, one asco-mycete, Gyromitra esculenta, and four homobasi-diomycetes, Amanita regalis, Heterobasidion anno-sum, Paxillus involutus and Suillus bovinus, representing saprophytic and mycorrhizal species, indicated that the gross structure of the microtubule cytoskeleton was comparable in spite of the multinucleate and dikaryotic cell conditions. In the apical cells of each species at interphase, longitudinally oriented microtubule tracks extended from the cross wall to the apex with strictly longitudinal orientation occurring in the apical part of the hypha. Immunoblots indicated that α- and β-tubulin mobilities of the dikaryotic species A. regalis, P. involutus and S. bovinus were identical, while the tubulin mobility patterns of the homobasidiomy-cete H. annosum and the ascomycete G. esculenta, both with multinucleate hyphae, differed slightly from each other and from those of the dikaryotic species. The use of HF microscopy made it possible to examine the relationship between the cytoplasmic microtubules and spindle formation in the long apical cells of the fungal hyphae. During nuclear division cytoplasmic microtubules disassembled in a region extending 40-60 μm around the dividing nucleus/nuclei. In multinucleate hyphae containing dividing nuclei close to the apex and in dikaryotic hyphae with short apical cells, disassembly of the cytoplasmic microtubules extended up to the apex, which may be an important observation when the function of cytoplasmic microtubules in the apical cells is considered. The disassembly of the cytoplasmic microtubules was first associated with an increase in the size of the spindle pole body (SPB), and then with the development of the spindle from the SPB. This suggested that the SPB had a central role in nucleating spindle microtubules from the pool of tubulin subunits built up through the disassembly of the cytoplasmic microtubules. The changes in structure of the SPB during nuclear division were clearly seen in the large nuclei of A. regalis and in the apical cells of H. annosum containing a row of non-synchronously dividing nuclei, each with an SPB at a different phase. In the dikaryotic and multinucleate hyphae of homobasi-diomycetes the development and structure of the spindle was similar, while in the ascomycete G. esculenta certain deviant features were recorded. The significance of the close orientation of the spindles, observed at anaphase, for the maintenance of the heterokaryotic nuclear condition in the hyphal cells of the homobasidiomycetes is discussed. Actin was detected in the apical region of the hyphae as well as at the developmental phase of the septum and was identified by immunoblot analyses for the first time in the homobasidiomycetes. The tips of the slow-growing hyphae of P. involutus and S. bovinus stained intensely with NBD-phallacidin as compared to the weak reaction seen in the fast-growing species. The detection of actin in all the species by a monoclonal anti-actin antibody, suggested that the stability of filamentous actin was greater at the hyphal apex in slow-growing than in fast-growing hyphae.