Mitochondria and Nuclei Move by Different Mechanisms in Aspergiilus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, ~-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal ~-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules. OrganeUar translocation in its many forms is essential to the growth and maintenance of most, if not all, eukaryotic ceils. In recent years organellar translocation has been studied extensively in many organisms (reviewed in reference 18) and while there is now good evidence that some forms of organellar translocation are microtubule-mediated (2, 19, 22, 23; and other data reviewed in reference 18) and others are mediated by actin microfilaments (reviewed in reference 18), the mechanisms of force production and the mechanisms by which force production is regulated are not known. We are studying organellar translocation in the filamentous fungus Aspergillus nidulans because this organism has a very good genetic system that makes it possible to apply the power of genetics to analyze the mechanisms of organellar translocation. Mutations in genes that encode the microtubule proteins a and/~ tubulin have been isolated in this organism (12, 15, 20) as have mutations that specifically inhibit nuclear movement (11, 14). These mutations have been used to demonstrate that nuclear movement is microtubule-mediated (14, 15). Our long-term goal is to identify the components of the organellar translocation apparatus(es) in A. nidulans and to determine how these components interact to produce organeUar movement. One of our initial goals has been to determine if there is 2392 but a single mechanism responsible for the translocation of organelles in A. nidulans or if there are two or more mechanisms as there apparently are in some organisms (reviewed in reference 18). We have, consequently, examined the movement of mitochondria from conidia (asexual spores) into germ tubes in the presence of the antimicrotubule agent benomyl and in strains that carry mutations that inhibit nuclear movement. We have found that while benomyl and these mutations inhibit the movement of nuclei from the conidium into the germ tube, mitochondrial movement occurs in virtually all germlings including those in which nuclear movement is blocked. These data demonstrate that there are at least two mechanisms of organellar translocation in A. nidulans. This work was presented in preliminary form at the 24th Annual Meeting of the American Society for Cell Biology, Kansas City, MO, November 12-16, 1984. MATERIALS AND METHODS Strains: Strains B3, BEN14, and BEN17 were originally constructed by Dr. J. M. van Tuyl (Agricultural University, Wageningen, The Netherlands) and were obtained by us from Dr. N. R. Morris (Rutgers Medical School, Piscataway, N J). Each of these strains carries biA 1. BEN 14 carries benA 16, and BENI7 carries benAl9. BRO2 was constructed by Dr. Bed R. Oakley in the THE JOURNAL OF CELL BIOLOGY • VOLUME 101 DECEMBER 1985 2392-2397 © The Rockefeller University Press • 0021-9525/85/12•2392•06 $1.00 on Jauary 8, 2018 jcb.rress.org D ow nladed fom laboratory of Dr. N. R. Morris and carries benA33 and yA2. The cold-sensitive (cs-) t B-tubulin mutants are revertants of BRO2. Each carries benA33 and each carries a different closely linked (recombination frequencies < 1.2 x l0 -4) intragenic suppressor that confers cold sensitivity. These revertants were isolated and characterized by Bed R. Oakley, C. Elizabeth Oakley, Kimbedy S. Kniepkamp, and Janet E. Rinehart (17). FGSCI54 was originally obtained from the Fungal Genetics Stock Center (Humboldt State University, Arcata, CA) and was obtained by us from Dr. N. R. Morris. It carries adE20, biA 1, wA2, cnxE, sC 12, methG l, nicA2, lacA l, choA l, and chaA 1. The nud mutants were isolated by Morris (1 l) and carry mutations nudAl-nudE5 in a FGSC154 background. We obtained these mutants from Dr. N. R. Morris. G r o w t h C o n d i t i o n s : All experiments were carried out in complete medium (YG medium, 5 g/liter yeast extract, 20 g/liter dextrose) containing 0.1% agar to inhibit conidial clumping. All incubations were carried out under constant agitation on New Brunswick gyratory shakers (New Brunswick Scientific Co., Inc., Edison, N J). Microscopy: A variety of fixation and staining procedures were tested. We obtained consistently good staining of nuclear and mitochondrial DNA with the following procedure. 0.9-ml samples were taken and fixed with 0. l ml of 10% glutaraldehyde for l0 min. The samples were then washed twice for l0 min in distilled water, once for 15 rain in acetone, and twice for l0 min in distilled water and resuspended in a staining solution containing 0.015 ug/ml 4',6-diamidino-2-phenylindole (DAPI) in distilled water. The acetone wash greatly improved the consistency of staining among germlings. DAPI was stored at 4°C as a 1.5 vg/ml solution in distilled water. Rhodamine 123 was dissolved immediately before use in distilled, deionized water to a concentration of l0 mg/ml and was added to samples at a ratio of I part rhodamine 123 solution to 99 parts sample, or 1 part rhodamine 123 to 199 parts sample. Samples were observed and photographed with a Zeiss standard microscope (Carl Zeiss, Inc., Thornwood, NY) equipped for epifiuoreseence. Neofluor optics were used for all observations and photographs. DAPI staining was observed with a G365 filter set (365-nm exciter filter, 395-nm beam splitter, and 420-nm barrier filter) and rhodamine 123 with a H546 filter set (BP 546/12 [546-nm narrow band] exciter filter, 580-nm beam splitter, and 590-nm barrier filter). To inhibit loss of rhodamine 123 staining, a 0.5 neutral density filter was sometimes used to reduce the intensity of the exciting illumination. Specimens were photographed with Tri-X pan film which was developed with D-76 or Diafine developers (Acufine, Inc., Chicago, IL). C h e m i c a l s : Benomyl (technical grade, 98% pure) was a generous gift from DuPont Co. (Wilmington, DE). DAPI was from Sigma Chemical Co. (St. Louis, MO). Rhodamine 123 was from Eastman Kodak Co. (Rochester, NY). Glutaraldehyde (10% solution, E. M. grade) and formaldehyde (16% solution, E. M. grade) were from Electron Microscopy Sciences (Fort Washington, PA).